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CytoJournal 2011,  8:16

Abstracts for the 59th Annual Scientific Meeting (November 2011) by American Society of Cytopathology (ASC) at Baltimore, MD, USA

Date of Submission12-Oct-2010
Date of Acceptance26-Mar-2011
Date of Web Publication16-Sep-2011

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1742-6413.84993

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 » Abstract 

These are peer-reviewed poster-platform submissions finalized by the Scientific Program Committee. A total of 153 abstracts (14 Platforms [PP1 through PP14] & 139 Posters [1 through 139]) were selected from 161 submissions to be considered for presentation during November 4 - 8, 2011, at the Hilton Baltimore Hotel, to pathologists, cytopathologists, cytotechnologists, residents, fellows, students, and other members of cytopathology-related medical and scientific fields.

Keywords: Abstracts, American society of cytopathology, ASC, cytopathology, cytology

How to cite this article:
. Abstracts for the 59th Annual Scientific Meeting (November 2011) by American Society of Cytopathology (ASC) at Baltimore, MD, USA. CytoJournal 2011;8:16

How to cite this URL:
. Abstracts for the 59th Annual Scientific Meeting (November 2011) by American Society of Cytopathology (ASC) at Baltimore, MD, USA. CytoJournal [serial online] 2011 [cited 2019 Dec 5];8:16. Available from: http://www.cytojournal.com/text.asp?2011/8/1/16/84993

Entire text can be searched in the uploaded PDF copy on the computer. Please press Ctr and F key simultaneously. Put text you want to search and press Enter key.

 » Platform Presentations Top

PP 1

Can high risk human papillomavirus positivity in a negative for intraepithelial lesion or malignancy in women 30 years of age and older be used to select cases for quality control review?

Sarah Wehmhoefer, CT(ASCP), Michael Beckmann, DO

Department of Laboratory Medicine and Pathology, Memorial Medical Center, Springfield, Illinois

Introduction: This study is aimed to evaluate if women aged 30 years or older, who are Pap and high-risk human papillomavirus (HR HPV) co-tested and found to be negative for intraepithelial lesion or malignancy (NILM) and positive for HR HPV, would benefit from a retrospective Pap re-screen.

Materials and Methods: Following institutional review board approval, a search of the cytopathology laboratory database from January to December, 2010, yielded 55975 Pap tests. One thousand seven hundred and six (3%) patients aged 30 years or older (range 30 - 61) were found to have been co-tested with a SurePath® Pap and HR HPV. High risk HPV testing was performed using Qiagen (Digene) Hybrid Capture 2 (HC2). Of those 1706, 72 (4%) patients who had an NILM Pap and were positive for HR HPV were re-screened by a Quality Control (QC) cytotechnologist (SMW) with the knowledge of the HR HPV status. Those cases determined to be abnormal upon re-screening were evaluated by a cytopathologist (MJB) who determined the final re-screen interpretation.

Results: Of the 72 re-screened cases, 12 cases (17%) were found to be atypical squamous cells (ASC) or higher on the final re-interpretation. Six cases were found to be atypical squamous cells of undetermined significance (ASC-US), two Atypical Squamous Cells, cannot exclude high grade squamous intraepithelial lesions (ASC-H), two low-grade squamous intraepithelial lesions (LSIL), two low-grade squamous intraepithelial lesions, Cannot exclude high-grade squamous intraepithelial lesions (LSIL-H), and 0 high-grade squamous intraepithelial lesions (HSIL) or atypical glandular cells (AGC). Given the cytopathology laboratory use of the focal point primary screening instrument, 40% of all Pap tests advanced to a QC queue. Of those QC Pap tests, 1.5% were upgraded to ASC or higher upon re-screening in 2010.

Conclusions: The focused re-screening of NILM Pap tests with positive HR HPV of women 30 years and older showed a higher detection rate of ASC or higher compared to routine QC screening. The current American society for colposcopy and cervical pathology (ASCCP) consensus guidelines recommend repeating both the Pap test and HR HPV test in one year if a woman aged 30 years or older has an NILM Pap and positive HR HPV test. If the follow-up Pap test is again NILM and HR HPV positive, colposcopy is suggested. This algorithm may change in the future, but focused re-screening in this patient population may provide an additional QC measure in the interim.

PP 2

The tahoe study: Bias in interpretation of Pap tests when HPV status is known

Ann Moriarty, MD 1 , Ritu Nayar, MD 2 , Andrew Renshaw, MD 3 , Nicole Thomas, MPH, CT(ASCP) 4 , Rhona Souers, MS 5

1 Pathology, AmeriPath, Indianapolis, Indiana; 2 Pathology, McGaw Medical Center of Northwestern University, Chicago, Illinois; 3 Pathology, Baptist Health, Miami, Florida; 4 Surveys, College of American Pathologists, Northfield, Illinois; 5 Biostatistics, College of American Pathologists, Northfield, Illinois

Introduction: The performance characteristics of cervical cytology as a primary screening test are well known. However, little data is available on the performance of Pap tests if the human papillomavirus (HPV) status is known prior to screening. The potential bias of the HPV status may affect the Pap test performance if HPV molecular testing is used as an initial screening test for cervical cancer followed by Pap testing.

Materials and Methods: Forty de-identified liquid-based Pap tests were selected from a quality assurance (QA) program that reviewed negative Pap tests after a positive HPV test. They were divided into two groups of 20 slides and circulated among two randomly assigned groups of 22 members, from the College of American Pathologist (CAP) Cytopathology Committee (CYP), who categorized them according to the Bethesda System (TBS). The slides were re-labeled and each batch was circulated to the opposite group, who also categorized them using TBS, after being told that all cases were HPV positive. Each response was evaluated against the general diagnosis category of negative for intraepithelial lesion (NILM) and epithelial cell abnormality (ECA), as well as, the descriptive diagnostic categories of atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL). Differences in the responses between groups were analyzed statistically by the chi-square and Cochran-Mantel-Haenszel tests at the 0.05 significance level.

Results: The unbiased group were more likely to identify the slides as negative for intraepithelial lesion or malignancy (NILM) and less likely to identify an epithelial cell abnormality (ECA) than the biased group (P < 0.001), as depicted in [Table 1]. There was an increase in each descriptive diagnostic category of epithelial cell abnormalities in the biased responses as compared to the unbiased responses (P = 0.002), as seen in [Table 2].

Conclusions: Knowledge of HPV status creates a bias in the interpretation of Pap tests. When the HPV status is known, observers are more likely to report the Pap test as abnormal and with greater frequency in all categories of epithelial cell abnormality (ASC-US, LSIL, and HSIL). If molecular testing is used as a primary screening test and Pap tests are used as secondary or reflex tests, it is more likely that the Pap test will be interpreted as abnormal, as compared to when a Pap test is used as a primary screening test.

PP 3

Risk profiling using HPV genotyping in women with mildly abnormal Pap results

Ming Guo, MD 1 , Jianping Wang, CT(ASCP) 1 , Marilyn Dawlett, CT(ASCP) 1 , Shobha Patel, CT(ASCP) 1 , Ping Liu 2 , Yun Gong, MD 1 , Therese Bevers, MD 3 , Nour Sneige, MD 1

1 Pathology, UT MD Anderson Cancer Center, Houston, Texas; 2 Biostatistics, UT MD Anderson Cancer Center, Houston, Texas; 3 Cancer Prevention Center, UT MD Anderson Cancer Center, Houston, Texas

Introduction: HPV DNA testing is the standard of care in triage of women with atypical squamous cells of undetermined significance (ASC-US) Pap results. Genotyping for HPV types16 / 18 was recommended by the American Society for Colposcopy and Cervical Pathology (ASCCP) for women with Pap- / HPV+ results. It is unknown whether adding a genotyping test for HPV 16 / 18 would improve patient management by risk profiling in women with mildly abnormal Pap results, that is, ASCUS, or low-grade squamous intraepithelial lesion (LSIL). To answer this question, we performed a study to evaluate the predictive value of HPV genotyping for cervical intraepithelial lesion / vaginal intraepithelial neoplasm (CIN / VAIN) 2+ in women with mildly abnormal Pap results and HPV DNA+ testing results.

Materials and Methods: We collected SurePath® Pap specimens from 351 women at the Department of Pathology, MD Anderson Cancer Center from 2005 to 2008 that met the criteria of this study. From these, 240 specimens had ASCUS Pap results and 111 specimens had LSIL Pap results, all with Hybrid Capture (HC2) HPV testing results. HPV genotyping was performed on these using the EasyChip HPV assay, which can detect 39 HPV genotypes. The Pap specimens with HPV genotypes 16 / 18, high-risk non16 / 18 (HR-HPV), and low-risk HPV types were compared with the follow-up biopsy results. Follow-up duration ranged from 1 to 36 months with a mean of 21 months.

Results: Of the 351 women, 36 (10.3%) had HC2- results. Genotyping showed low-risk HPV in 2 cases (5.6%, 2 / 36). In 315 women with HC2+ results, 18 (5.7%) tested negative using the polymerase chain reaction (PCR) assay. The distribution of HPV genotypes and a comparison with the results of follow-up biopsies appear in [Table 1]. Women with ASCUS or LSIL Pap results and HPV16 / 18 had significantly higher rates of CIN / VAIN2 / 3 on follow-up biopsies than did women with low-risk HPV types (P = 0.01-0.02). Higher odds ratios (OR) for CIN / VAIN2+ was found in women who had HPV16 / 18 genotyping and ASCUS (13.9, 95% CI, 1.7-113.4) or LSIL (5.7, 95% CI, 1.4-24.4) Pap results compared with women who had low risk-HPV genotypes.

Conclusions: HPV genotyping may be useful for risk profiling in women with mildly abnormal Pap results, especially ASC-US. Genotyping for HPV 16 / 18 may be valuable in patient management for predicting CIN / VAIN 2 / 3 in women with mildly abnormal Pap results.

PP 4

Performance characteristics of urinary tract Cytology: Observations from the College of American Pathologists interlaboratory comparison program in non-gynecological cytopathology (CAP NGC)

Guliz Barkan, MD, FIAC 1 , Manon Auger, MD 2 , Walid Khalbuss, MD, PhD 3 , Rodolfo Laucirica, MD 4 , Vijayalakshmi Padmanabhan, MD 5 , Rhona Souers, PhD 6 , Ann Moriarty, MD 7

1 Pathology, Loyola University, Maywood, Illinois; 2 Pathology, McGill University, Montreal, Ontario, Canada; 3 Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania; 4 Pathology, Baylor College of Medicine, Houston, Texas; 5 Pathology, Dartmouth Medical School, Lebanon, New Hampshire; 6 Statistics, College of American Pathologists, Northfield, Illinois; 7 Pathology, Ameripath Indiana, Indianapolis, Indiana

Introduction: Urinary tract cytology is currently used to evaluate patients with hematuria or as a management tool for those with urothelial neoplasia. This study investigates the performance characteristics of urinary tract specimens in the College of American Pathologists Educational Interlaboratory Comparison Program, CAP NGC, over an 11-year period.

Materials and Methods: The study evaluated participant responses between 2000 and 2010 from the CAP NGC with a reference diagnosis of 'positive' for malignancy (including high-grade urothelial carcinoma (HGUC), squamous cell carcinoma (SCC), or adenocarcinoma (ADC), and 'benign' diagnoses (such as Polyoma virus infection and ileal loop urine). The responses were analyzed with respect to the sample preparation type (conventional, liquid-based, and cytocentrifuged specimens) and participant type (laboratory, pathologist, cytotechnologist). The analysis was performed using a nonlinear mixed model, fitted with three factors - participant, preparation, and diagnosis types. The model also included the interaction terms between these factors and a repeated measures component to model the slide factor correlation structure. A P-value less than 0.05 was considered statistically significant.

Results: There were 96,093 responses from 1823 slides (46,637 pathologists, 29,976 cytotechnologists, 19,480 laboratories). Of the 74,821 responses for the 'positive' general diagnosis, 93.3% were concordant. Of the 21,272 responses for the 'benign' general diagnosis, 87.9% were concordant (P < 0.001). The main diagnostic difficulties in the malignant category were subtyping ADC and HGUC, and SCC versus HGUC. For the benign cases, the primary diagnostic pitfall was over-interpreting the negative cases, ileal loop specimens, and Polyoma virus specimens as HGUC. Within the positive reference diagnosis, cytotechnologists performed better than pathologists, whereas, pathologists performed better with the negative challenges (P < 0.001). Specifically, while pathologists were more likely to undercall HGUC as Polyoma, cytotechnologists were more likely to overcall Polyoma as HGUC (P < 0.001). Overall, the liquid-based samples performed significantly better than the other preparations (P < 0.001).

Conclusions: Urinary tract cytology preparations perform well in an interlaboratory comparison program. Liquid-based preparations performed the best. There were diagnostic difficulties subclassifying malignant cases as well as interpreting negative, ileal loop specimens, and Polyoma virus challenges as malignant.

PP 5

Malignant peritoneal and pleural fluid samples are adequate for molecular profiling

Raheela Ashfaq, MD, Yolanda Fong, MD

Caris Life Sciences, Irving, Texas

Introduction: Targeted therapy for cancer treatment is a growing aspect of clinical oncology and pathology. The Caris Target Now™ is a proprietary, evidence-based molecular profiling system for solid tumors providing specific and individualized molecular profiles for guidance of therapy in advanced stage and metastatic malignancies. The Target Now system is platform agnostic and utilizes data collected from a combination of immunohistochemical stains, Fluorescence in-situ hybridization (FISH), the Gene expression array, and sequencing tests, to give the final therapeutic guidance. A recent publication by Daniel Von Hoff et al. (JCO: 2010 Nov. 20; 28(33): 4877 - 83) shows improved progression-free survival with assay-guided therapy compared to physician-selected therapy. As acquisition of tissue in patients with advanced cancer can be challenging, we are reporting our experience with molecular profiling on malignant fluid samples.

Materials and Methods: A computer search was conducted to retrospectively identify malignant fluid samples or cell blocks submitted by various oncology service providers for the explicit purpose of molecular profiling of patients with advanced cancer. A cell block was either prepared or available for testing of all samples. A hematoxylin and eosin (H and E) slide was prepared from the cell block and reviewed by a pathologist before any testing. Malignant cell percentages were determined for the purpose of DNA microarray analysis and sequencing. Appropriate clusters and malignant cells were marked for FISH. The results were reviewed and data compiled, to calculate the yield of various molecular predictive tests.

Results: From January 2009 to April 2011, we studied 172 samples of peritoneal and pleural fluids. In order of frequency, the most common primary sites were, the lung (n = 49, 28.4%), ovary (n = 45, 26.1%), breast (n = 29, 16.8%), and pancreas (n = 5, 2.9%). The rest of the cases (44, 25.5%) included colon, peritoneum, endometrium, and others. We were able to perform more that 10 immunohistochemical stains in 123 samples (71.5%), 1 - 9 in three samples (1.7%), while 46 samples were insufficient for immunohistochemical analysis (26.7%). DNA microarray analysis was performed in 60 cases. FISH analysis was performed in 51 cases, and DNA sequencing for KRAS, EGFR or BRAF in 34 cases. The combined results of predictive markers from these various platforms were able to provide information on the therapeutic guidance for associated clinical benefit or lack of clinical benefit for various therapies in 129 of the 172 cases (75%).

Conclusions: Our study showed that cell blocks from malignant peritoneal and pleural fluid samples are informative and provide important therapeutic guidance in a large percentage of the cases. Molecular Profiling of malignant fluids offers opportunities for testing those patients where other tissue samples such as needle core biopsy or resection samples are not available.

PP 6

The optimal z-axis interval and focal planes to digitize 3-D gynecological SurePath® glass slides: Initial findings

Stanley Radio, MD 1 , Maheswari Mukherjee, MS, CT(ASCP) 1 , Najia Wright, MBA 1 , Jane Meza, PhD 2 , Amber Donnelly, PhD, MPH, SCT(ASCP) 1

1 Cytotechnology Education, School of Allied Health Professions, University of Nebraska Medical Center, Omaha, Nebraska; 2 College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska

Introduction: Virtual microscopy (VM) scans an entire glass slide and allows access to all areas of interest via a computer or digital device, without the use of a microscope. Application of VM to cytology has been dependent on the development of a three-dimensional focus. Our main objective is to determine the optimal 3-D scanning interval and focal planes necessary to achieve the best imaging resolution of the whole digitized SurePath® slide-prepared gynecological specimens, while maintaining a minimum file size. We are reporting the results of our pre-pilot study, which allows us to decrease the potential number of focal planes and interval levels.

Materials and Methods: [Table 1] Seventeen liquid-based preparations of gynecological (SurePath® ) glass slides were scanned, each at 40X magnification: First, with 13 focal planes (FP) at 1 micron interval (Group 1), second, with 13 FP at 0.8 micron interval (Group 2), and finally with 13 FP at 0.5 micron interval (Group 3) using an iScanCoreo Au scanner (BioImagene, California, USA). Thus a total of 51 virtual images (VI) were produced with an average file size of 12.8 gigabytes. A cytopathologist, cytotechnologist, and cytotechnology student used Image viewer software and diagnosed the pre-annotated cells of interest in Groups 1, 2, and 3. They also recorded the FP level, with which they were confident in giving the diagnosis. Subsequently, they diagnosed the corresponding 17 glass slides (Group 4) using conventional microscopy. The participants were selected from these diagnostic categories: Negative, Negative with organism, Low-grade dysplasia, and High-grade dysplasia. We evaluated the interobserver reliability by using the kappa statistics. We also calculated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for all interval levels.

Results: The interobserver reliability was found to be the highest for the glass slides (100%) followed by VI scanned at 1 micron (84%), 0.5 micron (83%), and 0.8 micron (72%). When compared with the glass slides, the sensitivity was the highest for the VI scanned at the 1 micron interval (96%), followed by 0.5 (93%), and 0.8 (85%). The NPV was highest for the 1 micron interval (96%), followed by 0.5 (92%), and 0.8 (86%). The specificity and PPV was 100% for all interval levels. The number of focal planes used to diagnose the VI was seven for the VI scanned at 1 micron and nine for either 0.5 or 0.8 microns. One of the main comments from the participants was the difficulty in evaluating the chromatin pattern on dark cell clusters leading to inability to accurately interpret the virtual images.

Conclusions: Our study found that VI scanned at the 1 micron interval potentially had the highest interobserver reliability, sensitivity, and NPV, while using a lower number of focal planes. With this pre-pilot study the number of focal planes was decreased from thriteen to seven and the interval level established at 1 micron. This study will be followed up by a pilot study in which 20 SurePath® gynecological glass slides are scanned, each at: seven focal planes at 1 micron, five focal planes at 1 micron, and three focal planes at 1 micron. A sample size calculated from the pilot study will be used to determine the optimal scanning focal plane and the interval necessary to digitize SurePath® prepared gynecological specimens. In response to participants' comments, an image enhancement option and an additional diagnostic category, 'Unsatisfactory for Diagnosis' will be added in the pilot study.

PP 7

Early clinical results on the implementation of ultrasound use in an academic fine needle aspiration service

Joseph Jakowski, MD, Celeste Powers, MD PhD, Alycia Reid, RT RDMS

Pathology, Virginia Commonwealth University Health Systems, Richmond, Virginia

Introduction: Ultrasound (US) has been successfully incorporated into the practice of many other medical subspecialties beyond radiology. In our pathology-based FNA service, we have seen superficial FNAs (SFNA) decrease substantially over the last five years in favor of image-guided FNAs. US and US fine needle aspiration (USFNA) are quickly gaining acceptance as a new tool for the interventional cytopathologist for real time evaluation and FNA of the target lesion. USFNA was recently incorporated into the SFNA service at our academic medical center and its initial impact and clinical utility are evaluated herein.

Materials and Methods: Retrospective evaluation of the first four months' experience (12 / 17 / 2010 - 4 / 19 / 2011) of our new USFNA service was performed. Prior to initiation of the USFNA service, a discussion was held with the clinicians. Electronic templates (E and M, US, and FNA) and an image database were developed. All FNA requests were screened by the cytopathologist for appropriateness. Criteria for USFNA included: Non-palpable or ill-defined lesions, < 1.5 cm, > 25% cystic, any thyroid or breast mass, specific request, prior failed radiologically guided FNA, and / or masses near critical structures. Our cytopathologists are, at minimum, certified from the College of American Pathology in USFNA. Our ultrasonographer joined the service in month four and is certified by the American Registry of Diagnostic Sonography, with five years of US experience. US scans were performed using a high resolution US instrument with a linear transducer. All USFNA were performed by the cytopathologist with graduated training: Initially on turkey phantoms, then FNA of palpable lesions, and finally FNA of non-palpable masses. Patient evaluation included review of previous radiographic studies, a focused history and physical examination, and a US scan for echolocation.

Results: The SFNA service averaged 23 FNA requests per month (93 total) over the four-month study period. US use in the first three months of implementation of our new service varied from 0 to 25% of the cases per month and increased to 64% in month four, with the addition of a full-time ultrasonographer. In 71% of the patients, a USFNA was performed after echolocation of the lesion; 37.5% of these lesions were non-palpable, identified only on prior radiographic studies (diagnostic US or CT). The FNA sites included soft tissue (28%), breast (28%), thyroid (22%), and lymph nodes (22%). The overall specimen adequacy rate was 95.2%. In the remaining 29% of the patients, no USFNA was performed after an initial US scan for reasons that included: (1) The lesion was too deep, (2) it involved important anatomical structures (e.g., near lung apex), (3) there was a benign clinical and US impression such that follow-up was preferred, and (4) no discrete lesion was identified on US.

Conclusions: We have observed a number of immediate and encouraging consequences by incorporating US into our SFNA service. First, our SFNA service capabilities have been expanded to include aspiration of non-palpable lesions of superficial sites and second, patient care has been improved by appropriate elimination of some procedures. We have also had positive responses from our patients, pathology residents, and referring clinicians; the latter have increased their requests for this service. Finally, the addition of a full-time ultrasonographer has increased our use of US for evaluation, and not surprisingly, the number of USFNAs performed.

PP 8

Virtual microscopy in cytotechnology education: Application of knowledge from virtual to glass

Maheswari Mukherjee, CT(ASCP) 1 , Elizabeth Lyden, MS 2 , Amber Donnelly, PhD, MPH, SCT(ASCP) 1

1 Cytotechnology Education, School of Allied Health Professions, University of Nebraska Medical Center, Omaha, Nebraska; 2 College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska

Introduction: Virtual microscopy (VM) is a technology in which glass slides are scanned and converted into digital images. This study reports the implementation of VM in the cytotechnology educational program at the University of Nebraska Medical Center. The main objective of this study was to determine if cellular morphology learned through virtual microscopy could be applied to glass slide screening.

Materials and Methods: A total of 142 glass slides (61 teaching and 81 practice) of breast, thyroid, and lymph node fine needle aspiration body sites, were scanned with a single focal plane (at 40X) using iScan Coreo Au (BioImagene, California, USA). Six students, including one distant student, used only digital images to learn cellular morphology and conduct daily screening. Subsequently, the students were tested on 10 glass slides using light microscopy (LM). At the end of the study, the students were asked to respond to a survey, anonymously and voluntarily, about their VM experience. The survey had a series of questions followed by an open comment section for the students to give any additional feedback. The glass slide screening test scores of the participating students who learned through VM and tested on glass slides (VMLM group) were compared with the last three classes of students who learned through LM and tested on glass slides (LMLM group).

Results: A non-parametric statistical analysis indicated no difference (P = 0.14) in the median test scores between VMLM (median = 94) and LMLM (median = 86) groups. The survey indicated that the students spent a longer time screening virtual images compared to their previous experience of glass slide screening. They strongly believed that faculty member guidance and interaction would have improved the outcome of their virtual slide screening. In general, the students preferred LM over VM. However, the annotated teaching slides and access to the VM slides off campus were well appreciated by the students.

Conclusions: Although the students preferred LM, they were able to apply the cytological criteria learned through VM to glass slide screening. One of the main reasons for their preference for LM was that it took less time to screen the glass slides than the VM images. Overall, VM was considered a great teaching tool in conjunction with LM, but not to be used as the sole method of instruction or for daily screening of practice slides.

PP 9

Validation of BRAF mutational analysis in indeterminate thyroid fine needle aspirations

K. Councilman, MD 1 , N. Thomas, BS 1 , J. Bohn, BS 1 , P. Chesnut, BS 1 , B. Haugen, MD 2 , J. Klopper, MD 2 , M. Said, MD, PhD 1 , W. Franklin, MD 1 , D. Aisner, MD, PhD 1

1 Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado; 2 Endocrinology, University of Colorado Anschutz Medical Campus, Aurora, Colorado

Introduction: Multiple studies have demonstrated the utility of BRAF V600E mutational analysis for refining the diagnosis of indeterminate thyroid lesions. In addition, some studies suggest that mutation testing can aid in the stratification of patients with papillary thyroid carcinoma (PTC) diagnosed by fine needle aspiration (FNA). There are multiple methodologies for sampling thyroid lesions for molecular testing and performing mutational testing on thyroid FNAs. Methods for mutational testing should either be sufficiently sensitive to detect mutation despite a significant population of non-lesional cells, or involve enrichment for lesional cells. Here we present the technical validation of an approach that utilizes the microdissection of lesional cells directly from Diff-Quik® stained slides followed by conventional sequencing for BRAF V600E mutation.

Materials and Methods: Thirty seven thyroid FNA cases were identified based on a retrospective review of the pathology information system. Eleven cases were diagnosed as negative for malignancy, nine were diagnosed as malignant and were all classified as PTC, and 17 cases were classified as 'indeterminate' based on the recent Bethesda classification system, including diagnoses of 'atypical cells', 'suspicious for malignancy', 'follicular lesion,' and 'follicular neoplasm'. Criteria for testing were, (1) minimum of one cluster of at least 10 lesional cells on a Diff-Quik® slide and (2) diagnostic material remaining in the archive if the identified Diff-Quik® slide was sacrificed for testing. Lesional cells were selectively microdissected from de-coverslipped Diff-Quik® slides under a dissecting microscope and DNA was extracted from the microdissected material. Direct sequencing of exon 15 of BRAF was performed to evaluate for the presence of BRAF V600E mutation. Of the 37 cases selected, 16 had corresponding follow-up resection material, which was also evaluated for the presence of BRAF V600E mutation.

Results: All cases yielded sufficient DNA to undertake mutational testing. Of the 37 cases, five had BRAF V600E mutations, all of which were diagnosed as PTC on cytology. None of the 11 negative or 17 indeterminate cases demonstrated a BRAF V600E mutation. Paired evaluation of the cytology specimen and follow-up resection specimen showed concordance in all cases except one, which indicated the absence of a mutation in the cytology specimen, but positive mutation status in the follow-up resection specimen. Additional testing of the surgical resection specimen demonstrated areas that were negative for mutation, suggesting heterogeneity for mutation within the tumor.

Conclusions: A high correlation between the BRAF mutation status and diagnosis was observed. Furthermore, a high correlation between the BRAF status on FNA and the subsequent resection specimen was seen. These findings indicate that selective microdissection of Diff-Quik® slides, followed by direct sequencing is an effective method to evaluate the BRAF mutational status of thyroid lesions. Although no mutation was identified in the indeterminate lesions tested, this finding is not unexpected, as several studies have required a larger number of indeterminate specimens in order to identify BRAF mutations. These findings further suggest that BRAF testing can be performed retrospectively, and does not typically require additional sampling or FNA passes to obtain material for testing.

PP 10

Assessment of Fine Needle Aspiration specimen adequacy for high risk HPV detection and genotyping in oropharyngeal squamous cell carcinoma

Charalambos Solomides, MD, Marluce Bibbo, MD,

Zi-Xuan Wang, PhD

Pathology Anatomy and Cell Biology, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania

Introduction: In patients with oropharyngeal squamous cell carcinoma (SCC), the presence of human papillomavirus (HPV) genotype 16 is a favorable prognostic indicator, with respect to recurrence and overall survival. Thus, fine needle aspirates (FNA) of these tumors should be properly handled to provide both morphological and molecular information. The aim of this study was to determine the adequacy of the archived and fresh FNA specimens for the molecular detection and genotyping of HPV.

Materials and Methods: Sample selection: A total of 37 specimens from 26 patients diagnosed with metastatic SCC during the last five years were available in the cytology laboratory of the TJU hospital to be included as retrospective specimens. Among these specimens, 19 had slides stained with Papanicolaou (Pap) and 18 with Diff-Quik® (DQ). Nine fresh FNA specimens from nine patients with metastatic SCC diagnosed during the last two months were included as prospective specimens. HPV analysis: Pap or DQ stained slides were soaked in xylene overnight to remove the cover slips. Cells on these slides were scraped into tubes, washed with ethanol, and dry pellets were resuspended in 2 ml of PreservCyt, a liquid-based cytology solution. The needles containing fresh residual FNA specimens were directly rinsed in 2 ml of PreservCyt solution. These specimens were then analyzed using the standard protocol for ThinPrep® cervical specimens, with the Cervista HR HPV detection kit. The positive specimens were then tested for the HPV 16 and 18 genotypes.

Results: The Cervista HR HPV detection assay used a housekeeping gene (human histone HIST2H2BE gene) as an internal control to assess if there were sufficient cells in the specimens for the detection of HR HPV. Overall, 36 of 46 specimens (78%) had sufficient cells to yield a valid HPV result. The adequacy rate for Pap-stained slides was 14 / 19 (74%), for DQ stained slides 14 / 18 (78%), and 8 / 9 (89%) for fresh needle aspirates. HR HPV was detected in 17 specimens. Identical HR HPV results were obtained from parallel specimens stained with DQ and Pap stains in six patients. Among the 17 HPV-positive specimens, 12 were genotyped as HPV 16, two were HPV 18, two contained both HPV 16 and 18. One specimen was unavailable for genotype analysis.

Conclusions: In our preliminary experience, HPV 16 detection and genotyping could be performed on FNA specimens prospectively collected in PreservCyt as well as archived slides stained with either Pap or DQ. This pilot study demonstrated the clinical utility of genotype testing for high-risk HPV strains when cytopathology materials, either fresh or archived, were available for analysis.

PP 11

Using Fluorescence in-situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fna material: Eight-year experience from a medical center

Xiaohong Wang, MD, PhD, Fleurette Abreo, MD, Jaiyeola Thomas, MD, Diana Veillon, MD, James Cotelingam, MD, Songlin Zhang, MD, PhD

Pathology, LSUHSC, Shreveport, Louisiana

Introduction: The role of fine needle aspiration in the assessment of lymphoproliferative disorders continues to be a subject of debate. Many studies have shown the accurate diagnosis and classification of lymphoma using FNA. Increasing publications in the past few years have shown the value of Fluorescence In-Situ Hybridization (FISH) using cytology preparation in the classification of lymphoproliferative disorders on FNA. Previously, we published two years of our experience and our algorithm for handling lymphoproliferative disorders on FNA. Here, we summarize the past eight years of our experience using FISH and Polymerase Chain Reaction (PCR) for the diagnosis and classification of lymphoma using FNA material.

Materials and Methods: A retrospective search from the pathology data base for FISH and PCR on FNA materials in the past eight years (2003 and 2010) was performed, and the results of the corresponding cytology diagnosis and flow cytometry results were reviewed.

Results: A total of 53 FNA cases had FISH and / or PCR testing during the study period, and the cytology / flow cytometry diagnoses included 32 lymphomas, 16 atypical, and five negative. Molecular tests included 33 FISH for targeted translocations, 11 FISH IgH, five PCR for B-cell clonality, and eight PCR for T-cell clonality. The 32 lymphomas were further classified as 12 follicular lymphomas, eight diffuse large B-cell lymphomas (DLBCL)-NOS, four Burkitt lymphomas, one peripheral T-cell lymphoma, one plasmablastic lymphoma, one indolent B-cell lymphoma, and five B-cell lymphomas without classification. The 16 atypical cases were further classified as two peripheral T-cell lymphomas, one MALToma, two DLBCL-NOS, two thymomas, and nine atypical / suspicious. The five negatives were further defined as one atypical and four negatives. Twenty-five cases had follow-up excisional biopsy, including 21 lymphomas, two thymomas, and two negatives. There were no false positive or negative cases based on the cytology / flow cytometry / molecular interpretation. For individual methods, there were four false negatives on flow cytometry, four false negatives on FISH IgH, and one false negative on PCR. All 16 lymphomas, with sub-classification, were confirmed on excisional biopsy. The remaining five cases were atypical diagnoses on cytology / flow cytometry and they could not be further defined or classified with FISH / PCR. The final surgical diagnoses of the five cases were three DLBCL, one nodal marginal zone lymphoma, and one Hodgkin lymphoma.

Conclusions: FNA cytology combined with flow cytometry immunophenotyping and targeted FISH on FNA material could accurately diagnose and sub-classify most of the non-Hodgkin lymphomas. Targeted FISH and PCR could further diagnose and then sub-classify atypical / suspicious cases. Due to the complexity of non-Hodgkin lymphoma classification, using the appropriate algorithm with a team approach, including hematopathologists, is necessary to render an accurate diagnosis with a useful classification. Excisional biopsy required on some difficult cases should be performed without hesitation; however, the significant value of FNA cannot be underestimated.

PP 12

Detection of cytogenetically abnormal circulating cells in lung cancer patients in peripheral blood using an antigen-independent fluorescence in-situ hybridization (FISH) assay: A case control validation study

Jeff Wang 1 , Tanweer Zaidi 1 , Weigong He 1 , Jieqing Chen 1 , Pathak Aditya 1 , Jeena Vaid 1 , Margaret Spitz 2 , Carol Etzel 2 , Randa El-Zein 2 , Ruth Katz 1

1 Pathology Department, MD Anderson Cancer Center, Houston, Texas; 2 Epidemiology, MD Anderson Cancer Center, Houston, Texas

Introduction: Detection of circulating tumor cells using a simple blood test may provide a minimally invasive method to assist in early diagnosis of indeterminate lung nodules representing lung cancer (LC) and to monitor the results of the therapy. In a pilot study, using 12 DNA probes known to be affected in LC, we previously demonstrated via an automated FISH scanner that an antigen-independent FISH assay was a sensitive and quantitative method to detect cytogenetically abnormal cells (CACs) in the peripheral blood (PB) of non-small cell lung cancer patients (NSCLC) 1 . The different DNA probe sets could distinguish cancer patients from controls, as also the number of CACs correlated with the stage of disease, with early stage patients showing a lower number of CACs than the advanced-stage patients 1 . In contrast to the existing methods of using EpCAM-coated beads to isolate circulating tumor cells, our assay showed much higher numbers of CACs than previously reported. The current study was performed on a new cohort of LC patients and controls, to validate if by using only two DNA probe sets [centrometric 10 / 10q22.3 (SP-A) and centromeric 3 / 3p22.1 (GC20)], we could still detect lung cancer in the PB samples.

Materials and Methods: The PB samples from consented LC patients [adenocarcinoma (12), squamous cell carcinoma (six), small cell carcinoma (two) and carcinosarcoma (one)] and 18 low and high-risk controls were evaluated. The demographic data (age, smoking history, and cancer status) was anonymized so that reviewers of the FISH images were blinded. PB mononuclear cells (PBMCs) were isolated from 10 ml of PB and hybridized with two, two-color (3p22.1 / CEP3 and CEP10 / 10q22.3) FISH probes and quantified on an automated fluorescence scanner (Bioview Duet™, Il). For each sample, chromosomal abnormalities in 500 intact round or oval cells were classified by one reader and confirmed by a second reader as monosomies (mono), polysomies (poly) loss / gain, of 3p22.1 / CEP3 and 10q22.3 / CEP10, as previously described (1). All abnormalities (abn) of 3p and 10q were summed. CACs in PB were calculated (% chromosomal abnormalities X total PBMC / ml PB) / 1000), and expressed as CACs per microliter of blood. The mean ± S.D. of all chromosomal abnormalities and CACs were analyzed by the Kruskall Wallis test for significance (P < 0.05). A total of 39 samples (21 patients and 18 controls) were evaluated.

Results: Compared to the control group, the LC group showed significantly higher percentages of CACs with del 3p22.1 and 10q22.3 (P < 0.001,0.002), and abn 3p22.1 and 10q22.3 (P < 0.001, P < 0.001); poly, gain, abn of 10q22.3 (P = 0.005, P = 0.004, P = 0.007); mono, poly, gain, and abn 3p22.1 + 10q22.3 (P = 0.04, P = 0.000, P = 0.001, P = 0.002); poly, gain, and abn3p22.1 (P = 0.004, P = 0.003, P = 0.008).

Conclusions: In a validation study, comprised of 39 blinded PB samples, we have demonstrated that it is possible to detect LC using an automated FISH test, which uses four DNA probes known to be important in the pathogenesis of LC. We showed highly significant correlations between the numbers of CACs as well as percentages of chromosomal abnormalities in LC patients compared to controls. We plan to accrue larger numbers of samples.

Reference: 1. Genetically Abnormal Circulating Cells in Lung Cancer Patients: An Antigen-Independent Fluorescence In-situ Hybridization-Based Case-Control Study, Katz, RL et al. Clin Cancer Res; 16(15); 3976-87, 2010

PP 13

Accuracy of the cytology specimen and needle core biopsies for detection of KRAS mutation in non-small cell carcinoma: Comparison with resection specimen

Ismatun Swati, MD, Shengle Zhang, MD, Jamie Tull, MS, Kamal Khurana, MD

Department of Pathology, SUNY Upstate Medical University, Syracuse, New York

Introduction: KRAS mutational analysis is critical for predicting the anti-EGFR therapeutic response in non-small cell lung carcinoma (NSCLC). Needle core biopsy and a cytology specimen from endobronchial ultrasound-guided transbronchial needle aspiration as well as CT-guided fine needle aspiration (FNA) play an important role in the diagnosis and staging of NSCLC. However, the concordance rate of KRAS mutation analysis between the cytology specimen / needle core biopsy and resected NSCLC is unknown. We evaluated the extent to which the KRAS mutation detection of a cytology specimen or needle core biopsy of primary lung tumor by comparing the results with the resected NSCLC from the same patient.

Materials and Methods: Twenty-five samples including eight cell blocks, seven cytology smears, and 10 needle core biopsies, and the corresponding 22 resection specimens of the primary lung tumor were correlated for KRAS mutational analysis. Formalin-fixed paraffin embedded cell blocks, needle core biopsies, resected tumor sections, and alcohol-fixed smears were used for isolation and amplification of DNA. Direct sequencing was applied to detect KRAS mutations in codon 12 and 13. In cases where the cell block material did not correspond with the results on the resected specimens, cytology smears of the corresponding cases were used for isolation of DNA, using microdissection.

Results: Sufficient DNA for PCR was successfully isolated on all cytology and surgical (biopsies and resection) specimens except for one cell block. KRAS mutation was detected in seven out of 10 needle core biopsy specimens and the corresponding surgically resected specimens, with 100% concordant results. KRAS mutation was detected in four out of eight cell blocks, compared to seven out of the eight corresponding surgically resected samples. Lack of correlation in three cases with cell blocks could be attributed to low cellularity (two cases) and failure to retrieve DNA (one case). However, use of cytology smears in these three cases confirmed the KRAS mutation noted in the corresponding surgically resected samples. In addition, four more smear samples were tested and showed 100% concordance with the corresponding surgically resected samples.

Conclusions: KRAS mutation analysis on the cytology specimen and needle core biopsies is highly accurate and comparable with that of the resected NSCLC. In cases with low cellularity on cell blocks, cytology smears provide a better substitute for KRAS mutation analysis, when using the microdissection technique.

PP 14

Identification of epidermal growth factor receptor mutation, K-ras mutation, and EML4-ALK translocation in the cytological specimens of primary and metastatic non-small cell lung cancers

Guoping Cai 1 , Gillian Levy 1 , David Chhieng 1 , Robecca Wong 1 , Scott Gettinger 2 , Jonathan Puchalski 2 , Robert Homer 1 , Pei Hui 1

1 Pathology, Yale University School of Medicine, New Haven, Connecticut; 2 Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

Introduction: Although its carcinogenesis is not fully understood, non-small cell lung cancer (NSCLC) has been shown to be associated with certain molecular alterations. Identification of molecular changes such as epidermal growth factor receptor (EGFR) mutation, K-ras mutation, and EML4-ALK translocation has therapeutic implications in the clinical management of NSCLCs. In case of lung cancers, the cytological specimens from pleural effusion or fine needle aspirates might be the only material available for molecular analysis. However, some factors encountered in the cytological preparations such as low cellularity may have a potential impact on the final outcome of the molecular tests. This study is to review our experience with molecular tests in the cytological specimens of primary or metastatic NSCLCs.

Materials and Methods: A total of 55 cases of NSCLCs that had molecular analysis were retrieved from the cytopathology archives of our institution. There were 12 primary and 43 metastatic NSCLCs, including 50 cases of adenocarcinomas. The metastatic sites included pleural / pericardial effusion (19 cases), lymph node (16 cases), soft tissue (3 cases), bone (2 cases), adrenal gland (2 cases), and liver (1 case). Molecular tests were performed on the cell-block materials of effusions (19 cases) or fine needle aspirates (36 cases). EGFR mutation was evaluated by PCR-sequencing analysis of exons 18, 19, 20, and 21. K-ras mutation was tested using PCR-single strand conformational polymorphism analysis of codons 12 and 13. EML4-ALK translocation was evaluated by FISH study using the Anaplastic Lymphoma Kinase (ALK) break-apart probe.

Results: The patients were 25 males and 30 females with ages ranging from 28 to 90 years (average 68). Molecular tests were successful in 50 of 55 cases (91%). The remaining five cases yielded no results due to insufficient material. Evaluation of EGFR mutation, K-ras mutation, and EML4-ALK translocation were performed in 42, 14, and 22 cases, respectively. EGFR mutations were found in 14 of the 42 cases (33%), including deletion mutation in exon 19 (six cases), L858R mutation in exon 21 (five cases), and T790M mutation in exon 20 (three cases). K-ras point mutation was detected in three of 14 cases (21%) including TGT mutation in codon 12 in two cases and AGT mutation in codon 12 in one case. EML4-ALK translocation was evident in three of the 22 cases (14%). All the molecular alterations detected were mutually exclusive for the cases tested. Six of the twelve primary tumors showed EGFR (five cases) or K-ras mutation (one case). EGFR mutation, K-ras mutation, and ELM4-ALK translocation were identified in nine, two, and three of the 38 metastatic tumors, respectively.

Conclusions: The current study demonstrates the feasibility of molecular tests in the cytological specimens of both primary and metastatic NSCLCs. The presence of EGFR mutation, K-ras mutation or EML4-ALK translocation, appears to be mutually exclusive in NSCLCs.

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Cytological assessment of estrogen receptor, progesterone receptor, and HER2 status on distant metastases of breast carcinoma

Ryan Des Jean, MD, Marcia Edelweiss, MD, MaryAnn Friedlander, MPA, CT(ASCP), Silvia Babore, CT(ASCP), Muzaffar Akram, HT, Melissa Murray, DO, Edi Brogi, MD, PhD

Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York

Introduction: The assessment of the estrogen receptor (ER), progesterone receptor (PR), and HER2 status of metastatic breast carcinoma (MBC), using a cytology material, is relatively uncommon. Furthermore, some reports indicate that ER, PR, and HER2 status of breast carcinoma (BC), as determined by immunohistochemistry (IHC), can change in metastatic lesions compared to the primary tumor (PBC), with discordance in up to 30 - 40% of the cases. To address these issues, we compared the results of ER, PR, and HER2 stains (IHC) on paired surgical samples of PBC and cytology material (CYT) from metastatic breast cancer (MBC).

Materials and Methods: We searched our files for patients (pts) with CYT of MBC with IHC results, between 2007 and 2009. CYT IHC was compared to PBC IHC. Any discrepant IHC result was retested on the CYT of the MBC, if available. Patient demographics, and HER2 FISH status of the PBC and / or MBC, if available, were also recorded. HER2 scoring followed the ASCO / CAP guidelines [Table 1].

Results: Eighty-seven patients with median age of 55 years (range 28 - 80) fulfilled the study criteria. The PBC was invasive ductal (72 / 87; 83%), invasive lobular (6 / 87; 6%), and mammary (9 / 87; 10%). Nineteen MBCs involved the liver, 19, the lymph nodes (only two axillary), 16, the bone, 14, the lung, nine, the soft tissue, two, the adrenal, two, the mediastinum, two, the pleura, and four, the body fluids (two pleural, one CSF, one ascite). IHC was performed on ThinPrep® (20 / 87; 23%) and cell block (CB) (67 / 87; 77%). [Table 1] summarizes the IHC results on MBC and PBC. There were 27 ER discrepant cases, of which four were positive on CYT, and 29 PR discrepant cases, with six positive in CYT. HER2 FISH on CYT, performed in 21 cases, allowed to definitively classify 13 MBCs, of which 12 were equivocal and one was negative on IHC, and five with HER2 gene amplification and eight without. Three patients had CYT and core biopsy from the same MBC; IHC on the core biopsy yielded the same results as CYT.

Conclusions: IHC performed on the cytology material of distant metastases of breast carcinoma has good concordance with IHC of the primary tumor. The biological significance of the discordant IHC profiles remains unclear and must be explored clinically. Our results also demonstrate that cytology material is suitable for HER2 FISH evaluation and its use can provide the relevant information for patient treatment.


Sclerosing adenosis of the breast: A restrospective review of fine needle aspiration features and correlation with the corresponding core-needle biopsy features

Uma Kundu, MD, Yun Gong, MD

Department of Pathology, Section of Cytopathology, MD Anderson Cancer Center, Houston, Texas

Introduction: Although the histological diagnosis of sclerosing adenosis (SA) of the breast is usually straightforward, the cytological diagnosis may be difficult due to the lack of well-defined features in the current literature. Published cytological studies on SA are sparse and are almost always represented by small cohorts or case reports. To improve on the diagnostic accuracy of SA on fine needle aspiration (FNA), we reviewed the cytological features of SA on FNA samples in conjunction with the histological findings on the corresponding core-needle biopsy (CNB) samples.

Materials and Methods: We searched our pathology database for breast lesions with a diagnosis of SA on CNB between January 2003 and February 2010. We subsequently searched for the corresponding FNA cases that were previously or concurrently sampled from the same lesions. We identified a total of 27 SAs that were sampled both by FNA and a concurrent / subsequent CNB of the same lesion. Clinical and radiographic data for each case was also obtained.

Results: The patients' ages ranged from 40 to 68 years (average, 51.2 years). Radiographically, the target lesions ranged in size from 0.4 to 4.2 cm (average, 1.16 cm). The lesions were described as ill-defined hypoechoic in 12 cases, hypervascular in one case, a well-circumscribed mass in four cases, an ill-defined mass in five cases, mass / NOS in four cases, and suspicious for carcinoma in one case. A CNB slide review identified SA in 10 (37%) cases, SA with radial scar / complex sclerosing lesion or other benign lesions in 11 (41%) cases, and SA with atypia in six (22%). FNA diagnoses were as follows: Benign lesion (mostly fibrocystic changes or fibroepithelial lesions) in 21 (78%) cases and atypical / suspicious in six (22%) cases. Of the 22 FNA cases available for slide review, low cellularity was found in 13 (59%), fibrotic stromal fragments in 20 (91%), and dyscohesive / isolated epithelial cells in five (23%). Small tubules (some with angulated configuration and open-ended lumens) and solid nests / cords of epithelial cells were seen in all (100%) cases and myoepithelial cells within these cell groups were often diminished or lacking (n = 19, 86%), raising a concern of low-grade ductal / tubular carcinoma or lobular neoplasm. However, these cell groups / tubules were seen focally in most cases along with overtly benign cells. The epithelial cells were uniform and bland in 20 (91%) cases.

Conclusions: Sclerosing adenosis can pose diagnostic dilemmas in cytological and radiological interpretations. Although the configuration and architecture of some cell groups may resemble that of low-grade carcinoma, the focality and paucity of these groups along with minimal to no nuclear atypia are suggestive of SA. Familiarity with the cytological features of SA can minimize false-positive interpretations on FNA and tissue confirmation is recommended for the challenging cases.


Atypical epithelial cells in fine needle aspiration biopsy of breast lesions: Histological follow-up and sources of equivocal cytological diagnosis

Gene Landon, MD, Qiusheng Si, MD, Ming Guo, MD, Nour Sneige, MD, Yun Gong, MD

Pathology, MD Anderson Cancer Center, Houston, Texas

Introduction: An indeterminate diagnosis made on fine needle aspiration (FNA) samples of breast lesions can cause dilemmas in the clinical management. Identifying the histological follow-up and potential sources of indeterminate diagnoses will improve diagnostic certainty.

Materials and Methods: In this retrospective study, we identified 143 FNA cases that had originally been diagnosed as 'atypical' or 'suspicious for carcinoma' between July 2002 and June 2010, and for which concurrent or subsequent core needle biopsy (CNB) samples of the same lesions were available. The histological follow-up information was recorded, and the cytological slides reviewed (available in 131 cases).

Results: The patients' mean age was 52 years (range, 19 - 85 years). The CNB diagnoses included carcinoma in 66 cases (46.1%), benign in 75 (52.5%), and indeterminate in two (1.4%); subsequent excision was performed in 57, 13, and two lesions of each category, respectively. Of the 66 carcinomas, 39 (59.1%) were diagnosed as invasive ductal carcinoma (IDC), 11 (16.7%) as invasive lobular carcinoma (ILC), five (7.6%) as mixed IDC and ILC, one (1.5%) as metaplastic carcinoma, eight (12.1%) as ductal carcinoma in-situ (DCIS), and two (3.0%) as lobular carcinoma in-situ. Nuclear grade 1 was found in 29 (44.0%) tumors, grade 2 in 33 (50.0%), and grade 3 in three (4.6%). Of the 75 benign lesions, 13 had been excised; one was diagnosed as atypical ductal hyperplasia (ADH) on CNB, and turned out to be grade 1 IDC on excision. Of the remaining 74 lesions, 15 (20.3%) were fibroadenoma, 13 (17.6%) benign epithelium (with or without stromal fibrosis), 10 (13.5%) ADH, nine (12.2%) adenosis (including two sclerosing adenosis and one atypical microglandular adenosis), 10 (13.5%) fibrocystic change or ductal hyperplasia, five (6.7%) papilloma, two (2.7%) pseudoangiomatous hyperplasia, and 10 (13.5%) others. Two CNB indeterminate lesions were found to be low-grade DCIS on excision. Cytologically, scant cellularity, loss of cohesion, morphological distortion due to fibrosis, small tubular architecture, diminished myoepithelial cells, nuclear crowding or enlargement, and prominent nucleoli were the common contributory features.

Conclusions: Approximately half of the cytologically indeterminate lesions are malignant, indicating a necessity for further histological confirmation. Low-intermediate-grade carcinoma, fibroadenoma, ADH, adenosis, and stromal fibrosis constitute most of such cases. Familiarity with the cytological features of these lesions will improve diagnostic certainty.

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Using ASC: SIL ratio, HPV, and interobserver variability to assess and monitor cytopathology fellow training performance

Ivan Chebib, MD, FRCPC 1 , Rema Rao, MBBS 2 , David Wilbur, MD 3 , Rosemary Tambouret, MD 3

1 Department of Pathology, University of Calgary, Calgary, Alberta, Canada; 2 Department of Pathology and Laboratory Medicine, University of California, San Francisco, San Francisco, California; 3 Division of Cytopathology, James Homer Wright Pathology Laboratories, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Introduction: The goal of the cytopathology fellowship is to advance the resident beyond minimal competency of independent sign-out to a level of expert proficiency. We assessed whether routine cytopathology monitors can be applied to gauge the progress of the cytopathology fellows.

Materials and Methods: The laboratory information system was searched for all cases of cervical cytology interpreted by five cytopathology fellows who trained at MGH between 2007 and 2010. The patient demographics, final pathology result, HPV-test data, and the fellow's original interpretation were recorded. The ASC: SIL ratio was calculated and compared with those of the cytopathologist. Inter-observer variability between the fellow and the pathologist was determined by using the Cohen κ-coefficient.

Results: The five fellows reviewed an average of 490.8 cases per annum. The average κ-value between the Fellow and pathologist interpretation was 0.74 (95% CI 0.69 - 0.79, range 0.67 - 0.80). The percentage of cases of ASC-US with agreement between the pathologist and the fellow ranged between 64.4 and 77.0%. The Fellows had a tendency to interpret ASC-US cases as NILM (range 13.2 - 22.7%) or LSIL (range 2.2 - 13.2%). The average ASC : SIL ratio for the Fellows was 1.15 (95% CI 0.92 - 1.38, range 0.84 - 1.40) with four Fellows having lower ASC : SIL ratios than the cytopathologist, whose average ASC : SIL for the same case cohort was 1.30 (95% CI 1.08 - 1.52, range 0.97 - 1.61). Of cases interpreted as NILM by the Fellow and ASC-US by the pathologist (13.2 - 22.7% of cases), between 25.9 and 44.0% were HPV+, intermediate between that expected for ASC-US (40+%) and for NILM (5 - 10%), possibly because the Fellows were more aggressive in interpreting NILM and / or pathologists were more cautious, rendering an ASC-US interpretation and maximizing the sensitivity. Those cases interpreted as LSIL by the Fellow and ASC-US by the pathologist ranged between 2.2 - 13.2% of the cases, with HPV+ between 61.1 - 100%, indicating that The Fellows could be more aggressive in their interpretations of borderline ASC-US cases as LSIL.

Conclusions: Cytopathology Fellows tend to be more aggressive in interpreting NILM and LSIL, based on subsequent HPV results, than pathologists who render more cautious interpretation of ASC-US, in borderline cases. Using this data, a framework can be applied to measure and improve a Fellow's performance and maturity during their training year by not only assessing the performance on a case-by-case basis, but also measuring ASC : SIL, HPV-status, cyto-histo correlation, κ-value, and review of discrepant cases.


iPad and cytopathology: Content creation and delivery

Brian Collins, MD

Department of Pathology and Immunology, Washington University, St. Louis, Missouri

Introduction: The iPad is a tablet-style computer device gaining wider use. It represents a newer style of hardware device with high quality visual capabilities and thin physical design. With abundant memory, storage space, portability and wireless connectivity, it is an excellent platform for storing and easily accessing dynamic medical information. With a high resolution 9.7 inch screen, it is particularly suitable for displaying images. A long battery life of ten hours enhances its portability as a point-of-care information access model. The epub 3.0 standard permits the creation of electronic books (e-books), which can be accessed across a variety of different platforms, including the iPad. Cytopathology-based data in the epub e-book format was developed and designed for utilization on the iPad.

Materials and Methods: The epub standard is designed for the creation of electronic books with text and images that can be accessed across a variety of different hardware platforms. To create this, the Adobe InDesign CS5 software was used, to design and organize the previously created text and image content on a MacBook Pro. The InDesign created a table of contents, organizing the content into chapters, and inserting jpg photomicrographs. Within the text, references were embedded and hyperlinked to online resources. Interactive elements and hyperlinks were designed to interface with the native software and wireless iPad connectivity. InDesign exported the final design in the epub file format as a single, complete file. This file was transferred to the iPad via iTunes software by a simple drag and drop method. The iPad required the iBook app to be installed, in order to access the e-book.

Results: The cytopathology e-book provided searchable, interactive text and references with high resolution cytopathology photomicrographs, which were conveniently stored and accessed on an iPad. Interaction with the content occurred by opening the textbook in the iBook app, and reading and interfacing with the data by touch. A single location or page was bookmarked electronically, which permitted a future reference after exiting. The video was embedded directly. Individual medical references were accessible through embedded hyperlinks, and the iPad seamlessly downloaded them via the wireless connection and built-in web browser for an instant review with a single touch of a finger. Hyperlinked online web resources were available in the iPad through the e-book, including web sites (ASC web site), video (YouTube), and audio. Individual images were viewed with the text and as stand-alone full screen images. The native zoom function of the iPad permitted closer examination of image details. The content could be copied, pasted, printed, and emailed. Video mirroring allowed the iPad to connect to a larger, external HDTV screen for presentation purposes. A single e-book can vary in size from 1MB to 100s MB, and with a capacity up to 64 GB, there was ample storage space.

Conclusions: The iPad is a dynamic, interactive tablet-style computer device with significant potential for improved access and organization of medical information. By using the epub format, a cytopathology based e-book has been created, which has taken advantage of its strengths and capabilities. The iPad, with the epub e-book format, demonstrates its capacity to provide interactive cytopathology content in a portable, easily accessible device.


The use of journaling in cytotechnology education: Live experience of a cytotechnology educator

Linda Hoechst, MA, SCT(ASCP), CT(IAC)

Clinical Laboratory Science, Cytotechnology Program, Saint Louis University St. Louis, Missouri

Introduction: Adult education tenets hold that learning be more learning-centered than teaching-centered. Toward this goal, this researcher has implemented the use of journaling within a Cytotechnology Education Program. In order to establish rapport and mutual trust between the learner and the instructor various methods may be used to accomplish this goal. Among these is the use of journaling.

Materials and Methods: Journaling as a self-reflective method has been explored in the literature. The purpose of this study is to determine how it performs as a self-evaluative method in Cytotechnology Education.

Students are required to submit journal entries weekly during the Program. Information requested in these entries include: The material they understand; the material that is challenging to them; the material they find difficult; how they would like the material to be presented; what works and does not work for them; any personal issues that may be influencing their ability to grasp the content; and any other comments that may provide information to determine an action plan toward their academic success. Every one of these items need not be included in every journal entry. Over the span of thirteen years, this researcher has used journaling as a means of assessing student educational needs, challenges, and impediments. Through an evaluation of the information provided by the students and their feedback about this method, data have been generated identifying the benefits of its use in Cytotechnology education.

Results: The data support the use of this method in Cytotechnology education. It has been an important generator of trust and rapport among students and the instructor. It has identified the best teaching methods and strategies for each individual student. It has provided an explanation of outside influences that impact learning. It has identified issues before they become problems. It has indicated student dissatisfaction in a timely matter, to be dealt with before course evaluations are written at the end of the course.

Conclusions: Journaling in Cytotechnology Education is an important and useful tool in the academic success of Cytotechnolgy students. It is a cost-effective method to assess Cytotechnology Education as a dynamic process and enables timely response to issues that arise in the teaching and learning process.


The cytotechnologists and their role in tissue analysis at the mayo clinic

Lindsey Kane, SCT(ASCP) 1 , Kandelaria Rumilla, MD 2 , Angela Sorenson, MA, SCT(ASCP) 1

1 Cytopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, Minnesota; 2 Molecular Genetics, Department of Laboratory Medicine, Mayo Clinic, Rochester, Minnesota

Introduction: Over time, the Cytotechnologists' (CT) workload has decreased, with a significant change in the cervical Pap volumes, while the pathologists' workload has seen a significant increase, specially in the area of molecular genetic testing. Our laboratory wanted to find a way to utilize the CTs' morphological skills to fill the void left by the decline of Pap volumes and a way to reduce the pathologists' workload. It was determined that the CTs' cytology skills could convert to the review of the histological sections of a tissue. This would allow a group of CTs' to collaborate with the molecular genetics laboratory, by being a part of the pre-analytic process of molecular tumor testing and documentation.

Materials and Methods: Meetings began with two consultants from the molecular genetics laboratory, to determine the responsibilities of the CTs. Documentation processes were modified to incorporate CTs into the tissue review process. CT responsibilities would include documentation for materials received, tumor-type and source, appropriateness of the material for the test ordered, including the presence / absence of the normal estimating percent of tumor nuclei in the tissue sample, and immunohistochemical stain interpretation, all of which would then be subject to review by the pathologist. To train for this new workflow there were group scope sessions and individual sign-out sessions with pathologists. Collaborating with the pathologists one-on-one allowed the CTs to quickly gain an understanding of the testing, tissue review, to estimate the percent of the tumor nuclei.

Results: To determine if the CTs were providing significant time saving for the pathologists, the times were recorded along with documenting any changes made to the case. The changes were classified as major (could impact test performance or result), moderate, minor (not likely to impact test), and judgment calls. When time recordings were reviewed it was found that the average pathologist's full-time equivalent (FTE) used per case was 0.000355 prior to the CT review and after the CTs were introduced to the process the average FTE used per case was 0.00038. When the overall average time per case for each pathologist was reviewed it was found that two pathologists saw substantial time improvements, by greater than 50%, while two saw only minor improvements. When the changes were charted out the two pathologists with substantial time saving had significantly fewer minor changes made to the cases. It also showed that those showing little time improvement had considerably more minor changes made on each case. Quarterly a set of de-identified cases circulate internally to the whole group and the results are compared, particularly percent tumor nuclei. A majority of the minor changes fall within our gold standard for QA case review. With this in mind the group went to minimizing those changes that would not impact the test results, in the hope of seeing a more significant time saving. When the time saving was recorded again a year later and compared to the time taken prior to the CT's reviewing the cases, it was seen that the average time saving was 35%.

Conclusions: We found the updated time saving to be valuable and helpful for the pathologists. One reason we may not be maximizing efficiency is the turnover of the Fellows rotating through the area. Our average pathologist FTE per case will continue to be monitored over time and the QA cases will also continue to be used, to hone both the pathologist and CT review skills.


Telecytology for immediate assessment of endoscopic ultrasound-guided fine needle aspiration: Improved pathologist efficiency, and a valuable educational tool

Jonathan Marotti, MD, Vickie Johncox, CT(ASCP), David Ng, MD, Vijayalakshmi Padmanabhan, MBBS, MD

Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire

Introduction: In a tertiary academic medical center, onsite evaluation of fine needle aspiration (FNA) procedures can take anywhere from 30 minutes to two hours of the cytopathologist's time, given the distance from the Department, and the training and the experience of the performer. Telecytology is being increasingly used as an alternative for onsite cytopathologist evaluation. One of our department's quality incentives for 2010, was the reduction of cytopathologist procedure time by 50%, improving the efficiency. The aim of this study was to evaluate the role of telecytology for immediate assessment of endoscopic ultrasound (EUS)-FNA procedures, to improve the efficiency.

Materials and Methods: Following an adaptation period of one week, which included performing onsite evaluation in tandem with the transmitted image review, telecytology was incorporated and evaluated over a 10-month period. Using an Olympus BX41 microscope and Olympus DP72 camera with Olympus CellSens software, dynamic images of the air-dried Diff-Quik® stained smears were transmitted by the cytopathology Fellow or the cytotechnologist, using a secure internet connection. The cytopathologist remotely accessed the real-time images on a computer and rendered immediate assessments, while communicating over the telephone with the cytopathology Fellow, who would communicate with the clinicians. Cytopathology Fellow / cytotechnologist procedure time and cytopathologist procedure time for each EUS-FNA was recorded.

Results: Two hundred and forty-nine EUS-FNA procedures were performed during the 10-month period. Overall, 164 (66%) procedures utilized telecytology. By the end of the 10-month period, the utilization rate was greater than 95%. The average cytology fellow / cytotechnologist procedure time, with and without telecytology was 1.1 hours. The average cytopathologist procedure time without telecytology was 0.74 hours; the average procedure time with telecytology was 0.2 hours. A total of 88.56 cytopathologist hours was saved. The Fellows greatly appreciated the autonomy and the ability to communicate with the clinical staff. The immediate assessment diagnosis matched the final diagnosis in all but one case, where the final diagnosis was suspicious and the immediate assessment positive for malignancy.

Conclusions: Incorporation of telecytology for immediate assessment of EUS-FNA successfully increased cytopathologist efficiency. The department's incentive goal was met and surpassed. It allowed the cytopathology Fellow to interact with the clinical staff as a junior consultant. Telecytology is now routinely incorporated in greater than 95% of all EUS-FNA procedures in our hospital, reducing cytopathologist time by at least 70%. The projected 2011 cytopathologist time saving is 174.96 hours. Furthermore, telecytology augmented the cytology fellowship training program.

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Non-urothelial carcinoma of the urinary bladder - Diagnosis on urine cytology

Manju Aron, MD, Maureen Croyle, CT(ASCP), Donna Hansel, MD, PhD, Christine Booth, MD

Pathology, Cleveland Clinic, Cleveland, Ohio

Introduction: Urothelial carcinoma of the urinary bladder comprises 90% of all bladder carcinomas. Squamous cell carcinomas, adenocarcinomas, and small cell carcinomas of the urinary bladder are rare and account for less than 5, 2, and 0.5% of all bladder carcinomas, respectively. Urine cytology is an efficacious diagnostic tool, with a high specificity and sensitivity in the diagnosis of high-grade urothelial carcinomas. The role of urinary cytology in the preoperative diagnosis of the above-mentioned tumors has rarely been reported. We report our experience on these rare tumors.

Materials and Methods: Between the period of 1983 and 2009, there were a total of 25 cases of small cell carcinoma, 20 of squamous carcinoma, and 10 of adenocarcinoma, wherein prediagnosis urine cytology was available. However, the cytology slides were available for review only from 1997 onwards. Only those cases with available cytology slides were included in the study.

Results: There were eight cases (12 samples) of small cell carcinoma, four cases (6 samples) of squamous cell carcinoma and four cases (5 samples) of adenocarcinoma. The cytological diagnoses of these samples are listed in [Table 1]. Of the nine samples of small cell carcinoma, categorized as atypical, suspicious for carcinoma, three (33%) were categorized as suspicious for small cell carcinoma, while the others were not further classified, due to limitations of cellular preservation. In the squamous cell carcinoma subgroup, the samples with 'atypical, suspicious for carcinoma' diagnoses did not show any keratinization and were interpreted as atypical urothelial cells. All the adenocarcinomas categorized as negative by urine cytology had sparse cellularity and did not reveal any atypical cells on slide review.

Conclusions: None of the samples of small cell carcinoma had a false negative diagnosis on cytology, while only one case of squamous cell carcinoma was diagnosed as negative. However, only one case of adenocarcinoma was diagnosed preoperatively. Although the sample size is small, this study highlights the relevance of urine cytology in the preoperative diagnosis of these rare non-urothelial tumors of the bladder.


Does routine preparation of multiple ThinPrep® and cell block slides improve the detection of malignancy in effusion fluid?

Charles Beavers, MD, Dustin Woods, MD, Sunati Sahoo, MD

Department of Pathology and Laboratory Medicine, University of Louisville Health Sciences Center, Louisville, Kentucky

Introduction: Cytology is the most accurate and cost-effective method of evaluating spontaneous effusions to rule out malignancy. The overall sensitivity of detecting malignant cells in an effusion is 70 to 90%. In effusions multiple factors, including the presence of markedly reactive mesothelial cells or few groups of atypical cells, can pose a dilemma for a definitive diagnosis. At our institution, we recently adopted a policy of preparing a duplicate ThinPrep® slide and slides from cell block for every effusion specimen routinely, with the aim of improving the sensitivity of detecting malignancy and minimizing the rate of 'suspicious' and 'atypical' interpretations. Prior to the new policy, one ThinPrep® slide was prepared, unless additional ThinPrep® slides and cell block were requested, based on the initial examination.

Materials and Methods: The study population comprised of 50 consecutive cases of effusions after the new policy (groups 1 and 2) and 50 cases before the new policy (group 3) was implemented [Table 1]. All the slides (a total of 150 ThinPrep® slides and 67 cell block slides) were reviewed separately by a cytopathologist, who was blinded to the original method of examination and clinical history. In each ThinPrep® slide an interpretation of 'positive for malignancy,' 'negative for malignancy,' 'atypical,' or 'suspicious for malignancy' and 'scant cellularity' was rendered.

Results: The diagnostic categories from each group of 50 slides are summarized in [Table 1]. In the vast majority of cases, a definitive diagnosis of 'positive for malignancy' (up to 26%) or 'negative for malignancy' (up to 70%) was reached, based on the review of one ThinPrep® slide. The rate of 'atypical' and 'suspicious' interpretation ranged from 2 to 12% (average 7%) when one ThinPrep® slide was reviewed. However, that rate dropped to 6% when two ThinPrep® slides and cell block slides were available for interpretation. The rate of 'scant cellularity' did not improve significantly with an additional slide or with the cell block preparation.

Conclusions: Routine preparation of two ThinPreps® and cell blocks on effusion specimens is particularly useful in instances where the initial evaluation reveals atypical or suspicious groups of cells or when the differential diagnosis includes reactive mesothelial cells versus malignancy. Routine performance of multiple ThinPrep® slides or cell block on every effusion specimen is not cost-effective. Additional slides are also of little help in evaluating cases with inadequate fluid volume and scant cellularity.


Utility of special stains for opportunistic infections in bronchioalveolar lavage fluid

Ramneesh Bhatnagar, MD, Paul Staats, MD

Department of Pathology, University of Maryland Medical Center, Baltimore, Maryland

Introduction: Bronchioalveolar lavage (BAL) fluid can be analyzed for opportunistic infections by bronchial and sputum cultures, as well as molecular studies and serology. Along with microbiology, special and immunohistochemical stains for cytology are often performed. However, it remains uncertain whether special stains for organisms on cytology preparations provide substantial additional diagnostic sensitivity compared to microbiological tests and routine cytological preparations.

Materials and Methods: We retrospectively reviewed the cytology reports of all BAL specimens over a period of one year (2010). The results of routine stains (Diff Quick and Papanicolaou) as well as stains for microorganisms, Gomori methenamine silver stain (GMS), Acid Fast Bacilli (AFB), and immunoperoxidase stains for Cytomegalovirus (CMV) and Toxoplasma were recorded. All cases reported as positive for organisms were reviewed for confirmation by two pathologists, and the presence of organisms on routine stains, special stains, or both was noted. Such cases were also correlated with the microbiology results.

Results: A total of 476 cases were reviewed, of which 235 (49%) had special staining for GMS, 222 (47%) for CMV, 189 (40%) for Toxomplasma, and 111 (23%) for AFB. Of the 235 GMS-stained specimens, 19 (8%) were positive for Pneumocystis carinii (PCP). Evidence of PCP infection was visible on routine stains in all but one case (18 / 19), and it was more prominent with Papanicolaou staining. Candida was seen in 10 out of the 235 GMS stains (4.2%), and five out of the 10 cases had microorganisms readily identifiable on routine stains. There was one specimen (< 1%) positive for Aspergillus, and one other (< 1%) positive for Cryptococcus neoformans. Both of these cases required GMS staining, as the microorganisms were not visible on routine stains. All cases with positive cytology were also identified by microbiological methods (positive cultures or antigen detection). None of the specimens were positive for CMV, Mycobacteria, or Toxoplasma on cytology.

Conclusions: Special and immunohistochemical stains for microorganisms can be helpful in identifying specific pulmonary infections, particularly GMS staining for fungal organisms, compared to routine stains; however, microorganisms, when present, are usually visible on routine stains, and in this study have always been identified on microbiological studies of the BAL specimen, so the special stains have provided no improvement in the overall sensitivity for the detection of organisms. CMV, mycobacteria, and toxoplasma are relatively uncommon pulmonary infections, as supported by the lack of any positive immunostains in our study, so the utility of routine staining for CMV, mycobacteria, or Toxoplasma is limited, although cost-benefit analysis of routinely performing these tests is beyond the scope of this study.


Atypia in pleural effusions after lung transplantation

Maureen Croyle, CT(ASCP), Ghada Aramouni, CT(ASCP), Beverly Cash, CT(ASCP), Deborah Chute, MD

Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio

Introduction: Pleural effusions are frequent non-gynecological specimens. Many benign processes can cause significant reactive atypia in mesothelial cells, including infectious and inflammatory disorders. Pulmonary transplant patients are immunosuppressed, and therefore, at a higher risk for development of both malignant and infectious conditions. This study evaluates the clinical and pathological features of pleural effusions in patients, after lung transplantation.

Materials and Methods: All patients who underwent lung transplant and had post-transplant pleural fluid within the last five years were included. The initial cytological interpretation was recorded, and the slides were reviewed for cellularity, presence of inflammation, mesothelial cell features, and the presence of cell clusters. Follow-up clinical and pathological information was recorded from the time of effusion to the most recent transbronchial biopsy.

Results: Thirty-six (15%) patients with a total of 63 pleural effusions were included. The average time from lung transplant to pleural effusion was 10.7 months. Twenty-seven patients had bilateral lung transplants, and nine had a unilateral lung transplant. In nearly all patients (97%), the pleural effusion was ipsilateral to the transplanted lung. The original cytological diagnosis was negative in 59 (94%) effusions. Four effusions were suspicious for neoplasia. Mesothelial cellularity was low in almost all cases (94%). Inflammation was a frequent finding, chronic in 46 cases (73%) and acute in 12 cases (19%). Mesothelial cells often had prominent nucleoli (38%) and were vacuolated (35%). Mesothelial cell clusters were uncommon (25%), and clusters with community borders was only seen in one case. On follow-up, one effusion suspicious for malignancy was diagnosed with adenocarcinoma on pleural biopsy. In this case, there was high cellularity, the cell clusters had community borders, and the effusion occurred contralateral to the transplanted lung. The other three atypical effusions were from one patient, concerning a lymphoproliferative disorder, but follow-up was negative. Two patients with negative fluids were diagnosed with post-transplant lymphoproliferative disorder within one year of the pleural effusion. All other patients were negative for malignancy on all subsequent biopsies.

Conclusions: Reactive atypia and chronic inflammation are frequent findings in pleural effusions of lung transplant patients. However, cell clusters with a community border, as well as effusions involving the contralateral lung, should raise concern for a diagnosis of malignancy.


Cytomorphology of atypical teratoid / rhabdoid tumor in cerebrospinal fluid

Eric Huang, MD, PhD 1 , Miguel Guzman, MD 2 , Edmund Cibas, MD 1

1 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; 2 Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts

Introduction: Atypical teratoid / rhabdoid tumor (AT / RT) is an extremely rare and highly aggressive intracranial neoplasm with a predilection to spread along the cerebrospinal fluid (CSF) pathways. The cytopathological characteristics of this tumor in the CSF specimens, however, have not been extensively studied. Here we report the pattern of CSF cytomorphology from a series of patients with histologically documented AT / RT.

Materials and Methods: We conducted a retrospective review of 36 malignant CSF specimens from eight patients with histologically confirmed AT / RT (all females; age range 0.8 to 9.6 years at initial diagnosis; mean age = 2.4 years) from 2001 to 2009. Cytospin preparations were made in all cases. In most cases, at least two slides were prepared with the Papanicolaou and / or Wright Giemsa stains.

Results: The CSF samples were predominantly sparsely cellular (67%; 24 / 36 cases) and the malignant cells were accompanied by lymphocytes and monocytes (69%; 25 / 36 cases). Two principal malignant cell types were seen: The most striking and recognizable form was a large, usually rhabdoid cell with an eccentrically placed nucleus, prominent nucleolus, and abundant cytoplasm, seen in 72% (26 / 36) of the cases. Some, but not all of these large cells contained the dense condensation of cytoplasm, characteristic of rhabdoid morphology. The second type of neoplastic cell had a 'small cell' appearance and was seen in isolation in 28% (10 / 36) of the cases. Both large and small malignant cells were seen in 31% (11 / 36) of the cases.

Conclusions: A significant proportion (28%) of AT / RT cases metastatic to CSF consisted of only the small cell component, without the large rhabdoid cells that were characteristic of this neoplasm. Familiarity with this pattern of spread was important in the differential diagnosis, with other small cell malignancies like medulloblastoma, which have a high propensity for spreading along the CSF pathways.


Immunoreactivity of the IMP3 antibody in malignant epithelial tumors and reactive mesothelial cells in serous effusions

Semra Karaburun, MD, Saverio Ligato, MD

Pathology, Hartford Hospital, Hartford, Connecticut

Introduction: The distinction between benign and malignant effusions can be a major diagnostic challenge in cytology. Several immunohistochemical / cytochemical antibodies have been studied as ancillary tools in the differentiation between benign / reactive and malignant effusions. The insulin-like growth factor II messenger RNA binding protein-3 (IMP3, also known as L523S) is an oncofetal RNA-binding protein, expressed during embryogenesis and in various malignancies. In this study, we investigate the use of IMP3 to help in the differentiation between benign and malignant cells in effusion cytology specimens.

Materials and Methods: A total of 33 (17 pleural, 15 peritoneal, and 1 pericardial) effusions, including 15 negative, 15 positive, and three atypical, but not diagnostic, effusion specimens were selected and stained with antibody to IMP-3 (clone 69.1; Dako, Carpinteria, California, USA). The intensity (scale 0-3+) and percentage of cells staining were evaluated, and a diffuse (>75%) staining with 2 - 3 positive intensity was considered positive.

Results: The immunohistochemical stain for IMP-3 / L523S showed positive staining in 11 (73%) of 15 malignant, two (66%) of three atypical, and one (7%) of 15 reactive effusions. The overall specificity for the diagnosis of malignancy with IMP-3 was 93%, whereas, the sensitivity was 73%. Positive and negative predictive values were 92 and 78%, respectively.

Conclusions: IMP3 / L523S antibody is a useful marker for the detection of malignant cells in serous effusions and it can be helpful in distinguishing neoplastic cells from reactive mesothelial cells in serous effusions. Our results are similar to the study previously performed by Ikeda Katusuhide in Japan.


The cytological appearance of a mixed malignant mesodermal tumor in pelvic washings: Correlation with histological findings

Ann Leathersich, MD, Kay Kasal, CT(ASCP), DengFeng Cao, MD, PhD, Hannah Krigman, MD

Pathology and Immunology, Washington University, St. Louis, Missouri

Introduction: A mixed malignant mesodermal tumor (MMMT) is an aggressive uterine tumor that typically occurs in women between the age of 50 and 80. MMMT accounts for approximately 2% of the endometrial primary tumors. As this tumor is rare, the findings on pelvic washing cytology are not well described. For this reason, we performed a study of pelvic washings obtained at the time of definitive resection for an MMMT. The goal was to identify the features specific to MMMT and to examine the spectrum of findings in this histologically diverse tumor population.

Materials and Methods: The pathology database was searched for all patients with a diagnosis of MMMT from 1995 to 2010. All cases with pelvic washing cytologies obtained at the time of definitive resection were selected for review. Pap stained, monolayer preparations from washings were reviewed and evaluated for the glandular component, stromal elements, and inflammation. The histological subtype of the MMMT, on definitive surgical resection, was also reviewed and compared with the elements shed into the pelvic fluid.

Results: Twenty-six positive pelvic washings were identified. Eight cases had a predominantly endometrial adenocarcinoma pattern. Three cases had a mixture of serous and endometrioid adenocarcinoma patterns. Two washings had a mixture of endometrioid and undifferentiated malignant cells. Five cases had predominantly small, undifferentiated malignant cells. In no cases, were definitive malignant stromal elements identified. The type of epithelium did not correlate completely with the reported histological subytpes. Five MMMTs with serous epithelium, on hysterectomy, had groups more reminiscent of adenocarcinoma, on the washings. Three patients with endometrial type adenocarcinoma on hysterectomy had serous appearing groups on washing, including one with psammomatous calcification.

Conclusions: A predominant histological pattern in uterine resections is not necessarily predictive of the pattern of malignant elements in pelvic washing cytology, for MMMT. This shift in pattern reflects the pluripotent nature of this neoplasm. A mixture of malignant epithelial types suggests an MMMT and warrants additional sampling, to demonstrate the presence of this particular subtype of malignancy.


Pericardial fluid cytology: Etiology and diagnostic accuracy. An analysis of 128 specimens from 113 consecutive patients over a six-year period

Lina Liu, MD, Ema Dragoescu, MD

Pathology, Virginia Commonwealth University Health System, Richmond, Virginia

Introduction: Pericardial effusion is a common finding in clinical practice with causes including: Infections, malignancy, connective tissue diseases, injury (post-trauma, postmyocardial infarction), metabolic causes (hypothyroidism, uremia), heart diseases (pericarditis, myocarditis, heart failure) or idiopathic causes. Pericardiocentesis (PC) is performed based on the clinical scenario, effusion size, and hemodynamic instability. This procedure is concurrently diagnostic and therapeutic. Although systematic evaluation of pleural and peritoneal fluid cytology is abundant in the literature, large series focusing on pericardial fluid (PF) cytology are sparse. This study aims to analyze a large cohort of PF specimens from a single institution, to determine the etiology of PF, the diagnostic utility of cytology, and correlation with the histological and microbiology results.

Materials and Methods: The PF specimens collected between January 2005 and December 2010 were retrieved. Clinical history, laboratory and radiological results, cytological and all pertinent histological diagnoses were recorded. The PF was collected through ultrasound or fluoroscopic-guided PC or via the pericardial window. The PF was sent for chemistry, microbiological, and cytological evaluation. The specimens were received fresh from Cytology. For each specimen one Diff-Quik® and three Papanicolaou stained cytospin slides were prepared and if more than 10 ml of PF was received a cell block was performed.

Results: One hundred twenty-eight PF specimens were obtained from 56 males and 57 females with age ranging from 6 to 85 years (mean 52.5 years). During the study period 1292 pleural and 1396 peritoneal fluid specimens were processed (4.54% incidence of PF specimens). The amount of fluid received ranged from 1 to 1,150 ml (mean 60.5 ml). [Table 1] summarizes the cytological diagnoses of all PF cases. Thirty-one cases (24.22%) were malignant. With regard to males, the most common malignancy was lung carcinoma (75%) followed by digestive system carcinomas (25%), while for females the most common malignancy was lung carcinoma (52.17%) followed by breast carcinoma (39.13%), [Table 2]. The underlying etiology of negative PF specimens was broad [Table 3]. The two cases diagnosed as suspicious for malignancy had few atypical cells present in the PF and no ancillary studies were performed. Pericardial biopsy was performed in 32 cases. In 25 (78.1%) the cytological diagnosis correlated with the histological one. Cytology failed to diagnose lymphoma (two cases) and metastatic sarcoma (one case) (9.4% false negative rate), whereas, biopsy failed to sample four carcinomas (12.5%), diagnosed by cytology and confirmed by clinicoradiological findings. Ten PFs were culture-positive for bacteria; one case lacked marked acute inflammation on cytology (confirmed on biopsy), and the culture-positive result was interpreted as contamination (clinical impression favored minoxidil-associated pericardial effusion).

Conclusions: PF specimens are uncommon and the vast majority of them are the result of a benign process (74.22%). The most common metastatic malignancy is lung carcinoma in both males (75.0%) and females (52.17%), followed by digestive system carcinomas in males (25.0%) and breast carcinoma in females (39.13%). The cytological / histological correlation is excellent (78.1%) and the cytological evaluation is superior to biopsy in identifying carcinomas, as biopsy carries a sampling error. The cytological diagnosis correlates 100% with the microbiology cultures in infectious cases.


Pediatric effusion cytology: Ten-year retrospective analysis at a children's hospital

Minoti Magotra, MBBS, MS, Nariman Gobara, MBChB, MS, Morris Edelman, MD, Nora Morgenstern, MD

Department of Pathology and Laboratory Medicine, North Shore Long Island Jewish Health System, New York, New York

Introduction: Background: Information on pediatric effusion cytology is limited. Some studies have shown that the pleura are the most common site of pediatric serosal effusions, primarily due to infectious etiologies. However, these studies have not focused on the cytopathology.

Materials and Methods: We report data of pediatric serosal effusions submitted for cytology from 2000 to 2010. One hundred and ten serosal effusions were categorized based on anatomical location, cytology, and age group (A: 0 to < 1 year, B: 1 to < 5 years, C: 5 to < 12 years, and D: 12 to < 20 years).

Results: Pleural effusions constituted 49% of the cases, ascites 33%, and pericardial effusions 18%. A majority of the effusions belonged to group D (48% pleura, 61% ascites, and 60% pericardial) followed by group C. Males had 55% pleural effusions and 21% were malignant. Pleural malignancies included neuroblastoma in groups A and C, Wilms' tumor in group B, lymphoproliferative disorders in groups C and D, and rhabdomyosarcoma in group D. For ascites, 72% were female, 8% were malignant (ALL, dysgerminoma, and Ewing's sarcoma), 39% had an associated malignant condition (one neuroblastoma, five lymphoproliferative, two CNS tumors, six ovarian, and one pancreatic cancer), and 14% had benign ovarian tumor. Sixty percent of the pericardial effusions occurred in males, 10% were malignant (all lymphoproliferative) and 10% were benign.

Conclusions: Among pediatric serosal effusions, pleural effusion was the most common and the most likely to be malignant. Teenagers had the highest prevalence of all effusions (54%). However, children in groups C and D had the highest prevalence of malignant effusions (27%), mostly lymphoproliferative disorders. The majority of malignant effusions in groups A and B were pleural, associated with neuroblastoma and Wilms' tumor. Ascites occurred mostly in females, typically due to ovarian tumors.


Cytomorphological and immunohistochemical assessment of metastatic breast carcinoma with apocrine features in fluid cytology

Sara Monaco, MD, Walid Khalbuss, MD, PhD, Payam Arya, MD, Liron Pantanowitz, MD

Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Introduction: Breast carcinoma is one of the most common metastases identified in fluid cytology. Immunocytochemical evaluation in these cases is often necessary and involves a panel of immunostains for mesothelial cells and adenocarcinoma. However, interpretation of immunostain results can sometimes be very difficult, particularly with apocrine breast carcinomas, where calretinin expression has been described in about 25% of these cases. The aim of this study is to report our experience with metastatic breast carcinomas showing apocrine features in fluid cytology specimens.

Materials and Methods: A retrospective search was conducted for surgical biopsies and excisions of primary breast carcinomas with apocrine features in our computer database. We then reviewed these cases to find any corresponding fluid cytology specimens. The fluid cytology cases were then evaluated for patient demographics and clinical features, cytomorphology, and available immunostain results. The cytospins, ThinPrep® slides, and cell block sections with immunostains were then reviewed in each case.

Results: A total of 20 fluid cytology cases were identified in 10 patients (mean age, 64.6 years; age range, 50 - 86 years) with a history of a primary breast carcinoma with apocrine features. Of these 20 cases, five were positive, two suspicious, four atypical, and nine negative for malignant cells. On retrospective slide review, 12 cases (60%) were positive for metastatic adenocarcinoma (six peritoneal fluids, six pleural fluids), including the five cases initially called positive, two called suspicious, three of the atypical cases, and two cases previously diagnosed as negative. The primary breast carcinomas in these cases included six cases of classical lobular carcinoma, four cases of ductal carcinoma with apocrine features, and two cases of pleomorphic lobular carcinoma. Cytomorphology revealed a predominance of single cells in a mesothelial-like pattern in eight cases (67%), and small clusters in four cases (33%). The tumor cells had moderate-to-abundant homogeneous cytoplasm and prominent nucleoli in all cases. The immunostain results were available in eight cases (78%). ER and mammaglobin immunoreactivity confirmed the presence of metastatic adenocarcinoma of breast origin in six out of eight cases (75%). Five cases (63%) showed positivity for calretinin in the tumor cells, but WT-1 was negative in the tumor cells. Four cases were negative for BerEP4 (50%).

Conclusions: Our results demonstrate that the cytomorphological and immunohistochemical evaluation of metastatic breast carcinomas with apocrine features can be challenging. These tumor cells often mimic benign or reactive mesothelial cells in fluid cytology, leading to non-definitive (atypical or suspicious) or negative diagnoses in some cases, and repeat sampling. In addition, these carcinomas may show unusual positivity for calretinin, and negativity for BerEP4, which can make their assessment even more difficult. Nonspecific cytoplasmic staining with calretinin may be due to the apocrine granules present in these tumor cells. Thus, nuclear stains like WT-1 (for mesothelial cells) and ER (for breast carcinoma) are more likely to be helpful in these difficult cases.


Pleural effusion in women with a known adenocarcinoma: The role of immunostains in uncovering another hidden primary

Haitham Nasser, MD, Tomi Kuntzman, DO

Cytology, William Beaumont Hospital, Royal Oak, Michigan

Introduction: Pleural fluid metastasis is often secondary to a known primary. We aim to identify the percentage of adenocarcinomas metastatic to pleural fluid and the frequency of a second primary being the origin of the effusion in women; and also assess the role of immunostains in uncovering the second primary.

Materials and Methods: We reviewed 131 cases of metastatic adenocarcinoma to pleural fluid from the archives of William Beaumont Hospital between June 2007 and November 2010.

Results: A total of 123 cases (94%) had a well-documented primary. In this group, the breast was the most common primary site (34%). Less common were lung (30%) and ovary (20%). Ten cases (7.9%) had two primaries. Seven cases, detected with the aid of immunostains at the time of effusion, proved to be secondary to an adenocarcinoma other than the one identified in the patients' records. Lung would have been the most commonly overlooked tumor (5 / 7 cases). Eight cases (6%) remained of inconclusive origin due to an occult primary, lack of cells on cell blocks, discordance with radiology or cytology of a known primary. Morphology, in association with a small panel of immunostains, including TTF1, Estrogen Receptor, Mammaglobin, WT1, CA125, and CDX2, proved helpful in identifying the primary origin in 94% of the cases.

Conclusions: Due to the major implications of the therapeutic strategy, even in the presence of a known primary, a small panel of immunostains is recommended on the available cell block material, to verify the origin of the effusion. Otherwise, a comparison with the previous available material is highly recommended to avoid overlooking another primary.


Cytomorphological features of metastatic sarcomas in effusions

Julieanna Constantine, BSc, Walid Khalbuss, MD, PhD, Sara Monaco, MD, Liron Pantanowitz, MD

Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Introduction: Metastatic sarcomas are an infrequent finding in the practice of effusion cytology. Making a diagnosis of sarcoma in fluid cytology can be challenging and often requires knowledge of a known primary tumor and / or use of ancillary studies. Moreover, a variety of different sarcomas can manifest with a malignant effusion. The morphology of sarcomas in effusion preparations may differ from those of the original sarcoma and may also mimic other poorly differentiated neoplasms. The aim of this study was to evaluate the cytomorphology of sarcomas in effusions and to determine the useful characteristic features for their recognition.

Materials and Methods: A nine-year retrospective review of cytology cases was performed, which included effusions with a final cytological diagnosis of metastatic sarcoma. Data regarding patient information (age, gender), effusions (body cavity involved), and sarcoma findings (primary site, presentation in months to effusion, and ancillary studies such as immunocytochemistry and FISH) were recorded. The cytology samples included direct smears, cytospins, ThinPrep® slides, and cell blocks. The following cytomorphological features were evaluated: Cellularity, cell arrangement, cell size and shape, cytoplasm quality and quantity, nuclear detail (nuclear: cytoplasmic ratio, chromatin, nucleoli), and background.

Results: A total of 27 malignant effusions (15 pleural, 56%; 12 peritoneal, 44%) due to metastatic sarcoma were identified. In three cases (11%), effusion was the initial presentation, and in 24 (89%), sarcomas were diagnosed, on an average, 11 months (range, 1 - 48) prior to obtaining the fluid specimen. A wide variety of sarcomas were diagnosed including Ewing sarcoma (four), MMMT (four), epithelioid sarcoma (four), osteosarcoma (three), clear cell sarcoma (three), leiomyosarcoma (three), rhabdomyosarcoma (two), pleomorphic sarcoma / MFH (one), chondrosarcoma (one), liposarcoma (one), and high-grade sarcoma NOS (one). The specimens were of variable cellularity. The cytological features shared by these sarcomas in effusions included single cell arrangement (93%), multinucleation (52%), nuclear pleomorphism (37%), indistinct cell borders (225), and a bloody proteinaceous background (63%). Loose clusters were infrequent (26%), including one Ewing sarcoma with rosettes. In almost all cases, the tumor cells were round, and rarely spindle-shaped (one osteosarcoma, one MFH) or bizarre (one rhabdomyosarcoma, one high-grade sarcoma NOS) in appearance. The matrix material was identified in the chondrosarcoma case, with a tigroid background in the case of metastatic clear cell sarcoma.

Conclusions: This series indicates that a wide variety of sarcomas may be diagnosed in effusion specimens. Although their cytomorphological findings are diagnostic of malignancy, very few specific characteristics appear to be unique to these metastatic sarcomas in effusions. Further sub-classification of these malignant effusions relies largely on a comparison of metastatic tumor cells with the primary sarcoma and / or ancillary testing.


Peritoneal washing cytology in patients with ovarian serous tumors of low malignant potential

John Thomison, III, MD, Anais Malpica, MD, Nour Sneige, MD

1 Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas

Introduction: At presentation, up to 30% of patients with ovarian serous tumors of low malignant potential (OvSeLMP) demonstrate extra-ovarian disease, further categorized as non-invasive or invasive implants. Patients with implants are at a higher risk of developing recurrences than patients with OvSeLMPs only. Although peritoneal washing cytology (PW) is generally recommended as part of the staging workup of OvSeLMP, to date, no study has evaluated the role of PWs in predicting the presence of peritoneal implants and their correlation with invasive versus non-invasive status.

Materials and Methods: A total of 110 patients diagnosed with OvSeLMP, ages 21 to 89 years, with 155 corresponding PWs, met the inclusion criteria, based on the medical record review. Surgical diagnoses were divided into four categories: OvSeLMP (72 patients), OvSeLMP with microinvasion or micropapillary features (30 patients), and focal low-grade serous carcinoma arising from OvSeLMP (LGSC / LMP) (eight patients). Staging biopsies were diagnosed as negative, endosalpingiosis, non-invasive implant, invasive implant, or low-grade serous carcinoma (LGSC). The corresponding PWs were reviewed and categorized as negative, endosalpingiosis or serous neoplasm, using the previously published criteria. These results were correlated with the histological diagnosis and statistical analysis was done using the Fischer's exact test.

Results: Staging biopsies showed peritoneal disease in 35 patients (32%) (18 non-invasive implants, 12 invasive, and five LGSC) and endosalpingiosis in 22 patients (20%). Sixty-nine percent of the patients with peritoneal disease demonstrated PWs with serous neoplasm (P < 0.0001). However, there were no definitive cytological features separating invasive or LGSC from non-invasive implants. PWs were positive for serous neoplasm in 11 of the 75 patients (15%), without the corresponding histological tumor implants (results summarized in [Table 1]. Of these, two patients had recurrent disease within one year (one with OvSeLMP and one with LGSC / LMP). A total of 11 patients had recurrent disease, with 10 having either microinvasion or LGSC / LMP in the primary tumor, or invasion or LGSC in the peritoneal implants, and eight were with PWs showing serous neoplasm (median time to recurrence, three years). Of the 110 patients, 93 were alive at the time of data collection, with nine lost to follow-up, five deceased from other causes, and only three dead from their disease, each with LGSC / LMP (median follow-up, four years; mean, 4.9 years).

Conclusions: PW is a sensitive indicator for the presence of peritoneal implants and may detect a subset of patients with subclinical peritoneal involvement in the staging of OvSeLMP. The distinction between invasive and non-invasive implants requires tissue sections, and the role of PW is limited in this distinction. However, in a small percentage of patients without histological implants, a positive PW may be useful in predicting disease recurrence.


Rapid evaluation of Diff-Quik® stained corneal scrapings for acanthamoeba cysts

Cora Uram-Tuculescu, MD1, Charlie Thompson, MD2 , Joseph Iuorno, MD2 , Ema Dragoescu, MD1

1Pathology, Virginia Commonwealth University Health System, Richmond, Virginia; 2Opthalmology, Virginia Commonwealth University Health System, Richmond, Virginia

Introduction: Acanthamoeba is a ubiquitous protozoan found in soil and freshwater. Acanthamoeba keratitis (AK) is an intractable, sight-threatening infection of the cornea. In the United States (US), 85% of the AK cases occur in contact lens wearers, usually as a result of exposure to contaminated water. In the US, the incidence of AK is estimated to be one-to-two cases / million contact lens users. Currently, there is no approved drug for the treatment of AK in the US. An early diagnosis and appropriate management of AK are critical for ensuring the best visual outcome. Clinically, AK should be suspected in all cases of corneal trauma complicated by exposure to soil or contaminated water and in all contact lens wearers. Tests used to confirm the diagnosis are corneal biopsy and culture (which require at least few days of processing until the final result is reported) or in vivo tandem scanning confocal microscopy (which is an expensive technology, available only in select centers). The aim of this study is to report our experience with rapid evaluation of Diff-Quik® stained corneal scrapings in suspected AK cases. Diff-Quik® stain, a modified Wright Giemsa stain, has the advantage of reducing the staining time to 15 seconds and offers better visualization of cytoplasmic details and extracellular materials, including microorganisms.

Materials and Methods: Corneal scrapings collected between January 2005 and April 2011 were retrieved. Demographic data, results of corneal biopsy and culture and calcofluor white stain, if available, were recorded. The cornea was gently scraped with the blunt side of a scalpel blade and the material was smeared on a glass slide, air dried, Diff-Quik® stained, and rapidly evaluated. If enough material was obtained, a second slide was stained for calcofluor white (a fluorochrome stain used to detect fungi and parasites in microbiology).

Results: Fourteen corneal scrapings from 12 females (85.71%) and two males (14.29%) with age ranging from 18 to 78 years (mean 39) were retrieved. Seven cases (50%) had a concomitant calcofluor white stained slide. In 11 cases (78.57%), the material was sent for microbiology culture and in three cases (21.42%), corneal biopsy was performed. One corneal scraping was positive for Acanthamoeba cysts on Diff-Quik® , with the result subsequently confirmed with a positive calcofluor white stain and corneal biopsy. The cysts were extracellular, large-sized and double-walled [Figure 1]. The background showed reactive corneal epithelial cells and acute inflammation. Another case was negative on corneal scraping and culture, however, the clinical suspicion for AK persisted and the confocal microscopy confirmed the diagnosis, followed by positive corneal biopsy one month later. The corneal scraping slide was re-reviewed and no cysts were seen. Their absence was attributed to the deep penetration of corneal stroma by the cysts and the superficial nature of the scraping. The other 12 cases were negative on Diff-Quik® , confirmed by their clinical evolution. Fungal hyphae were identified in one case, and in six cases, various bacterial organisms were identified on cultures.

Conclusions: Acanthamoeba cysts can be identified in Diff-Quik® stained corneal scrapings. This has not previously been reported in literature. The cysts are extracellular, large, and double-walled. A rapid positive result of AK helps in immediate treatment and a better visual outcome. In the case of a negative result, if clinical suspicion for AK persists, further tests are needed.


PAX2 and PAX8 : Efficacious markers for evaluating effusions from metastatic tumors

Lindsay Waters, MD, Donna Coffey, MD, Luan Truong, MD, Dina Mody, MD

Pathology, The Methodist Hospital, Houston, Texas

Introduction: PAX2 and PAX8 are transcription factors of the paired box gene (PAX) family that serve as important developmental regulators. Prior studies of surgical specimens demonstrate that PAX2 plays an important role in renal and female genital tract maturation; similarly, PAX8 is understood to influence thyroid, renal, and female genital tract development. Knowledge of PAX2 and PAX8 immunohistochemical staining can be applied to reach diagnoses in challenging metastatic malignant effusions. Distinguishing Mullerian and non-Mullerian carcinoma may be difficult; therefore, these adjunct diagnostic markers for tumors of Mullerian origin are valuable. Additionally, identifying renal cell carcinoma (RCC) in effusion cytology, albeit uncommon, is essential. PAX2 and PAX8 immunohistochemical staining has not been extensively studied in cytology specimens, specifically pleural and peritoneal effusions from both renal cell and Mullerian carcinomas.

Materials and Methods: We evaluated PAX2 and PAX8 expression in gynecological carcinoma, non-gynecological carcinoma, and renal cell carcinoma effusion specimens. These specimens had prior panels of immunohistochemical staining, that is, CA-125, WT1, CK7, CK20, ER, PR, TTF-1, and CDX2, performed to correctly identify the primary tumor. Sections from cell blocks of 89 pleural and peritoneal effusions were selected from 2003 to 2011 at The Methodist Hospital (18 controls, 10 renal cell carcinomas, 21 Mullerian carcinomas, and 40 non-Mullerian carcinomas). PAX2 and PAX8 immunohistochemical staining was performed. The stained slides were blindly reviewed, and immunohistochemical positivity was defined as at least 25% strong nuclear staining of tumor cells.

Results: PAX2 stained 0 / 18 (0%) control cases, 9 / 9 (100%) RCC cases, 7 / 20 (35%) Mullerian carcinoma cases, and 1 / 40 non-Mullerian carcinoma cases (2%). PAX8 stained 1 / 18 (5%) control cases, 10 / 10 (100%) RCC cases, 20 / 20 (100%) Mullerian carcinoma cases, and 1 / 40 non-Mullerian carcinomas (2%). PAX2 is 35% sensitive to Mullerian carcinomas and 100% sensitive to renal cell carcinomas. PAX2 is 98% specific to Mullerian carcinomas and renal cell carcinomas. PAX8 is 100% sensitive to Mullerian and renal cell carcinomas. PAX8 is 98% specific to Mullerian and renal cell carcinomas. One pleural effusion from a metastatic breast carcinoma showed weak PAX2 staining, and a second pleural effusion from a metastatic breast carcinoma showed PAX8 staining.

Conclusions: Cell blocks from metastatic malignant effusions are often limited due to low tumor cellularity. Therefore, arriving at an accurate diagnosis and identification of the primary tumor requires a small panel of highly sensitive and highly specific immunohistochemical stains. In pleural and peritoneal effusions from metastatic Mullerian and renal cell carcinomas, PAX2 and PAX8 are expressed. We observed that PAX8 stains more tumor cells with high nuclear intensity. PAX2 stains tumor cells less strongly, thus it is often challenging to interpret. PAX8 is more sensitive to metastatic effusions from Mullerian carcinomas, and it appears superior to PAX2 for this purpose. We recommend adding Pax8 in a panel of immunohistochemical stains when the differential diagnosis includes tumors of Mullerian origin.

 » FNA-General Top


An analysis of fine needle aspirations with atypical diagnoses

Leigh Ann Cahill, BS, CT(ASCP)CMIAC, Joseph Bergeron, MD, Adnan Siddiqui, MD, Michael Idowu, MD, Celeste Powers, MD, PhD

Cytopathology / Anatomic Pathology, Virginia Commonwealth University Health System, Richmond, Virginia

Introduction: The development of advanced imaging techniques over the last decade has been paralleled by equally advanced image-guided fine needle aspiration (FNA) techniques. This has created a paradigm shift from a clinic to an intensivist mode. A definitive diagnosis may not always be rendered. The use of the term 'atypical' in the interpretation occurs for various reasons including limited material, or absence of distinctive cytomorphology, and / or patient inability to tolerate the procedure. The aim of this study is to review the final FNA diagnoses of superficial FNAs (SFNA) and image-guided FNAs (IGFNA), to determine the factors that precluded a definitive interpretation.

Materials and Methods: A five-year retrospective review of 6880 FNA cases from January 2006 to December 2010 resulted in 123 FNAs with a final diagnosis of 'atypical'. Only cases with follow-up specimens that resulted in a definitive diagnosis were included in this analysis. Follow-up procedures included surgical biopsies, repeat FNAs and / or non-gynecological specimens. The presence of cell blocks and ancillary testing modalities (immunohistochemistry, flow cytometry) as well as specimen adequacy was noted. The rate of atypical FNA diagnosis was compared with the total FNAs and the rate of atypical diagnosis with a definitive subsequent diagnosis was determined.

Results: From a five-year total of 6880, 123 (1.8%) cases were diagnosed as 'atypical'. Seventy-one of 123 (57.7%) cases were SFNA and 52 / 123 (42.3%) were IGFNA. [Table 1] illustrates the subsequent diagnosis from the follow-up procedure for each FNA type. Sixty-nine of 71 (97.2%) SFNA cases and 45 / 52 (86.5%) IGFNA cases had either non-contributory / inadequate or no cells blocks. Although most atypical diagnoses were made in the absence of a useful cell block, other factors also played a role, including, but not limited to, low cellularity smears, bland cytomorphology, and the need for flow cytometry in suspected hematological cases. Smears with low cellularity in this study resulted from a difficult FNA procedure, and were, not surprisingly, also associated with non-contributory cell blocks and no ancillary testing.

Conclusions: The 'atypical' diagnosis is unavoidable as it is most often attributed to low specimen yield. At our institution, only 1.8% of all FNAs had an 'atypical' diagnosis and 79.7% of these ultimately proved to be malignant. This suggests that 'atypical' diagnoses are rendered very sparingly and most prove to be malignant. For those atypical diagnoses where sampling is adequate, it may be possible to reduce this diagnosis by conducting a peer review of atypical cases before verification, thus reducing or avoiding unnecessary follow-up procedures.

 » Gastrointestinal Tract Top


Evaluating immunophenotypes of normal stomach, duodenum, ampulla, pancreas, and colon

Haiyan Liu, MD1, David Lucas, MD1, Yajue Huang, MD2, Fan Lin, MD1

1 Laboratory Medicine, Geisinger Health System, Danville, Pennsylvania; 2 Department of Pathology, Temple University Hospital, Philadelphia, Pennsylvania

Introduction: Distinction of mucin-producing neoplasms of the pancreas from gastrointestinal contaminants on fine needle aspiration biopsy specimens can be very challenging. Many biomarkers have been reported to be useful in this regard. However, a reliable diagnostic panel is still not available. To address this issue, the immunophenotypes of potential contaminants, including normal tissues of gastric antrum and fundus, duodenum, pancreas, ampulla, and less likely colon, are studied.

Materials and Methods: We immunohistochemically evaluated the expression of, (1) epithelial markers (CK7, CK20, CK17, CK19), (2) mucin gene products (MUC1, MUC2, MUC4, MUC5AC, MUC6), (3) tumor suppressor genes and transcription factors (p53, CDX2, pVHL), and (4) tumor-associated proteins (S100P, IMP3, maspin, MOC31, CEA, CA19-9, CD10, CD15, HepPar1, villin, and P504S), on 20 cases of each of the aforementioned normal tissues. The staining intensity and the distribution were recorded.

Results: The positive staining results are summarized in [Table 1]. CK7 was positive in the mucosa of the duodenum and ampulla, focally positive in about 40% of the cases of gastric mucosa (both antrum and fundus), and negative in the colonic mucosa. CK20 was positive in mucosa of the duodenum, ampulla, and colon, and only positive on the surface epithelium of the gastric mucosa. Gastric mucosa mainly expressed MUC5AC [Figure 1], while colonic and duodenal mucosa expressed MUC2 (mainly Goblet cells) [Figure 2], with the exception of Brunner's glands, which demonstrated strong reactivity to MUC6 [Figure 3]. EMA reactivity was seen in 100% of the stomach tissue, but rarely in colonic and duodenal tissue, with the exception of Brunner's glands. Cytoplasmic staining for HepPar1 was seen in the duodenal mucosa (except Brunner's glands) and it was non-reactive in the stomach and colon tissues. Vimentin was non-reactive in all the tissues tested.

Conclusions: These data provide some important information in building an antibody panel for differentiating mucin-producing neoplasms of the pancreas from the gastrointestinal contaminants. Evaluation of the immunostain profile of MPN in a large series of cases is being undertaken.


CINtec® PLUS as a triage tool for anorectal  Pap smear More Detailss with atypical squamous cells of undetermined significance (ASC-US) / low- grade squamous intraepithelial lesions: A pilot study

Ann Walts, MD, Shikha Bose, MD

Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California

Introduction: CINtec® PLUS (CINtec), a dual immunostain for p16 and Ki67, has shown promise for a triage of women, with abnormal findings on a cervical Pap smear. In both anal and cervical cytology, atypical squamous cells of undetermined significance (ASC-US) / low- grade squamous intraepithelial lesion (LSIL) are the most frequent abnormal diagnoses rendered and require further triage for optimal patient management. This study was designed to assess the utility of CINtec for the detection of the underlying or subsequent (U / S) anal high-grade squamous intraepithelial lesions (AIN2 / 3) in individuals with ASC-US / LSIL, on anorectal Pap smear.

Materials and Methods: Forty-four anal SurePath® Pap smears (19 ASC-US, 18 LSIL, seven controls (four negative, three HSIL)) with histological and / or cytological follow-up (range, 1 to 72 mos; median, 5.5 mos) were retrieved from our departmental files. The Pap-stained slides were de-stained and then immunostained using the CINtec® PLUS Kit (mtm laboratories, Inc. Westborough, MA) that detected the overexpression of p16 as a brown / cytoplasmic and Ki67 as a red / nuclear reaction product. As recommended by the manufacturer, the presence of ≥ 1 dual stained cell was interpreted as a positive test result. P16 or Ki67 staining alone was recorded as a negative test result. Dual stain results were correlated with follow-up diagnoses. Sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) for detection of U / S AIN2 / 3 were calculated. Sixteen of the LSIL cases had also been tested for high-risk human papilloma virus (HR HPV) (Third Wave Technologies, Hologic Inc, Madison, WI). Results of the CINtec dual stain and the HR HPV screen were compared.

Results: Dual stain results and the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), of a positive CINtec test result in ASC-US / LSIL anal smears for detection of U / S AIN2 / 3 are shown in [Table 1]. Use of a minimum threshold of 1 CINtec+ cell has the highest sensitivity for the detection of U / S AIN2 / 3, and is therefore, the optimal number of cells for triage purposes. Increasing the minimum number of CINtec positive cells required for a positive test result dramatically increases the specificity of the test, while only minimally decreasing its negative predictive value. The sensitivity, specificity, PPV, and NPV of CINtec and the HR HPV screen in LSIL anal smears for detection of U / S AIN2 / 3 are shown in [Table 2]. The two test results were concordant and correctly detected the presence (or absence) of U / S AIN2 / 3 in seven of sixteen (43.5%) LSIL cases. The test results were concordant, but incorrect in three (18.8%) cases and discordant in the remaining six (37.5%) LSIL cases. Among the LSIL with discordant test results, the CINtec dual stain and HR HPV screen each accurately detected the AIN2-3 in three cases.

Conclusions: - CINtec® PLUS can be applied to routinely prepared and previously screened liquid-based anal Pap smears without loss of cytological detail.

  • CINtec is less expensive, less labor intensive, and offers a shorter turn-around time than HPV testing.
  • In a cohort of ASC-US / LSIL anal Pap smears, a positive CINtec test result has a sensitivity of 82%, a specificity of 35%, a PPV of 52%, and an NPV of 70% for detection of U / S AIN2 / 3, suggesting that CINtec could help stratify ASC-US / LSIL cases for clinical management.
  • In LSIL cases, a positive CINtec test result is less sensitive and slightly more specific than the HR HPV screen for detection of U / S AIN2 / 3.
  • A large prospective study is warranted to confirm our results.

 » Genitourinary (Kidney and Bladder) Top


Impact of cytodiagnosis on triaging of patients with renal lesions: An eleven-year retrospective study

Israh Akhtar, MD, Anwer Siddiqi, MD, Rhyne Flowers, MD, Mithra Baliga, MD

Department of Cytopathology, University of Mississippi Medical Center, Jackson, Mississippi

Introduction: The use of image-guided fine needle aspiration (FNA) and core biopsy (CB) in the diagnosis of renal lesions has a considerable impact on clinical management. We undertook a retrospective study to evaluate the treatment approach based on the cytological diagnoses of these lesions.

Materials and Methods: A retrospective review of the renal FNAs and CBs performed at our institution during the past 11 years (from 2000 to present) was conducted. The accuracy of the cytological diagnoses and treatment approach based upon the follow-up data was then evaluated.

Results: Eighty-two FNAs and / or core biopsies were performed on suspected renal lesions during this time period. These were performed on 81 patients, 49 males and 32 females, ranging in age from 4 to 87 years. The most common indication for performing these procedures was the presence of a renal mass, either solid or cystic. The diagnoses were broadly classified into five main categories: Positive for malignancy, suspicious / suggestive of malignancy, benign neoplasm, negative for malignancy, and non-diagnostic. Forty-four cases were positive for malignancy, seven cases suspicious / suggestive of malignancy, one benign neoplasm, 28 negative for malignancy, and two non-diagnostic. The most common malignant neoplasm was renal cell carcinoma (RCC) and its variants, that is, clear cell type (n = 22), papillary RCC (n = 4), sarcomatoid RCC (n = 1), and collecting duct / medullary carcinoma (n = 1). Other malignant neoplasms were Non-Hodgkin's Lymphoma (n = 4), leiomyosarcoma (n = 1), small round blue cell tumor (n = 1), and metastatic malignancies (n = 10). One Non-Hodgkin's Lymphoma case was false positive, with the follow up of the resected transplanted kidney showing chronic rejection and no evidence of lymphoma. In the suspicious category, one case, suspicious for RCC, was positive for RCC on follow up. Five cases were suspicious / suggestive of oncocytoma, two were suggestive of angiomyolipoma and a differential was provided in these cases with no available follow-up. The most common cause of negative diagnosis was a renal cyst. A majority of these patients were not re-biopsied and were routinely followed up as per clinical protocol. However, one case that was negative by cytology, but highly suspicious by imaging, was resected and the histopathological diagnosis was cystic RCC. Out of the two non-diagnostic cases, one was re-biopsied and diagnosed as RCC. In two RCC cases, the previous cytology specimen of voided urine was highly suspicious for malignancy. Tumors metastatic to the kidney most commonly originated from the lung (n = 5, four non-small cell and one small cell), one from the cervix, one metastatic osteosarcoma, one metastatic urothelial carcinoma, and two poorly differentiated carcinomas, which could not be further classified. Thirty out of 81 patients had either a histopathological correlation or previously confirmed diagnoses. Only 17 patients had surgical resection and two had open biopsy.

Conclusions: From the study undertaken, we conclude that image-guided FNA / CB is a safe, cost-effective, and a fairly accurate diagnostic mode for triaging suspicious renal lesions. Based on the cytological diagnoses, out of 81 patients, only 17 (20.9%) had a major surgical intervention, two (2.4%) had biopsy confirmation, and 62 (76.5%) were followed up as per clinical protocol.


Concurrent polyoma virus infection is common in allograft transplant patients with urothelial carcinoma

Ramneesh Bhatnagar, MD, Cinthia Drachenberg, MD, Paul Staats, MD

Pathology, University of Maryland, Baltimore, Maryland

Introduction: Polyoma virus infection is a frequent finding in the urine cytology of immunocompromised patients, especially renal transplant patients. The presence of polyoma virus infection with concurrent high-grade urothelial carcinoma is uncommon, but has been reported in rare cases. The distinction between degenerated polyoma virus infected cells and those of high-grade urothelial carcinoma can be challenging, and the possibility that a patient with a history of polyoma infection may develop urothelial carcinoma should not be overlooked.

Materials and Methods: We retrospectively reviewed the reports of all voided urine cytology cases that were interpreted as 'Positive for Malignancy' or 'Suspicious for Malignancy' at our institution over a six-year period, from 2005 to 2011, to identify cases of urothelial carcinoma. These patients' medical records were reviewed for evidence of prior organ transplantation or immunosuppression and prior evidence of polyoma virus infection. The cytological features of all cases of high-grade urothelial carcinoma with reported concurrent polyoma virus infection were reviewed by two pathologists, to confirm the interpretation.

Results: During a six-year interval, 33 cases of high-grade urothelial carcinoma were diagnosed on urine cytology, 22 of which were confirmed by tissue histology. Five of the 22 patients were renal allograft recipients. Four patients with high-grade carcinoma also had concurrent polyoma virus infection; and all had a history of renal transplant (4 / 5; 80%). In two of the cases, immunostain for SV-40 confirmed the cytological impression of a coexistent polyoma virus infection. Distinction of polyoma virus infected cells from malignant cells, on cytology, could be challenging. Features that favored the polyoma virus included: Single cells, round nuclei with smooth to slightly irregular nuclear membranes, and granular or homogenous cytoplasm. The presence of a single large, smooth, granular or vesicular nuclear inclusion was the most specific finding. Features that favored urothelial carcinoma included clusters of cells, marked size variation, coarse chromatin texture, marked nuclear contour irregularities, prominent nucleoli, and cytoplasmic vacuolization.

Conclusions: Even as concurrent urothelial carcinoma and polyoma virus infection have been reported only rarely in literature, it has been a relatively common finding in our study population, particularly among renal transplant recipients. Awareness of this overlap is important to avoid underdiagnosis of either entity, particularly urothelial carcinoma, in immunocompromised patients. Due to the significant overlap in the morphological features, the recognition of both components may be challenging, but there are several features in addition to the intranuclear inclusions of polyoma virus that can aid in making the diagnosis. Polyoma virus infection has demonstrated oncogenic potential by inactivating p53; however, its specific role in urothelial carcinoma is not well known. Although a causative relationship between polyoma virus and high-grade urothelial carcinoma cannot be inferred from our data, our results indicate that the polyoma virus may be present in high-grade carcinoma more frequently than previously proposed. It may be appropriate in certain circumstances to exclude the coexisting polyoma virus with SV-40 immunohistochemical staining and / or PCR viral load.


Clinical outcome of patients with trisomy fluorescence in-situ hybridization results using UroVysion™

Shannon Brankley, CG(ASCP) CM , Jesse Voss, CT(ASCP), MB(ASCP), Michael Campion, BS, Emily Barr Fritcher, CT(ASCP), MB(ASCP), Kevin Halling, MD, PhD

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Introduction: Fluorescence in-situ hybridization (FISH) with the UroVysion™ probe-set (consisting of chromosome enumeration probes to 3, 7, and 17 and a locus-specific identifier probe to 9p21) has been shown to have a high sensitivity for the detection of bladder cancer in patients with hematuria. Even as several studies have shown that a polysomy (i.e., ≥ 3 copies of ≥ 2 probes) FISH result is highly predictive of malignancy, very little data has been published showing a clinical correlation of patients with a trisomy FISH result (i.e., exhibiting three copies of a single probe). The aim of this study is to examine the clinical outcome of patients with a trisomy result on urine specimens analyzed by UroVysion™ FISH.

Materials and Methods: Electronic medical records were utilized to identify patients with a urine specimen diagnosed as the trisomy of chromosome 3, 7, or 17 (containing ≥ 10 cells) in the absence of multiple gains (i.e., polysomy or tetrasomy) that was analyzed by UroVysion™ FISH, as part of the clinical practice. Forty-five patient specimens, obtained between February 2001 and May 2008, were identified. Of the 45 patients, 18 patients (16 males and 2 females; median age, 71 years; range, 44 - 86 years) had follow-up data, including cytology and / or biopsy results. A positive cytology result and / or a histopathological diagnosis of carcinoma were considered to be evidence of malignancy.

Results: Trisomy of chromosome 7 was observed more often (67%; 12 / 18) than the trisomy of chromosome 3 (33%; 6 / 18). Four of the 12 (33%) patients with trisomy 7 and 2 / 6 (33%) patients with trisomy 3 were found to have malignancy at the time of FISH or on follow-up. All six patients presented with a histopathological diagnosis of superficial urothelial carcinoma (median, 26.5 days to diagnosis; range, 0 - 378 days), while 3 / 6 patients also had a positive cytology result on follow-up. One patient was diagnosed with superficial urothelial carcinoma 52 days after FISH and was later found to have invasive grade 2 (of 3) papillary urothelial carcinoma (389 days). Patients without evidence of malignancy were followed for a median of 580.5 days (range, 26 - 2014 days).

Conclusions: Even as the findings of this study indicate a positive predictive value of a trisomy FISH result, in the urine specimens it is low (33%). Our data also suggests that patients with a trisomy FISH result are unlikely to present with muscle invasive bladder cancer. Due to the infrequency of trisomy FISH results using the UroVysion™ probe-set, further studies are needed to fully understand the nature of this anomaly.


Fine needle aspiration of renal lesions in adults: A five-year retrospective study of 101 cases

Shaoxiong Chen, Harvey Cramer, Howard Wu

Pathology, Indiana University, Indianapolis, Indiana

Introduction: Fine needle aspiration (FNA) is a useful diagnostic tool for the assessment of patients presenting with renal tumors. With advances in neoadjuvant targeted therapy, the accurate subclassification of renal cell carcinoma by FNA is critical for selecting the appropriate treatment and for devising optimal strategies for patient follow-up. The aim of this study is to determine the accuracy of the subclassification of renal neoplasms by FNA.

Materials and Methods: A computerized search of our laboratory information system was performed for the five-year period, extending from January 2006 through March 2011, to identify all the FNA cases of renal lesions. The cytology reports, correlating the surgical pathology reports and related clinical histories were reviewed. The slides from all cases in which there was a discrepancy between the initial FNA diagnosis and the final histopathology were re-examined.

Results: A total of 101 renal lesions diagnosed by FNA were identified. The age of the patients ranged from 22 to 96 years (mean, 63 years). The male to female ratio was 1.2:1. The size of the renal lesions ranged from 1.2 cm to 20.8 cm (mean, 4.9 cm). The FNA diagnoses were classified as follows: Clear cell renal cell carcinoma (RCC) (15), papillary RCC (8), RCC, type not specified (21), urothelial carcinoma (9), angiomyolipoma (3), oncocytic neoplasms (8), other diagnoses (7), negative (14), and nondiagnostic (16). Histological correlation was available in 65 cases (64%), of which, eight of eight cases of clear cell RCC, six of six cases of papillary RCC, and seven of seven cases of urothelial carcinoma were confirmed histologically. Of the 20 cases of RCC, type not specified, the histological follow-up revealed clear cell RCC (nine), papillary RCC (four), unclassified RCC (four), chromophobe RCC (two), and non-diagnostic (one). Of the two cases of angiomyolipoma diagnosed by FNA, only one was confirmed histologically and the other revealed only benign renal parenchyma. All four cases of oncocytic neoplasms diagnosed by FNA revealed a discrepancy with the final histology including one case of carcinoid tumor, one case of unclassified RCC, and two cases of normal renal parenchyma. In one case, diagnosed as leiomyoma by FNA, the follow-up histology was angiomyolipoma. In two cases, the FNA diagnosis was poorly differentiated carcinoma and the correlating tissue showed renal cell carcinoma. In four cases, the FNA diagnosis was spindle cell neoplasm and the follow-up histology revealed one case of angiomyolipoma, two cases of sarcoma, and one case of sarcomatoid RCC. There were two false negative FNA cases that proved to be clear cell RCC by histology.

Conclusions: Overall, 72% (47 / 65) of the cases with histology follow-up were diagnosed correctly by FNA. A correct subclassification of the renal neoplasm was rendered in 21 cases (32%) including clear cell RCC (eight), papillary RCC (six), and urothelial carcinoma (seven). The false negative and nondiagnostic cases were attributed to sampling error. In two cases, misinterpretation of the proximal tubular epithelial cells contributed to the false positive diagnoses of oncocytic neoplasm. The cytological diagnosis of angiomyolipoma was challenging, with only one of the four cases being accurately diagnosed by FNA. To maintain the clinical relevance of this diagnostic technique in the era of modern targeted therapy, further improvements in the accuracy of the cytological diagnosis and classification of renal tumors by FNA will be necessary.


Urothelial cell carcinoma cytology-histology correlation: Are we missing hypochromatic high grade urothelial carcinomas?

Ghada Aramouni, CT(ASCP), Debbie Sabo, CT(ASCP), Deborah Chute, MD

Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio

Introduction: Urine cytology is the primary screening test for urothelial carcinoma. It has a high sensitivity for high-grade urothelial carcinomas, but is less useful in the diagnosis of low-grade urothelial carcinoma. This is due to the improved criteria for the diagnosis of high-grade urothelial carcinoma: High nuclear to cytoplasmic ratio, hyperchromasia, coarse chromatin, and nuclear membrane irregularities. However, some cases of biopsy-proven high-grade urothelial carcinoma are not identified by urine cytology.

Materials and Methods: A cytological-histological correlation search was performed over five years for all urine cytology specimens (voided, instrumented, or washing) with a urothelial tract biopsy accessioned within three days of the index urine. Only cases with biopsy-proven high-grade urothelial carcinoma were included. The original cytology slides (all ThinPrep® ) were reviewed by two cytotechnologists, for the presence or absence of atypical urothelial cells and acute inflammation. The number of atypical urothelial cells was quantified as < 5 or ≥ 5. The atypical urothelial cells were qualitatively assessed for nuclear chromatin quality and considered as either hyperchromatic [Figure 1] or hypochromatic [Figure 2]. Photomicrographs of examples of hyperchromatic and hypochromatic atypical cells were provided to the reviewers for comparison and standardization. For statistical analysis, suspicious and positive urine cytology cases were combined into a 'positive' category, as this would trigger further management at our institution. All other diagnoses were combined into a 'negative' category.

Results: A total of 132 urine cytology cases with a concurrent positive biopsy were available for review. The breakdown of original cytology diagnosis was: Negative 25%, atypical 25%, cannot exclude urothelial carcinoma 5%, suspicious 14%, and positive 30%. Twelve cases (9%) lacked atypical cells, all of which were originally interpreted as negative, and excluded from further analysis. There was no significant association of cytology diagnosis with the method of collection or the presence of inflammation. Thirty-eight cases (31%) lacked hyperchromatic atypical cells (hypochromatic only), of which 10 cases were originally called suspicious or positive. The absence of hyperchromatic atypical cells was significantly associated with a 'negative' diagnosis (P < 0.001) [Table 1]. Twenty-two cases (18%) had < 5 atypical urothelial cells present, none of which were originally called suspicious or positive. The presence of > 5 atypical urothelial cells was significantly associated with a 'positive' diagnosis (P < 0.001) [Table 2]. When all cases with < 5 atypical urothelial cells were excluded from the analysis, the absence of hyperchromatic atypical cells trended toward a significance for association with a 'negative diagnosis (P = 0.07).

Conclusions: In patients with known high-grade urothelial carcinoma, at the time of urine cytology sampling, low numbers of atypical cells are the strongest predictors of a 'negative' urine cytology diagnosis. However, a significant number of high cellularity samples contain atypical urothelial cells with hypochromatic chromatin. We propose that a subset of urothelial cell carcinomas exfoliate hypochromatic cells, which may be missed out on urine cytology.


Lymphoepithelioma-like carcinoma of the urinary bladder: Cytopathological findings and differential diagnosis on urinary cytology

Hui Guan, MD, PhD1 , Syed Ali, MD 2

1 Pathology, The Johns Hopkins Hospital, Baltimore, Maryland; 2Pathology and Radiology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Lymphoepithelioma-like carcinoma (LELC) of the urinary bladder is a rare variant of high-grade urothelial carcinoma (UC) accounting for less than 1.3% of all bladder carcinomas. As the name suggests, LELC displays phenotypic resemblance to nasopharyngeal lymphoepithelioma. There are only rare published accounts of the cytopathological features of LELC of the bladder.

Materials and Methods: All urinary cytology cases of biopsy / resection-proven LELC from a 14-year period (1997 - 2011) were retrieved from the archives of a large university hospital. Slides were prepared by SurePath® methodology. Both the cytological and histological material was reviewed and the morphological characteristics were analyzed.

Results: A total of eight patients were identified with an age range of 63 to 88 years (mean age, 77) with a male : Female ratio of 2 : 1. None of the cases was diagnosed on cytology as LELC. Retrospectively, in six of eight cases, the lesional cells were identified. The cytological diagnoses were 'atypical cells' (5) and 'suspicious for urothelial carcinoma' (1). The salient cytomorphological features included rare large, pleomorphic cells with high N / C ratios and nuclei with an open / vesicular chromatin pattern containing single prominent nucleoli. The predominant pattern was single cells with only two cases showing cellular fragments. All cases showed a variable presence of intermingled lymphocytes. Necrosis was not seen.

Conclusions: A definitive urinary cytology diagnosis of LELC is not possible and most of the cases are interpreted as 'atypical'. LELC shows only rare malignant cells and a variably increased lymphocytic population. A definitive diagnosis is only established on histological material. LELC may be included in the differential diagnosis of atypical urothelial cells with vesicular chromatin and prominent nucleoli, particularly when these cells are intermixed with lymphocytes.


Cytological and cystoscopic predictors of recurrence and progression in patients with low- grade urothelial carcinoma

Julie Jackson, MD, Güliz Barkan, MD, Umesh Kapur, MD, FASCP, Eva Wojcik, MD, MIAC

Pathology, Loyola University Medical Center, Maywood, Illinois

Introduction: Patients with low-grade urothelial carcinoma (LGUC) are at risk of recurrence and must undergo lifelong surveillance. To date, cytology and cystoscopy are the gold standard for the detection of de novo and recurrent urothelial carcinoma (UC). Our aim is to further characterize the role of cytology and cystoscopy in determining the risk of recurrence and progression in these patients.

Materials and Methods: Patients with a diagnosis of LGUC on bladder biopsy specimens or transurethral resection of bladder tumor (TURBT) specimens with corresponding urine cytology performed within two months prior to diagnosis were identified between 2006 and 2010 at our institution. Electronic medical records were reviewed for cystoscopic findings, and histological and cytological follow-up data, from the time of biopsy to the present. Chi-square and Fisher Exact tests were used for statistical analysis as appropriate.

Results: The study was comprised of 106 cases from 86 patients (mean age, 71; range, 27 - 89; 57 males, 29 females). Cytology was negative in 68 (64%), positive for UC in 10 (9%), atypical in 20 (19%), suspicious in six (6%), and unsatisfactory in two (2%) cases. On cystoscopy, a single lesion was found in 55 (52%) cases, multiple lesions in 48 (45%), and no lesions were seen in three (3%). In 58 cases with documented lesion sizes in a cystoscopy report, 22 (38%) showed lesions ≥ 2 cm. Of 94 cases with follow-up histological data, recurrence of UC (either high-grade or low-grade UC) was demonstrated in 51 (54%), recurrence of LGUC in 40 (42%), progression to high-grade urothelial carcinoma (HGUC) in 28 (29%), and no histological recurrence was demonstrated in 40 (42%) cases, during the study period. The initial urine cytology tended to be positive / suspicious when cystoscopic lesions were ≥ 2 cm rather than < 2 cm (5 / 22 (23%) cases, versus 2 / 36 (6%), respectively, P = 0.092). Of 96 cases with cytological follow-up data available, patients tended to have abnormal cytology (atypical or above) on future surveillance when > 1 lesion rather than when ≤ 1 lesions were present (28 / 42 (67%), versus 26 / 54 (48%), respectively, P = 0.070). Patients more often developed recurrent UC (either HGUC or LGUC) when > 1 lesions rather than ≤ 1 lesions were seen on cystoscopy (30 / 42 (71%), versus 21 / 52 (40%), respectively, P = 0.003). Finally, recurrence of UC (either HGUC or LGUC) was more likely when initial urine cytology was positive / suspicious rather than negative or atypical (12 / 15 (80%), versus 40 / 79 (51%), P = 0.048). More specifically, development of HGUC was more likely during the follow-up period when initial cytology was positive / suspicious rather than negative or atypical (8 / 15 (53%), versus 20 / 79 (25%), P = 0.030).

Conclusions: In conclusion, our data shows that the cystoscopic findings, including size of bladder lesions ≥ 2 cm, presence of multiple lesions, as well as positive urine cytology, at or shortly prior to the time of diagnosis of LGUC, predict recurrence or progression of the disease.


Correlation of UroVysion® FISH test results with histological findings

Dorota Rudomina, MBA, CT(ASCP), Oscar Lin, MD, PhD

Cytology, Memorial Sloan-Kettering Cancer Center, New York

Introduction: UroVysion® is a fluorescent in-situ hybridization (FISH) probe set, which has been approved by the FDA for use in monitoring tumor recurrence in patients with a history of urothelial carcinoma (UC) and screening for UC in patients with hematuria. The UroVysion® test probe set contains fluorescent labeled DNA probes to the centromeres of chromosomes 3, 7, and 17, and a locus-specific probe to the 9p21 band. The probes for the centromeres of chromosomes 3, 7, and 17 detect the hyperploidy of these chromosomes, a common feature of high-grade UC, in-situ and invasive; while homozygous deletion of the p16 gene at 9p21 is believed to be one of the most common alterations in UC and occurs early in the development of papillary UC and UC in-situ. The test has a reported sensitivity ranging from 65 to 73% for pTa and 95 to 100% for pT1-T4 urothelial carcinomas. The objective of this study is to evaluate the correlation of the cytogenetic abnormalities found in the cytology specimens that have positive UroVysion® results with UC grade and invasion in the surgical specimens.

Materials and Methods: The cases selected for this study included cases with positive UroVysion® tests and a positive biopsy within six months of the UroVysion® test. This cohort consisted of 159 cases represented by 15 low-grade UC and 144 high-grade UC (71 invasive and 73 in-situ). The UroVysion® results were considered positive according to the manufacturer's guidelines. A quantitative analysis correlating 9p21 deletions and CEP 3, 7, 17 gains in low-grade papillary UC (LGPUC), in-situ high-grade UC, and invasive UC was performed.

Results: The prevalence of 9p21 homozygous / heterozygous deletions and CEP 3, 7, 17 gains in low-grade papillary UC, in-situ high grade UC, and invasive UC are summarized in [Table 1].

Conclusions: Deletion of 9 p21 is noted in approximately 40% of the cases of LGPUC, while gains in chromosomes 3, 7 or 17 are present in approximately half the cases of LGPUC. Deletion of 9p21 is rarely seen simultaneously with gains in chromosomes 3, 7 or 17 in LGPUC. Furthermore, the presence of 9p21 deletion in the absence of gains in chromosomes 3, 7 or 17 suggests LGPUC. The presence of gains for chromosomes 3, 7 and 17 does not predict UC grade or invasion status. High-grade UC, either in-situ or invasive, is cytogenetically similar when using the UroVysion® test probe set.


Dysplastic squamous cells in urine predict malignancy in the absence of urothelial atypia

Beatriz Sanchez, MD, Umesh Kapur, MD, Güliz Barkan, MD, Grazina Chatt, CT(ASCP), Eva Wojcik, MD

Cytopathology, Loyola University Medical Center, Maywood, Illinois

Introduction: The presence of dysplastic squamous cells (DSCs) in the urine in the absence of urothelial cells, although an uncommon finding, may indicate an underlying malignant process either in the urinary or gynecological tract. Finding DSCs in the urine may precede a de novo histological diagnosis of malignancy or be the first sign of a recurrence. The aim of our study is to demonstrate the clinical significance of urine cytology with dysplastic squamous cells.

Materials and Methods: Cytology specimens with diagnoses of dysplastic squamous cells, without urothelial atypia, were identified over a period of four years (2007 - 2010). Electronic medical records (EMR) were examined for clinical follow-up, age, gender, follow-up biopsy or cytology (if applicable), and the time interval between the diagnosis of DSCs and biopsy.

Results: Thirty-eight cases of DSCs were identified (voided urine-18, bladder barbotage-19, and neobladder-1). The male : female ratio was equal and the age ranged from 21 to 81 years (mean, 53 years). Hematuria (n = 36 / 38) was the most common presenting symptom. Surgical biopsy diagnosis was available in 17 / 38 (45%) of the patients. Fifteen of the seventeen had a urinary bladder biopsy that showed a wide spectrum of squamous lesions, including squamous metaplasia with low-grade dysplasia (5), infiltrating squamous cell carcinoma of urinary bladder (3), and high-grade papillary urothelial carcinoma with squamous differentiation (3). In addition, infiltrating squamous cell carcinoma was identified in three other patients (vulva-1, penile-1, and anus-1). The remaining two patients had a benign diagnosis. Twelve patients who followed up with repeat urine cytology alone (two weeks up to one year) had a benign diagnosis. Nine of 38 (24%) patients who only received clinical follow-up had negative cystoscopy and negative fluorescence in-situ hybridization.

Conclusions: Dysplastic squamous cells, even in the absence of atypical urothelial cells, are a strong predictor of urinary tract malignancy. Follow-up of DSCs should include genitourinary tract examination.


The significance of cellular clusters in urine cytology after cystectomy

Leah Schultz, CT(ASCP)CM, Jamie Covell, CT(ASCP), Kristen Atkins, MD

Pathology, University of Virginia Medical Center, Charlottesville, Virginia

Introduction: Cystectomy for bladder cancer is typically done for invasive high-grade urothelial carcinoma. Routine surveillance by urine cytology from the neobladder or ileal conduit usually demonstrates numerous degenerating glandular cells and histiocytes. Occasionally larger papillary-like clusters of low-grade cells are seen, but the significance of these findings as an indicator of recurrent disease in this setting is uncertain.

Materials and Methods: A computer search of the Cytology Laboratory records (July 1998 - February 2010) identified 424 post cystectomy urine specimens: 351 (83%) were interpreted as negative for malignancy, 56 (13%) were with clusters of atypical cells, and 17 (4%) were diagnosed as recurrent carcinoma. This study focused on urine specimens from the atypical and malignant categories, which were reviewed for cytomorphological features and follow-up. Records of the original tumor diagnoses for these cases were noted. Immunohistochemistry was performed on some recent cases in an attempt to identify the origin of the clusters of atypical cells.

Results: Review of the pathology reports of primary tumors showed high-grade invasive urothelial carcinoma in all patients; however, a few patients also had concurrent low-grade papillary urothelial carcinoma. Sixteen of the seventeen urine samples classified as malignant had high-grade cytology: Enlarged nuclei, high N : C ratios, hyperchromasia, and marked anisonucleosis. One specimen showed numerous papillary-like clusters of cells with a more bland cytological appearance. This case had no follow-up confirmation of malignancy and could represent a false-positive interpretation. Specimens reported with atypical clusters showed two patterns. The most frequently seen pattern was clusters of cells characterized by cells with relatively low N : C ratios, eccentrically-placed nuclei, crinkled nuclear contours, and cytoplasmic vacuoles. The second, less common pattern, contained rare atypical cells with some features similar to the overtly malignant cells, but the findings were insufficient for a definitive interpretation of recurrence. Two cases from the atypical group had documented recurrent carcinoma. Review of their cytology showed clusters of cells with minimal nuclear atypia in one case and rare markedly atypical single cells in the second case. The remaining cases had benign follow-up and / or negative imaging. Immunohistochemistry performed in four recent cases demonstrated strong CK 20 and CD 68 positivity in the papillary-like cell clusters.

Conclusions: Papillary-like clusters of cells with relatively low N : C ratios, crinkled nuclear contours and vacuolated cytoplasm most commonly represent degenerated small bowel glandular cells rather than neoplastic urothelial cells. These cell clusters are potential pitfalls in the overdiagnosis of atypical urothelial cells and can lead to increased unnecessary screening. Most cystectomy procedures are performed for high-grade urothelial carcinoma and recurrences are typically similar high-grade lesions, which present with high-grade cytological features in urine specimens.


Category performance and morphological criteria of atypical urothelial cells, cannot exclude high-grade urothelial carcinoma

Christopher VandenBussche, MD, PhD 1 , Christopher Owens, MD 2 , Frances Burroughs, SCT(ASCP) 1 , Douglas Clark, MD 1 , Dorothy Rosenthal, MD 1

1 Pathology, The Johns Hopkins Hospital, Baltimore, Maryland; 2 Pathology, UMass Memorial Medical Center, Worcester, Massachusetts

Introduction: In most cytopathology laboratories, urinary tract (UT) samples are second only to Pap tests in annual volume. We previously designed a template in order to standardize our UT diagnostic categories to enable our clinicians to uniformly manage their patients. We now examine the clinical utility of one of the two atypical categories, atypical urothelial cells, suspicious for high-grade urinary carcinoma (AUC-H), as well as specific cytological features found in these specimens that are most predictive of high-grade urothelial carcinoma (HGUC).

Materials and Methods: Of the 725 patients diagnosed with atypical cells (atypical urothelial cells of uncertain significance (AUC-US or AUC-H) on urine cytology, between July 2007 and June 2009, 58 were diagnosed by our faculty as having at least one sample with AUC-H; eight patients had two samples. A total of 62 specimens were available for review. A junior and senior pathologist blindly and separately evaluated each of the specimens for individual cytological criteria and predicted whether each sample represented HGUC. Nine cytological; criteria were selected, which were thought to be the most predictive. The predictions were then matched with the follow-up biopsy outcomes, which were tracked over 18 months following the July 2009 cutoff, for inclusion in the study. The hospital laboratory information system was searched for cases that met the following criteria for the period from July 1, 2007 to June 30, 2009: (1) All cytological specimens with a diagnosis of AUC-US or AUC-H; (2) any follow-up surgical specimens resulting from the diagnosis rendered in (1); the specimens in (1) and (2) were then matched following a blind review of the cytological specimens; (3) all surgical pathology specimens with a diagnosis of high-grade urothelial carcinoma, regardless of invasion status; (4) all preceding cytological specimens from patients identified in (3); and (5) total urinary cytology samples for the same period.

Results: Of the nine cytological criteria that were selected, the four most commonly seen in AUC-H specimens were: Hyperchromatic nuclei (71%), irregular nuclear borders (66%), increased N / C ratio (56%), and anisonucleosis (55%). A large proportion of specimens (74%) contained abnormal cells meeting these multiple criteria. Enlarged nuclear size, a characteristic feature of HGUC specimens, was not a common feature in AUC-H specimens. Once the follow-up data were unmasked, all but two patients (97%) with AUC-H specimens were ultimately diagnosed with HGUC in the corresponding surgical pathology material. The small number (n = 2) of AUC-H patients with a benign follow-up diagnosis did not allow for statistical comparison of how predictive the selected criteria were for HGUC at this stage in our study.

Conclusions: Patients with an AUC-H diagnosis had a high rate (97%) of having HGUC on follow-up biopsy. The AUC-US category has a much lower predictive value of HGUC than AUC-H, but is a more common diagnosis. We are currently examining how the cytologic criteria identified in the AUC-H cohort will perform in this larger group (n = 667) of patients with an AUC-US diagnosis, to predict an outcome diagnosis of HGUC.

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Presentation of papillary serous carcinoma in Pap smears: A 15-year experience in a community hospital setting

Nyasha Bullock, MD, Vasavi Kaliki, MD, Srinivas Mandavilli, MD

Pathology and Laboratory Medicine, Hartford Hospital, Hartford, Connecticut

Introduction: Glandular lesions are often difficult to characterize in Pap smears and a significant portion of cervical glandular atypias are flagged as AGC (atypical glandular cells) with 'adenocarcinoma' (ACA) diagnosis, used usually with high-grade glandular atypia. We had the anecdotal experience of diagnosing ACA in a Pap smear and this represented the first diagnosis of 'papillary serous carcinoma' (PSC) for that patient. PSC are aggressive tumors, with early detection / treatment being the key in successful patient management. There is limited literature addressing this issue and the aim of this study is to evaluate whether the Pap smear diagnosis of ACA can be associated with PSC.

Materials and Methods: The pathology files were searched for the time period 1995 to present, for cases of ACA in Pap smear, for which there was tissue diagnosis available. The patient demographic information and pathological staging were also reviewed.

Results: There were a total of 75 cases of Pap smears with the diagnosis of ACA. Twenty-two cases (29.3%) were excluded (11 cases of metastatic carcinoma from a known extra-gynecological malignancy; 11 cases showing either primary cervical carcinoma or no available follow-up). Among the 53 cases included in this analysis, the follow-up showed 40 (75%) cases of PSC (31 cases pure PSC; 9 cases in which PSC was a component of MMMT or other mixed carcinoma) and 13 (25%) cases of non-serous endometrial adenocarcinomas. In 23 / 40 (58%) Pap smears, the diagnosis was the initial diagnosis of malignancy (i.e., no prior tissue or clinical diagnosis established). Ten of the 40 cases (25%) were in patients with a prior diagnosis of serous carcinoma. In 7 / 40 (17%) patients, there was a concurrent tissue biopsy at the time of Pap smear. Of the 40 cases, the endometrium was the primary site in the vast majority (29 cases, 73%), with the rest being ovarian (7 cases, 18%) and others (tubal-1, peritoneal-1, no clear primary source-2). The age range for patients with serous carcinoma was 40 to 92 years (mean age, 68 years).

Conclusions: This selected review of 'ACA' in Pap smears in this study shows that all women were > 40 years of age. PSC of the endometrium was the most common surgical pathology diagnosis on follow-up and in more than half the women, ACA in the Pap smear was the presenting pathological finding. This study suggests that the diagnosis of ACA in a Pap smear should raise the possibility of a 'high-grade adenocarcinoma' such as uterine and extra-uterine PSC and should warrant appropriate clinical follow-up.


Unsatisfactory ThinPrep® Pap tests in Mexico: Obscuring elements, formal cell counts, and reprocessing with glacial acetic acid

Kathryn Dyhdalo, MD 1 , Julie Shorie, CT(ASCP) 1 , Christine Booth, MD 1 , LucyBeth Nieves-Arriba, MD 2 , Jerome Belinson, MD 3 , Jennifer Brainard, MD 1

1 Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio; 2 Gynecologic Oncology, Cleveland Clinic, Cleveland, Ohio; 3 Preventive Oncology International, Inc, Cleveland Heights, Ohio

Introduction: The Michoacan Cervical Cancer Screening Study II (MECCS II) sought to determine the sensitivity and specificity of HPV tests compared to liquid-based cytology. In the cytology arm of MECCS II, a large number of Pap tests were unsatisfactory and these were studied in detail.

Materials and Methods: MECCS II was conducted in Michoacan, Mexico. Non-pregnant patients aged 30 to 50 years, with varied prior screening, and no prior hysterectomy or pelvic irradiation, participated. All patients had direct samples for HPV testing (Qiagen digene HC2, Gen-Probe Aptima) and a ThinPrep® Pap test (Hologic). Patients positive for any test were recalled. At the second visit, VIA was done to rule out large pre-invasive disease or cancer; colposcopy and biopsy followed using the POI biopsy protocol. All HC2 positive patients eligible by VIA triage were treated with cryotherapy. All unsat Pap tests were reviewed. Obscuring elements were recorded. Squamous cells were counted over 10 fields at 40X (adequate cellularity average, 3.8 cells / hpf). The presence of a transformation zone, endometrial cells, organisms and shift in vaginal flora was recorded. HPV results and biopsies were reviewed. After initial interpretation, ThinPrep® vials became available for reprocessing with glacial acetic acid (GAA).

Results: A total of 376 of 2503 Pap tests (15%) were called unsatisfatory. Fifty-nine Pap tests (2.3%) were reclassified as satisfactory after formal cell counts. Of the 374 Pap tests that were reprocessed, 125 (5.0%) remained unsatisfatory. Average cellularity increased from 2.51 to 7.61 cells / hpf after reprocessing. No squamous or glandular lesions were seen on review of the unsatisfatory cases, including those with biopsy-proven CIN. Thirty-eight patients were HPV positive and 15 had CIN on biopsy. Four patients with unsat Pap tests had CIN 2+ (12% of CIN 2+ in study). The remade Pap tests showed five ASCUS. None of these patients had CIN 2+. Transformation zone was present in 93.8%. Most Pap tests were unsatisfatory due to excess blood (69.4%); a significant subset showed marked acute inflammation (30.6%). Thirty-one Pap tests had endometrial cells, 17 a shift in vaginal flora, and seven had organisms.

Conclusions: Marked obscuring blood, inflammation, and low cellularity compromised Pap interpretations in our study. The transformation zone was sampled. A significant number of patients with an unsatisfatory Pap test and positive HPV test had CIN on cervical biopsy (39.4%). Reprocessing with GAA resulted in a marked decrease in unsatisfactory rates, although the average cellularity of those re-processed is still considered borderline. A 5% unsatisfactory rate after reprocessing remains increased.


Manual versus Imager: Comparison of full slide review using the ThinPrep® imaging system versus using a non-imager scope for full slide review

Carol Eisenhut, MD 2 , William Nelson, BS, CT(ASCP) 1 , Beth Eder, BS, CT(ASCP) 1

1 Cytology, and 2 Pathology, DCL Medical Laboratories, a wholly owned subsidiary of Laboratory Corporation of America Holdings, Indianapolis, Indiana

Introduction: Experience, internet chat, and word-of-mouth activity suggests that laboratories using the ThinPrep® Imaging System differ in their approaches to performing full slide review (FSR). There is also confusion as to whether these approaches are in regulatory compliance or if only the FSR performed on imaging system scopes would reflect full compliance with the FDA-approved methodology. Practical reasons (efficiency, availability of scopes, space, auto-overlap) and more philosophical reasons (attention span, comfort, use of 4x magnification) have created these disparate solutions. Off label use of any FDA-approved system would require additional validation to assure acceptable laboratory performance. This study attempts to demonstrate as a proof of concept that the two methods perform similarly in our laboratory.

Materials and Methods: An abnormal, enriched population of 102 previously diagnosed cases (17 HSIL, 46 LSIL, 18 WNL EC+, 12 WNL EC-, and 9 UNSAT) were divided equally among six slide sets that were subjected to 323 separate comparison events. Each of the 19 cytotechnologists (CTs) (with experience ranging from 2 years to 25+ years) reviewed one set of 17 comparison events representing a mixture of Bethesda categories. For the purpose of this study, ASCUS / ASC-H was removed from the population and the CTs were instructed to categorize any equivocal case into either SIL or Neg. They believed they were performing an efficiency study. The cases were pre-selected to avoid those with 'memorable' diagnoses or those with distinct cellular presentations. Cases that had few abnormal cells or that had poor representation in the 22 FOV were specifically included. Each CT reviewed their set of cases using 22 FOV. Those identified by the CT as needing FSR were first screened by their preferred method (either with a standard microscope or with the imager scope). The CTs were asked to perform the same task by using the other method of FSR, one-to-two weeks later. They were not informed that they would be receiving the same set of slides that were rearranged, relabeled, and re-imaged.

Results: The CT diagnoses were categorized numerically by increasing the clinical concern: 1 = WNL / EC+, 2 = WNL / EC-, 3 = Unsat, 4 = LSIL, 5 = HSIL. The differences of the outcomes were measured at three differing thresholds (detection of abnormality, differing degrees of abnormality, and the presence of a glandular component). The results showed that there was very little difference in the CT diagnoses, regardless of their method of FSR, at all three decision points. There were abnormal cases and EC+ cases identified by each method that were not detected by the other, with a slight bias toward the imager method for abnormal cases and a slight bias toward the manual method for a glandular component. None of the thresholds demonstrated a statistical significance [Table 1], [Table 2] and [Table 3].

Conclusions: This study should be viewed as preliminary, as ASCUS (with its lack of reproducibility) was deliberately not included. The population was enriched with abnormal cases to amplify any differences between the methods. Still, the methods appear to perform so similarly that it is doubtful if the inclusion of ASCUS cases would cloud the issue enough to significantly change the outcomes. The data seem to suggest no differences between methods strongly enough, to be able to proceed with that study.


Quantitative profiling of p16 / Ki-67 immunoreactivity in epithelial nuclei for automated screening of Pap smear tests

Arkadiusz Gertych, PhD 1 , Harsha Rajendra Prasad, BS 3 , Anika Joseph, MS 3 , Shikha Bose, MD 2

1 Surgery, Cedars-Sinai Medical Center, Los Angeles, California; 2 Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California; 3 Biomedical Engineering, University of Southern California, Los Angeles, California

Introduction: The ambiguity of Pap test results, substantial manual burden, as well as lack of cytochemical reporters in Pap staining compounds has led to an ongoing effort in cytopathology to incorporate human papillomavirus (HPV)-specific biomarkers into the diagnostic and screening workflows. CINtec® PLUS, a dual immunostain that provides for simultaneous detection of p16 and Ki-67 overexpression in Pap smears has recently been shown to have comparable sensitivity, but significantly higher specificity, when compared to HPV testing, for identifying high-grade cervical intraepithelial neoplasia in women with mildly abnormal Pap smears. Overexpression of p16 in cervical cells has been validated as a biomarker for transforming HPV infections and marks the earliest stages of cervical disease, while Ki-67 is a marker for active cell proliferation. The color and intensity of the nuclei is crucial in the visual determination of immunoreactivity in epithelial cells. Although the assessment of p16 or Ki-67 overexpressions alone can be straightforward, the evaluation of the superimposed p16 and Ki-67 signals may be challenging for the human eye. This study aims to evaluate an automated quantitative profiling of mixed p16 / Ki-67-related signals in cell nuclei as a potential strategy for improved screening of liquid-based Pap smears.

Materials and Methods: Five cervical smears with atypical squamous cells in the Pap smear were destained and then restained utilizing a CINtec® PLUS (mtm laboratories, Westborough, MA) kit that marks the overexpression of p16 and Ki-67 as brown / cytoplasmic-nuclear and red / nuclear reaction products, respectively. The biochemical processing of smears was followed by the high-resolution imaging of slides, and selection and recording of fields of view, where abnormal cells were found by a cytopathologist. Our image analysis began with a nuclei localization algorithm that aimed to isolate objects of high optical density and roundness. Next, a training set of patterns was formed. It consisted of 60 nuclei manually assigned by a cytopathologist to four types of immunoreactivity: Ki-67 positive, p16 positive, dual p16 / K-i67 positive or negative. Then, quantitative profiling (QP), a technique for formation of immunoreactivity profiles in subsequent nuclear areas, was carried out, utilizing the training data. Finally, the profiles were classified to investigate the method's efficacy.

Results: Collected image data was successfully processed by automated QP-based algorithms returning profiles extracted from the cell nuclei. The classification of profiles reached a good agreement with cytopathologist evaluation: 98.7% concordance in non-immunoreactive (p16 / Ki-67 negative), 78.4% in dual p16 / Ki-67 positive, and overall 91.2%. Success rate in nuclei detection was nearly 95% as measured in n = 4174 cells found in the analyzed fields of view.

Conclusions: Owing to the drawbacks of the Pap test screening methods, the idea to evaluate epithelial cells using a quantification of p16 / Ki-67 immunoresponses in digitized smears has emerged. This study manifests QP-based potential as an enhancing screening tool that can boost automated techniques or reduce the manual burden, especially in the screening of negative slides. Future studies will validate the efficacy of the combined approach on a larger patient cohort.


Comparison of atypical glandular cells (AGC) on conventional versus SurePath® preparations

Stephanie Hamilton, EdD, SCT(ASCP), IAC, MB(ASCP) 1 , Marilyn Dawlett, CT(ASCP) 2 , Savitri Krishnamurthy, MD 2

1 School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, Texas; 2 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas

Introduction: The improved sensitivity and specificity of liquid-based cytological preparations of gynecological specimens have been compared with conventional Pap smears for the detection of cervical intraepithelial lesions by several investigators. However, there are only rare reports that compared these two types of preparations for the detection of glandular lesions. We compared the utility of conventional Pap smears with SurePath® (SP) for the recognition of glandular lesions of the cervix. Even though the Pap smear was intended for the detection of cervical lesions and not for the detection of glandular lesions, which occur higher in the endocervical canal and uterus, the presence of atypical glandular cells (AGC) on cytological preparations warrants investigation as to the cause. Although there have been many studies comparing the sensitivity and specificity between conventional Pap smears and the ThinPrep® Pap Test, especially with respect to cervical intraepithelial lesions, there are scarce reports comparing the conventional with the SurePath® (SP) preparations for glandular lesions. In the present study, the cytological diagnosis of AGC on conventional Pap (CP) smears and SP tests was correlated with follow-up histological results.

Materials and Methods: The cytopathology records of MD Anderson Cancer Center were searched to identify all the gynecological specimens with a diagnosis of atypical glandular cells (AGC) on conventional Pap smears between January 1999 and December 2002 and on SP between January 2003 and December 2006. The cytological findings were compared with the histological diagnosis and / or clinical follow-up over a nine-month period. The finding of AGC in CP and SP was correlated with the subsequent significant findings, including squamous and glandular lesions (adenocarcinoma in-situ, glandular atypia / dysplasia), endometrial hyperplasia, fistula, polyp, Nabothian cyst, and leiomyoma / leiomyomata / adenomyosis statistically using the Chi-square test. This study has been submitted to the Institutional Review Board of The University of Texas MD Anderson Cancer Center (PA11-0403).

Results: AGC was reported in 180 CP smears and 120 SP preparations. Follow-up information was available in only 147 (82%) and 77 (64%) of these cases, respectively. A cytological diagnosis of AGC on CP was associated with significant lesions in 69 / 147 (47%) of the cases. Similar lesions were identified by SP in 45 / 77 cases (58%), however, the difference was not statistically significant (P < 0.1019). The sensitivity of SP for the recognition of significant glandular lesions was 41% in comparison to 36% for CP. The PPV of a diagnosis of AGC on SP for the recognition of glandular lesions was 50% in comparison to 41% with CP.

Conclusions: 1) A diagnosis of AGC on SP was associated more often with significant glandular lesions of the cervix in comparison to CP (58 versus 47%). 2) Sure path preparations were more sensitive than CP for the recognition of significant glandular lesions of the cervix.


Cytology of Pap tests in patients with benign endocervical polyps

Naima Kim, CT(ASCP), Kelisha Hendricks, CT(ASCP), Behzad Vakil, Cytology Supervisor, Andrew Schreiner, MD, Suzanne Brandt, MD, Rana Hoda, MD, FIAC

Department of Pathology and Laboratory Medicine, Papanicolaou Cytology Laboratory, New York Presbyterian Hospital, Weill Cornell Medical College, New York, New York

Introduction: Endocervical polyps (EC polyp) are the most common benign lesions of the uterine cervix. The mechanical pressure of the polyp may cause reactive and reparative changes in the EC epithelium. The cytology of the Pap tests in patients with benign EC polyp is not well-characterized.

Materials and Methods: Pap tests of a histologically proven benign EC polyp over a 7-month period (08 / 01 / 10 to 02 / 01 / 11) were retrospectively reviewed. All of them consisted of one Papanicolaou-stained ThinPrep® slide. Pap tests were either concurrent with the biopsy (bx) or up to six-months prior to the bx. Biopsies were performed for follow-up of an abnormal Pap or were clinically indicated for EC polyp removal. Results of human papillomavirus (HPV) tests were reviewed when pertinent.

Results: During the study period, there were 108 EC polyps from 108 women [age range, 21 to 75 years; mean age, 43.7 years]. Upon review of the Pap tests, no EC cells were seen in two cases. Significant findings were seen in 11 / 108 (10%) Pap tests and included: One case each of invasive squamous cell carcinoma and high grade squamous intraepithelial lesion (HSIL); Low grade SIL (LGSIL), four cases; and Atypical Squamous Cells of Undetermined Significance (ASCUS), five cases. Two of the five ASCUS cases were positive for HPV. Additional non-specific findings, in decreasing order of frequency, included: Acute inflammation (mostly present in clumps), reactive EC cells in flat sheets, squamous metaplasia, thick mucin (clumps and strands) associated with EC cells, reparative EC cells in flat streaming sheets, EC cells with cytoplasmic vacuoles with ingested neutrophils, altered blood, tubal metaplasia, EC atypia (enlarged vesicular nucleus with prominent nucleolus), and granulation tissue. Review diagnoses remained unchanged in 106 / 108 (98%) Pap tests. In the remaining two cases, that is, both the HPV negative ASCUS, the diagnosis was changed to EC atypia and reactive changes, respectively. Histologically, theEC polyps showed acute inflammation, squamous metaplasia, occasional repair, and rare ulceration only. Malignant and dysplastic abnormalities detected on the Pap tests were histologically present in the adjacent epithelium and not in the EC polyp. The ASCUS cases did not show any histological abnormality.

Conclusions: Significant squamous abnormalities were seen in 10% of the patients with benign endocervical polyp. HPV negative ASCUS may represent reactive changes in the EC polyp. No consistent combination of non-specific findings was noted in the Pap tests.


Identification of trichomonas vaginalis in different Pap test preparations: Trends over time in the college of American pathologist's educational inter-laboratory comparison program

Lydia Howell, MD 1 , Teresa Darragh, MD 2 , Rhona Souers, MS 3 , Nicole Thomas, CT(ASCP), MPH 4 , Ann Moriarty, MD 5

1 Pathology and Laboratory Medicine, University of California, Davis, Sacramento, California; 2 Pathology, University of California, San Francisco, San Francisco, California; 3 College of American Pathologists (CAP), Northfield, Illinois; 4 College of American Pathologists (CAP), Northfield, Illinois; 5 Ameripath, Indianapolis, Indiana

Introduction: Trichomonas is challenging to identify on Pap tests due to its small size, and the presence of distracting associated cellular changes resembling other inflammatory conditions or intraepithelial lesions. Clinically, the repercussions of misdiagnosis are discomfort, expense, and treatment complications. Artifacts of different slide preparations may amplify problems of Trichomonas detection. The College of American Pathologists Interlaboratory Comparison Program in Gynecological Cytology (CAP-PAP) has seen an increase in liquid-based challenges with a concomitant decrease in the enrollment for conventional Pap tests. This study evaluates CAP-PAP participant results for Trichomonas over 20 years, to ascertain if there is a change in performance with experience with different preparations types.

Materials and Methods: The concordance rates for the target diagnosis of Trichomonas vaginalis were evaluated for 167,956 responses on 3730 slides evaluated in the CAP-PAP, between 1990 and 2010. A nonlinear mixed model was fit with three factors participant type, preparation type, and a two-level program year (1990 - 1999, 2000 - 2010), and included interaction terms between these factors and a repeated measures component to model the slide factor correlation structure.

Results: The cytotechnologists (CT) showed higher overall concordance than pathologists (MD) (89.8% vs. 83.4%, P<.001). Both readers improved concordance in the second time interval, although the CTs showed higher concordance than the MDs (91.3 vs, 85.0%, P = 0.01). The lowest concordance rates were SurePath® preparations for MD and conventional smears for CT.

Conclusions: Detection of Trichomonas has improved with experience, with a liquid-based preparation. Awareness of differences between MD and CT and preparation types may help ensure accurate results in clinical practice.


Cervicovaginal cytology findings in uterine malignant mesodermal tumor (MMMT)

Kay Kasal, HT(ASCP), Hannah Krigman, MD, DengFeng Cao, MD, PhD

Pathology and Immunology, Washington University St. Louis, Missouri

Introduction: Mixed malignant mesodermal tumor (MMMT) is an aggressive uterine tumor that typically occurs in women aged 50 to 80 years. MMMT accounts for approximately 2% of the endometrial malignancies. The most common presentation is of a polypoid mass. These tumors are frequently necrotic, so biopsies are not informative. As this tumor is rare, the findings on cervicovaginal cytology are not well described. For this reason, we undertook a survey of preoperative cervicovaginal cytologies in an effort to identify features predictive of MMMT

Materials and Methods: Because MMMT is a relatively rare tumor, the database was searched for all patients with a diagnosis of MMMT from 1995 to 2010. The database was manually searched for cervicovaginal cytologies obtained within three months prior to resection. Pap tests were rescreened and examined for inflammation, tumor diathesis, and atypical cells, which were further classified as being of a glandular, stromal, undifferentiated or squamoid type.

Results: Sixteen monolayer cervicovaginal cytologies were identified in our files. Six were negative on rescreen, and one was considered unsatisfactory for evaluation due to paucicellularity. Nine cervicovaginal smears were abnormal. Four smears had glandular cells most typical of adenocarcinoma. Five had atypical endocervical type cells. We did not have samples with both atypical endocervical and endometrial type cells. Two samples with endometrial type groups and two smears with abnormal endocervical type epithelial cells had atypical squamoid cells as well. Of note, four samples had a population of small atypical, undifferentiated cells either singly or in groups. In two Pap tests, no other atypical cells were present. One sample had a striated muscle and one sample had fragments of stroma. Eleven samples had blood, inflammation or necrosis, including two samples without atypical epithelial cells.

Conclusions: Smears with abundant diathesis or inflammation should be carefully screened for small single or grouped poorly differentiated cells. In our series, that constellation seemed most predictive of MMMT. The admixture of two atypical epithelial cell types suggests a diagnosis of MMMT. The presence of striated muscle of abundant stroma also suggests a diagnosis of MMMT.


Cervista TM HPV HR, Cervista TM HPV 16 / 18 assays versus hybrid capture 2 assay: Comparative assay outcome evaluation in women with negative Pap smear cytology

Elizabeth Kurian, MD 1 , Mandi-Lee Caporelli 1 , Stephen Baker, MScPH 2 , Bruce Woda, MD 1 , Ediz Cosar, MD 1 , Lloyd Hutchinson, PhD 1

1 Department of Pathology, Memorial Medical Center, University of Massachusetts, Worcester, Massachusetts; 2 Departments of Cell Biology, Quantitative Health Sciences and Information Services, University of Massachusetts, Worcester, Massachusetts

Introduction: A controversial commentary by Kinney et al. (AJCP 2010, 134:193-199) highlighted the poor clinical specificity of the Cervista TM assay. In response, we directly compared the Hybrid-Capture 2 (HC2) and Cervista TM assays.

Materials and Methods: The consecutive specimens (n = 601) were tested using HC2, then Cervista TM HR and Cervista TM HPV 16 / 18, with subsequent analysis of cytology negative cases (n = 533).

Results: Results indicated no significant difference (P = 0.458) in the prevalence rates between HC2 (7.5%) and Cervista TM HR (8.5%). Cervista TM 16 / 18 prevalence was 1.6%. Negative percent agreement was 96.5% (468 / 485) versus 70% (28 / 40) positive percent agreement. The internal Cervista TM DNA control had minimal impact for limiting the false negatives. Low genomic DNA limited evaluation in 4.1% (22 / 533) of the specimens with scant cellularity. In specimens with sufficient material, re-extraction and retesting yielded negative results (19 / 19). Indeterminate rates were as follows: Manual Cervista TM HPV HR 6.94% (37 / 533), Cervista TM HPV 16 / 18 6.80% (35 / 514) versus 1.13% for automated HC2 HPV HR (6 / 533).

Conclusions: Our data shows 29 discordant positive results (12 HC2 or 17 Cervista TM HR), suggesting that some women with negative cytology may be triaged for unnecessary follow-up with either assay. For clinical screening, both Cervista TM HR and HC2 are comparable, and by extension should provide an excellent negative predictive value for histologically relevant disease.


Comparing two methods of detection for  Chlamydia trachomatis Scientific Name Search  using liquid-based specimens

Angelique Levi, MD, Danita Beckman, CT(ASCP), Kevin Schofield, CT(ASCP), Malini Harigopal, MD, David Chhieng, MD

Pathology, Yale University, New Haven, Connecticut

Introduction: Chlamydia trachomatis (CT) is a sexually transmitted infection that may remain asymptomatic in female patients. Because of the widespread use of liquid-based (LB) preparations, testing for CT as part of a Pap test examination is clinically useful, efficient, and convenient. This study compared the detection rate of CT in liquid-based gynecological specimens using the following two methods: The BD ProbeTec TM CT Q x -amplified DNA assay on the BD Viper TM System; and the Qiagen rapid capture system (RCS) CT DNA Test

Materials and Methods: A total of 1054 BD SurePath® and ThinPrep® specimens from female patients referred from multiple different referring practices in both an urban and suburban setting were tested for CT nucleic acids by the following two methods: The next generation assay BD ProbeTec TM CT Q x amplified DNA assay on the BD Viper TM System with XTR TM Technology (BD Diagnostic, Franklin Lakes NJ); and the Qiagen Rapid Capture System (RCS) CT DNA Test (Qiagen, Germantown MD). For all positive and any discrepant CT test results between the two methods, an additional PCR-based confirmatory test for CT was performed using TaqMan real-time PCR assay.

Results: Among the 1054 clinical LB Pap test samples, 13 (1.2%) specimens were positive for CT using the ProbeTec CT Q x assay on the Viper platform, while eight (0.76%) were positive for CT using the Qiagen RCS CT DNA Test. Twelve of the 13 CT-positive specimens using the ProbeTec CT Q x assay were confirmed by the real-time PCR assay. Therefore, among the 13 CT-positive cases using the Viper system methodology, there was one false positive case. There were no false positive cases using the Qiagen RCS methodology, but four of the 12 PCR-confirmed CT-positives cases were negative by the Qiagen RCS CT DNA Test.

Conclusions: In this study, the ProbeTec TM CT Q x assay detected more positive cases of CT from the LB Pap tests as compared to the Qiagen RCS CT DNA Test. Although the ProbeTec TM CT Q x assay on the Viper system may be more sensitive in the detection of CT from the LB Pap tests, confirmatory testing is suggested to exclude the possibility of a false positive test.


CINtec® PLUS for triage of women with low-grade squamous intraepithelial lesions on Pap smear

Sanam Loghavi, MD, Ann Walts, MD, Shikha Bose, MD

Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California

Introduction: Current practice guidelines recommend colposcopy, an invasive and expensive procedure, for women with low-grade squamous intraepithelial lesions (LSIL) reported on Pap smear. Colposcopic cervical biopsy has a sensitivity of 68 - 85% for detection of high-grade cervical intraepithelial neoplasia (HG CIN). CINtec® PLUS (CINtec) is a dual immunostain that provides for simultaneous detection of p16 and Ki67 overexpression in Pap smears. This study was designed to determine the utility of CINtec for detection of an underlying or subsequent HG CIN in women with LSIL on Pap smear.

Materials and Methods: Sixty-five cervical SurePath® Pap smears from women ranging from 17 to 75 years of age (median, 29.5 years) with LSIL and histological and / or cytological follow-up (range, 1 to 33 mos; median, 10 mos) were retrieved from our departmental files. The Pap-stained slides were destained and then immunostained utilizing the CINtec® PLUS Kit (mtm laboratories, Inc Westborough, MA) that showed p16 as brown / cytoplasmic and ki67 as red / nuclear reaction products. A ≥ 1 CINtec dual-stained cell was interpreted as a positive test result. Staining alone by p16 or Ki67 was recorded as negative. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detecting an underlying or subsequent HG CIN were calculated.

Results: On follow-up, 51 cases had a negative or LSIL biopsy and / or Pap smear and 14 cases had HG CIN. Initial LSIL Pap smears were CINtec positive in 10 of these 14 women. CINtec had a sensitivity of 71%, a specificity of 47%, an NPV of 86%, and a PPV of 27% for detection of an underlying or subsequent HG CIN.

Conclusions: CINtec® PLUS is an inexpensive, noninvasive tool that can be applied to routinely prepared Pap smears and can contribute to the triage of women with LSIL on Pap smear.


CINtec® PLUS immunostain is better than p16 or ki67 for triage of women with abnormal Pap smears

Sanam Loghavi, MD, Ann Walts, MD, Shikha Bose, MD

Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California

Introduction: p16 and ki67 have each been shown to be good biomarkers for high-grade cervical intraepithelial neoplasia (HG CIN). Of late, CINtec® PLUS (CINtec), a dual immunostain for p16 and ki67, has been proposed as a tool for the triage of women with atypical squamous cells of undetermined significance (ASC) and / or low-grade squamous intraepithelial lesions (LSIL) on Pap smear. We compared the utility of CINtec with that of p16 and ki67 alone to detect the underlying or subsequent high-grade cervical intraepithelial neoplasia (HG CIN).

Materials and Methods: One hundred and fifty-two cervical SurePath® Pap smears (87 ASC and 65 LSIL) with histological and / or cytological follow-up (range 1 to 83 mos; median 10 mos) were retrieved from our departmental files. The Pap-stained slides were destained and then immunostained utilizing the CINtec® PLUS Kit (mtm laboratories, Inc , Westborough, MA) that detected the overexpression of p16 and ki67 as brown / cytoplasmic and red / nuclear reaction products, respectively. Each smear was evaluated with respect to CINtec, p16, and ki67 separately. A smear was considered CINtec positive if ≥ 1 dual stained cell was present and p16 or ki67 were positive if 10 immunostained cells were present. Sensitivity, specificity, and positive and negative predictive values for detecting an underlying or subsequent HG CIN were calculated for each stain.

Results: The results are shown in [Table 1].

Conclusions: Compared to p16 or ki67 alone, CINtec® PLUS has a considerably higher specificity and comparable negative predictive value for detecting an underlying or subsequent HG CIN.


Significantly improved predictive value provided by ProExC immunostaining of Pap smears diagnosed as HSIL warrants leap to LEEP

Andrew Veyliotti, CT(ASCP) 1 , Sarah Wachs, CT(ASCP) 1 , Chris Rozelle, MD 1 , Robert Krum, MD 2 , Terry Morgan, MD 1

1 Pathology, OHSU, Portland, Oregon; 2 Pathology, Kaiser NW, Portland, Oregon

Introduction: Management guidelines now allow for a 'leap to loop electrosurgical excision procedure (LEEP)' in women with a pap smear diagnosis of high grade dysplasia (HSIL). This change was due to an improved positive predictive value (PPV) of HSIL (70 - 80) provided by liquid-based paps sufficient to bypass inefficient colposcopy. However, our clinicians are hesitant to leap to LEEP because of the remaining significant false positive rates. HPV testing is not useful in these cases because the PPV is no better than cytology. Our objective was to test whether a specific marker of neoplastic transformation, such as ProExCTM, provides sufficient PPV to warrant the 'leap to LEEP'.

Materials and Methods: SurePath® slides were prepared from the residual cell pellet from cervical Pap smears diagnosed as HSIL (n = 118) and immunostained for ProExC using a Ventana Benchmark XT. At least 5000 epithelial cells were required on each slide to be adequate for staining. Sufficient outcome data required either biopsy-proven high-grade dysplasia (CIN2+) or at least three years of negative follow-up. Biopsies were also stained for ProExC and a 'Consensus Diagnosis' between gynecological pathologists (TM and RK) provided the gold standard. Stained Pap smears were also independently scored by a cytopathologist (TM), pathology resident (CR), and cytotechnologists (AV and SW). Kappa statistics and predictive values were calculated and associations were tested by Chi-square analysis.

Results: We observed an excellent agreement between pathologists scoring ProExC (kappa statistic = 0.81) and between cytotechnologists (kappa = 0.82). Discordant scores were primarily in paps with fewer than 10 positive cells (52% discordance compared to 3 - 5% in negative or abundant cases). Surprisingly, cytotechnologists showed no additional improvement in their inter-rater reproducibility, also discordant in cases with few positive cells. The prevalence of CIN 2+ in HSIL was 81% with positive staining leading to a PPV of 95, NPV 78, and positive likelihood ratio of 4.5. Chi-square analysis revealed an association between positive ProExC staining and CIN2+ outcome (P < 0.0001).

Conclusions: We conclude by stating that ProExC staining may strengthen clinical confidence by significantly improving the positive predictive value in HSIL Pap smear diagnoses before leaping to LEEP. The weakness may be cases with only rare positive cells.


Proficiency testing of high-risk human papillomavirus DNA tests: The first three years of experience of the college of pathologists CHPV surveys

Ann Moriarty, MD 1 , Joel Bentz, MD 2 , Barbara Winkler, MD 3 , Andrew Fischer, MD 4 , Rodolfo Laucirica, MD 5 , Rhona Souers, MS 6 , Nicole Thomas, MPH, CT(ASCP) 7 , Chengquan Zhao, MD 8

1 Cytology, AmeriPath, Indianapolis, Indiana; 2 Pathology, Lab Med Consultants Ltd, Las Vegas, Nevada; 3 Director, Pathology, Mt Kisco Med Group PC, Mount Kisco, New York; 4 Pathology, University of Massachusetts Medical Center, Worcester, Massachusetts; 5 Pathology, Baylor College of Medicine, Houston, Texas; 6 Biostatistics, College of American Pathologists, Northfield, Illinois; 7 Surveys, College of American Pathologists, Northfield, Illinois; 8 Pathology, Magee-Womens Hospital, Pittsburgh, Pennsylvania

Introduction: Laboratories that test for high-risk human papillomavirus (HRHPV) DNA must participate in annual proficiency testing (PT) for HRHPV as required by the Clinical Laboratory Improvement Amendment of 1988. The College of American Pathologists (CAP) Human Papillomavirus (High-Risk) Survey for Cytopathology and Other Laboratories (CHPV) meets this requirement, with five samples sent three times per year. It is the only national HRHPV proficiency test available in the United States adapted to all the commercially available transport / preservative media types, and it can be used for any HPV testing method. This study analyzes the performance of laboratories using various transport media and testing methods,

Materials and Methods: The analysis examines the CHPV survey results from 2008 through 2010. Each survey offered either Digene™, SurePath® , ThinPrep® transport media or a mixture of different media types. A correct response was defined as a challenge that achieved participant consensus at 80%. A nonlinear mixed model with a significance level of 0.05 was used to analyze the survey year, media, method, and challenge type, to identify factors associated with performance.

Results: There were 476 laboratories that submitted 14, 911 responses; 80% consensus was achieved for all challenges. Overall, 14,620 (98%) responses were correct. There were no differences in performance between positive and negative challenges, or the rate of correct responses from year to year. Significant differences in performance were identified for transport media and testing methods as shown in [Table 1].

Conclusions: The CHPV surveys demonstrated consistently good laboratory performance of HR HPV DNA tests over the three years of the program. All challenges were concordant to the 80% threshold. Digene STM and ThinPrep® transport media performed better than SurePath® media. Digene, user developed test methods, and Cervista performed better than the other commercial kits and third wave invader assays. The CAP CHPV survey provided useful information for laboratories to assess their choices for HPV testing.


Analytic validation of the cervista HPV HR test using SurePath® cervical cytology specimens

Marilyn Nutter, BA, CT(ASCP), Brenda Sweeney, MS, SCT(ASCP) MB, David Wilbur, MD

Cytopathology, Massachusetts General Hospital, Boston, Massachusetts

Introduction: High Risk (HR) Human Papillomavirus (HPV) testing is important in the management of cervical cytological abnormalities and in assessing the risk in cytology-negative patients who are over 30 years of age. This study compared the prior validated Qiagen Hybrid Capture II HPV Test (HC II) with the Hologic Cervista HPV HR test in SurePath® specimens for the purposes of analytic validation prior to the initiation of the latter assay to routine use.

Materials and Methods: The tests were prospectively evaluated in a single center. DNA was extracted from the residual SurePath® cervical cytology specimens and assessed for the presence of HR HPV using the two tests. Results obtained were compared. Discordant results were adjudicated by PCR performed at an outside institution.

Results: One hundred and eighty-three cases had complete results for both tests (86 NILM, 87 LSIL, 5 HSIL, and 5 ASC-US). One hundred and sixty-six specimens showed concordant results between the two tests (91%). Of the 17 discordant cases, all were HC II positive and Cervista negative. PCR adjudication results on these cases showed that one case was positive for HR HPV (type 68), six were negative for HR HPV, and 10 were positive for low risk (LR) HPV. There was a single HC II positive / Cervista negative discrepancy in the HSIL group, which was found to be positive for LR HPV on PCR adjudication. Follow-up biopsy and endocervical curettage on this patient showed no pathology. Compared to HC II with PCR adjudication, Cervista HPV HR showed only one false negative and no false positive test, (1 / 183 = 0.6% error rate). The false negative case was in a specimen with ASC-US and follow-up showed no pathological abnormality.

Conclusions: The Cervista HPV HR Test could be used for the detection of HR HPV with residual cervical cytology specimens collected in the SurePath® media. The test did not show cross reactivity with LR HPV types as was noted in 10 of the 17 (59%) HC II specimens from the discordant test pairs.


Endometrial carcinoma: In need of a screening tool

Claudia Ormenisan 1 , Marina Mosunjac 1 , Bhagirath Majmudar 1 , Michael Koch 1 , Angie Earhart 1 , Victor Y Du 1 , Stefan Pambuccian 2 , Gabriela Oprea 1

1 Pathology, Emory University, Atlanta, Georgia; 2 Pathology, University of Minnesota, Minneapolis, Minnesota

Introduction: Endometrial carcinoma (EC) is the most common invasive neoplasm of the female genital tract and the fourth most frequently diagnosed cancer in the United States. In 2010, there were an estimated 43,470 new cases of endometrial carcinoma and approximately 7,950 deaths caused by EC. Even as the Pap test represents the most successful screening tool for squamous cell cervical carcinoma and its precursors, it is of limited value in the detection of EC. A possible explanation for the low sensitivity of Pap test for EC includes the low shedding rate of EC cells. This study intends to determine the efficiency of the Pap test in detecting Endometrial Carcinoma in our patient population. We also plan to evaluate the presence in the Pap test of diagnostic cells for glandular neoplasia

Materials and Methods: A retrospective review for surgically removed uterine carcinomas between 1997 and 2009 was performed. Demographic data, Pap test results, pathological type and stage were entered in a Microsoft Excel spread sheet. Trends and Chi-squared statistical analyses were performed. The likelihood of preoperative diagnosis of EC by cytology via Pap test was correlated with the type and grade of the tumor. The age range for the types of EC (Endometrioid, Serous / Mixed and MMMT) was assessed.

Results: One hundred and seventy-three patients were treated surgically for EC. [Table 1] details the type of endometrial carcinoma with age, stage, and relation to the cervical cytology.

Conclusions: Serous carcinomas and malignant mixed mullerian tumors (MMMTs) were more likely diagnosed at an older age. Patients with serous carcinoma and MMMT were more likely to present with a higher stage. MMMTs were the least likely to have a preoperative positive Pap test. Our data shows that early diagnosis still remains a challenge for endometrial carcinoma.


Cytological features of malignant mixed mullerian tumor (MMMT): A report of twenty-three cases

Michelle Reid, MBBS 1 , John Crosby, MD 2 , Sravankumar Kavuri, MBBS 3 , Preetha Ramalingam, MBBS 2

1 Pathology, Emory University School of Medicine, Atlanta, Georgia; 2 Pathology, Georgia Health Sciences University, Augusta, Georgia; 3 Pathology, Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia

Introduction: Malignant mixed mullerian tumor (MMMT) is a biphasic malignant tumor of the female genital tract that is often seen in the elderly. It may arise in the vagina, cervix, endometrium, ovaries or  Fallopian tube More Detailss, as well as the peritoneum. Although its histological features are well-established , its cytological features are only depicted in isolated reports. We describe the cytological features of 23 MMMTs, the largest such series to date.

Materials and Methods: We identified 23 cytologically sampled MMMTs from 21 patients who ranged in age from 45 to 97 years (mean 70 years.). The site of origin, specimen-type and cytological features of each case were analyzed and tabulated.

Results: There were eight primary and fifteen metastatic tumors. Primary sites included cervix (Cx) (1), endometrium (13), ovary (3), and peritoneum (P) (4); 20 / 21 patients had biopsied (2) or resected (18) primary tumors confirmed histologically, and 14 / 20 of these had heterologous elements (HE) on histology. These HEs included rhabdomyoblasts (6) and chrondrosarcoma (8). The cytological specimens included Pap smears, ascitic fluid (AF), pleural fluid (PF), pelvic washings (PW), and fine needle aspirations (FNAs) from liver and lung, as well as inguinal and iliac lymph nodes (LNs). The specimen types and sites of sampling are summarized in [Table 1]. The cytologic findings included carcinoma (CA), sarcoma (SAR), and less commonly HE [Table 2]. The most common CA was adenocarcinoma, not otherwise specified (AD, NOS), with a few less common cytomorphological subtypes, including papillary serous, squamous, papillary, and plasmacytoid carcinoma.

Conclusions: The majority of cytologically sampled MMMTs were metastatic and most represented pelvic washings and FNAs of peritoneal metastases. Primary tumors were most often endometrial and cervical MMMTs, sampled via Pap smear, and followed by FNAs of the primary MMMT of the peritoneum. Most cytologically diagnosed metastatic MMMTs had only CA cells and no SAR component, and in these cases, the cytological diagnosis was facilitated by the knowledge and review of the histology of the corresponding resection or biopsy specimen. The latter observation in our review represents a potential diagnostic pitfall that could lead to the erroneous misclassification of MMMT as CA, resulting in the under-staging and suboptimal management of patients. Not surprisingly, most primary MMMTs resembled their histological counterparts and had a mixture of both CA and SAR cells. Although HEs were identified frequently on histology, they were rarely identified on cytology in both the primary and metastatic MMMTs. As such, the identification of HEs is not helpful in the cytological diagnosis of MMMT, which relies instead on the identification of a mixture of sarcomatoid cells as well as malignant epithelial cells. The diagnosis of MMMT must be borne in mind by cytotechnologists and cytopathologists when evaluating cytology specimens, especially those from the gynecological tract and peritoneum.


Cytological parameters predicting neoplasia in atypical glandular cells with histological follow-up: A single institution experience

Jordan Reynolds, MD, Andrew Sciallis, MD, Jesse Voss, CT(ASCP), Ashley Johnson, CT(ASCP), Mohammad Dairi, CT(ASCP), Ocla Kigen, CT(ASCP), Jill Caudill, CT(ASCP), Brent Bedke, MD, Michael Henry, MD, Aziza Nassar, MD

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Introduction: Studies investigating the histological follow-up of the 2001 Bethesda system diagnosis of 'atypical glandular cells' (AGC) have been performed focusing on various screening methods, patient populations, and Pap preparations. We report the histological follow-up of women receiving an AGC diagnosis using liquid-based screening (ThinPrep® Hologic, Inc., Bedford, MA). We also look for specific cytological features that would predict benign versus malignant follow-up results.

Materials and Methods: A retrospective medical record search was performed to identify liquid-based cervical cytology tests interpreted as AGC. The AGC diagnoses were stratified into five groups: Atypical endometrial cells (AGC-EM), atypical endocervical cells (AGC-EC), atypical glandular cells favor neoplastic (AGC-FN), atypical glandular cells not-otherwise-specified (AGC-NOS), and atypical glandular cells favor reactive (AGC-FR). Histological follow-up, either as a biopsy or resection specimen with a diagnosis of cancer, complex endometrial hyperplasia, or high-grade squamous dysplasia, was considered as an evidence of disease. Available slides were blindly reviewed by three cytotechnologists and a Fellow, and the specific, pre-determined cytological features were recorded for each case. The cytological parameters evaluated are shown in [Table 1]. Available cases were reviewed at a consensus session with a cytopathology Fellow and cytopathologist. Statistical analysis included a Pearson chi-square test, using a JMP 8.0 (SAS, Cary, NC), to compare the cytological factors, which would predict benign versus malignant follow-up.

Results: We analyzed 123,763 liquid-based cervical cytology specimens in our laboratory between 2005 and 2009. Of these, 264 (0.2%) samples were diagnosed as AGC. Patients without subsequent biopsy were excluded from the statistical analysis (n = 16). Of these remaining 248 patients, 60 (24%) were diagnosed with AGC-EM, 36 (15%) with AGC-EC, 28 (11%) with AGC-FN, 122 (49%) with AGC-NOS, and two (<1%) with AGC-FR. Of the 248 patients with AGC, 80 (32%) had a clinically significant lesion represented as: Severe squamous dysplasia (n = 9), endometrial hyperplasia (n = 4) , ADCA (n = 48), MMMT (n = 1), serous carcinoma (n = 7), endocervical ADCA (n = 3) , AIS (n = 3), metastatic carcinoma (n = 4), and endometrial stromal sarcoma (n = 1). Patients with evidence of neoplasia were significantly older (P < 0.001) than those without (59.6 and 49.2 year-olds, respectively). Two hundred and eighteen cases had available slides for review. Multiple parameters were assessed . [Table 1] summarizes the cytological parameters that are significantly more or less frequently associated with malignant follow-up. Neoplastic cases showed significantly increased numbers of single cells, cells in three dimensional clusters, gland formation, engulfed neutrophils, nuclear enlargement, increased N : C ratio, irregular nuclear borders, reniform nuclei, loss of polarity, and macronucleoli.

Conclusions: These results are in accordance with the previous studies highlighting the clinical significance of a diagnosis of AGC on liquid-based screening. Cytological parameters can be used to predict benign from neoplastic follow-up conditions. Close clinical follow-up is warranted in the findings of AGC, especially in older women, by virtue of the frequency of significant neoplasia. Moreover, biopsy follow-up is necessary to correlate the cytological findings with the histology, and this should be addressed in the pathology reports.


Adequacy criteria for papanicolaou tests in postmenopausal women: Should we be content with less cells?

Melissa Rodgers, CT(ASCP), Ana Alvarez, MD, Swati Mehrotra, MD, Grazina Chatt, SCT(ASCP), Eva Wojcik, MD, Guliz Barkan, MD, FIAC

Cytopathology, Loyola University Medical Center, Maywood, Illinois

Introduction: The 2001 Bethesda System for reporting cervical cytology includes two categories for specimen adequacy, regardless of age or menopausal status: Satisfactory (SAT) and unsatisfactory (UNSAT). Liquid-based preparations require an estimated minimum of 5000 well-visualized / well-preserved squamous cells for the specimen to be considered adequate. Postmenopausal women frequently have hypocellular Pap tests (PAP) due to atrophy; therefore, the current criteria for cellular adequacy may be too stringent. The aim of this study is to evaluate the criterion of optimal cellularity in postmenopausal women.

Materials and Methods: A retrospective review from 2001 through 2008 showed that 170,094 ThinPrep® PAP tests were diagnosed at our institution; 68,821 of which were from women 45 years and older. Of these cases, 792 PAP (1.15%) were diagnosed as UNSAT. The following specimens were excluded from our study: Specimens that lacked follow-up, specimens from pre / perimenopausal patients, inadequate samples due to acellularity and inadequate samples due to obscuring blood / inflammation. This left 235 PAP (29.67%) UNSAT, due to low cellularity. These cases were evaluated for cellularity with follow-up frequency and diagnoses recorded. Follow-up was defined as a repeat PAP or biopsy performed within one year or more.

Results: Of the 235 UNSAT PAP, 189 (80.43%) had follow-up within one year (184 PAP and five biopsies). Of these, 163 (86.24%) were diagnosed as negative for intraepithelial lesion or malignancy (NILM), 21 (11.11%) as UNSAT, and five (2.65%) as atypical cytology or higher. Of the 21 repeated UNSAT PAP, further follow-up was 16 NILM (76.19%), one ASCUS (4.76%) (which reverted to NILM within one year), and four (19.05%) were lost to follow-up. The five UNSAT PAP that had atypical cytology or higher were diagnosed as one LGSIL on PAP, and one atypical endometrial cell, and three carcinomas (one endometrial adenocarcinoma and tow poorly differentiated carcinomas) on surgical biopsy. Both the LGSIL and atypical endometrial cells cases had NILM PAP within the following two years. Due to past medical history, all three carcinoma cases were clinically classified as high risk. Rescreen of the UNSAT PAP for these three cases revealed interpretive error (one case) and sampling error (two cases). In 46 cases, follow-up within two to three years was NILM. In total, 232 (98.72%) of the cases originally diagnosed as UNSAT were resolved to be NILM by follow-up over time. The mean squamous cell count per high power field for the cases was 1.5, leading to a mean cellularity of 2000 cells / PAP.

Conclusions: The knowledge of menopausal status is important in older women, for screening of their PAP. Postmenopausal women frequently have hypocellular samples due to atrophy. Upholding the same adequacy criteria in clinically low-risk postmenopausal women for PAP as pre / perimenopausal women, may lead to increased unnecessary medical procedures and increased healthcare costs. Our findings support lowering the cellularity criterion for adequacy in PAP of clinically low-risk postmenopausal women. However, if a postmenopausal woman is deemed to be clinically high risk, the current standard of cellularity should be observed and their cases should be targeted for a 10% prospective rescreen, to avoid / reduce interpretive error.


Clinical experience with Cervista™ HPV HR as a function of cytological classification: Comparison with retrospective Hybrid Capture (Hc2) data

Elizabeth Schroeder, Sandra Balzer, Lynn Kroeger, Jolanta Czarnecka, Judy Griep, Robert Amrhein, Connie Yauck, Kevin Ross, Erik Munson

Wheaton Franciscan Laboratory, Wauwatosa, Wisconsin

Introduction: Molecular high-risk human papillomavirus (HPV) detection has utility within the appropriate triage algorithms in the context of cervical cancer screening, particularly in women with a cytological diagnosis of atypical squamous cells of undetermined significance (ASCUS). Although hybrid capture technology (Hc2; Qiagen) has been a mainstay in molecular high-risk HPV-augmented triage, published laboratory validation and clinical trial data (e.g., Am. J. Clin. Pathol. 130: 401-408, 2008; J. Clin. Microbiol. 46: 1641-1646, 2008; Gynecol. Oncol. 118: 116-122, 2010) have suggested that the Invader® chemistry-based Cervista™ HPV HR (Hologic) possesses equivalent performance characteristics. However, one recent publication (Am. J. Clin. Pathol. 134: 193-199, 2010) raises caution over a purported increased high-risk HPV detection rate for Cervista™ HPV HR.

Materials and Methods: An audit of cytological diagnoses was conducted nine months prior to and following the in-house introduction of Cervista™ HPV HR for molecular HPV detection. The high-risk HPV molecular result (formally requisitioned by the clinician or indicated by cytology) was paired with cytological diagnosis. In a subset of patients with cytological diagnosis of ASCUS or absence of atypical cellularity, tandem results were stratified by patient age.

Results: In the nine months prior to the implementation of Cervista™ HPV HR, 2634 liquid-based cytological studies were subsequently forwarded for Hc2 performance; 72.6% of these studies yielded no significant cytological findings, while 15.1% were classified as ASCUS; 2539 cytological collections were forwarded for subsequent Cervista™ HPV HR within the first nine months of test implementation. Significantly more ASCUS cytological results (21.5%) were reported in this second testing interval (P < 0.0002), with fewer specimens yielding non-significant cytological findings (69.6%; P = 0.02). In spite of the increased ASCUS cytological findings during the Cervista™ HPV HR testing interval, the rate of molecular high-risk HPV detection (40.3%) did not exceed that observed in ASCUS-diagnosed collections subjected to Hc2 testing in the corresponding interval (43.0%; P = 0.41). In a similar fashion, the Hc2 high-risk HPV detection rate in 1913 cytological studies, without significant findings (7.7%), did not differ from the Cervista™ HPV HR detection rate (9.1%; P = 0.14) in 1752 analogous collections. A calendar-matched five-month subset of data, individualized to each molecular assay, revealed no difference in the non-significant finding - and ASCUS-based high-risk HPV detection rates with age delineations of < 30 years (P ≥ 0.24) and ≥ 30 years (P ≥ 0.10) were analyzed. Rejection rates on the basis of insufficient specimen volume were 9.4% for Hc2 and 5.5% for Cervista™ HPV HR (P < 0.0002).

Conclusions: Performance characteristics of Cervista™ HPV HR were largely unchanged from those of Hc2. When taken together with a decreased minimum specimen volume requirement and incorporation of a nucleic acid-based internal control, the Cervista™ HPV HR provided a viable nucleic acid hybridization alternative for high-risk HPV-augmented cervical cancer screening.


Evaluation of CINtec PLUS® testing as an adjunctive test in ASC-US diagnosed SurePath® preparations

Neil Edgerton, Cynthia Cohen, Momin Siddiqui

Department of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, Georgia

Introduction: The CINtec PLUS® system is an immunohistochemical cocktail composed of antibodies against p16INK4a and Ki-67, meant to improve the detection of high-grade dysplasia (HGD). Specifically, it has been examined as an adjunctive test that would serve to increase the overall specificity. In the presence of dysplasia, a red chromogen marks the Ki-67 expression in the nucleus and a brown chromogen marks the cytoplasmic p16INK4a expression. Only cells showing dual staining were interpreted as positive. This retrospective study examined the performance characteristics of CINtec PLUS® testing, when performed on ASC-US diagnosed samples, prepared by SurePath® preparations.

Materials and Methods: A total of 63 SurePath® Pap test slides diagnosed as ASC-US, with correlative colposcopic biopsy, were evaluated. HR-HPV testing was performed at the time of cytology diagnosis in all cases. Correlative colposcopic biopsy diagnoses were also derived from the medical records. In the event, multiple biopsies were taken, the most severe dysplasia (highest CIN) was recorded. Slides were analyzed by a single pathologist without adjudication. The pathologist knew the samples were composed of ASC-US-diagnosed cytology specimens, but was blinded to the HR-HPV results and surgical biopsy findings. The slides were interpreted as positive or negative for CINtec PLUS staining based on  Atlas More Details and guidelines provided by the manufacturer. In the presence of dysplasia, there was red chromogen-marked Ki-67 expression in the nucleus and a brown chromogen-marked cytoplasmic p16INK4a expression. The testing was considered positive if a minimum of one cell showed simultaneous dual staining. The utility of CINtec staining on SurePath® slides was determined using the sensitivity and specificity calculations performed by the JavaSTAT online contingency table calculator.

Results: Within our sample set, 44% of atypical squamous cell diagnoses were found to have a CIN lesion (CIN I-III). Fourteen cases (22%) of our sample set of ASC-US cases had high-grade dysplasia (CIN II / III) at the time of cervical biopsy. No cases of cervical carcinoma were present. Staining with the CINtec PLUS® system showed modest sensitivity (64%) and specificity (53%) in identifying the presence of HGD at surgical biopsy. Positive and negative predictive values for HGD were found to be 28% and 84%, respectively. In contrast, HR-HPV DNA testing yielded a sensitivity of 100% and specificity of 21%.

Conclusions: Overall, the combined specificity gained by the performance of CINtec and HR-HPV testing was an improvement over current practice. The increased specificity of this test, besides simply creating another triage option, could conceivably lead to a reduction in colposcopic procedures. Cost effectiveness of this test remains to be demonstrated, as it would require the addition of both HR-HPV testing and immunohistochemical staining. The cost benefit lay in avoiding colposcopy, which carried both an evaluation and management fee along with the associated procedural cost. Although more difficult to calculate, the time and discomfort the patient is saved, is the biggest argument for increasing the specificity. Our study, although admittedly small, demonstrates an improvement in specificity above HR-HPV testing alone.


HPV positive / PAP negative diagnoses in women ages 30 and older: Looking to correlate morphological cues to determine QA / QC modifications

Rachel McMahon, BS 1 , Amber Schneider, BS 1 , David Tritle, MS 1 , Lynn Meyers, BS 1 , ChangHong Ye, BS, SCT(ASCP) 1 , Traci Arts, BS, SCT(ASCP) 2 , Michele Smith, MS, SCT(ASCP) 1 , Daniel F, Kurtycz, MD 1

1 Cytology Program, Wisconsin State Laboratory of Hygiene at UW-Madison, Madison, Wisconsin; 2 Cytology Laboratory, Wisconsin State Laboratory of Hygiene At UW-Madison, Madison, Wisconsin

Introduction: The Wisconsin State Laboratory of hygiene (WSLH) provides gynecological services (Pap Tests, HPV, and limited biopsies) to under- and uninsured women in Wisconsin. The WSLH and its clinical partners modified a previous cost savings agreement to meet the 2006 ASCCP guidelines for women 30 years and older. The two-tiered model allowed liquid-based cytology (LBC) collection for all women of ages 30 years and older, to incorporate Pap and HPV testing. Women 29 years and younger were screened using conventional Pap (CP) tests with abnormal follow-up tests collected via LBC. During 2009, the WSLH performed 5530 HPV tests from a mix of 34,805 Pap tests. In this study we were interested in the cases where HPV was detected, but the cytology Pap test was diagnosed as Negative for Intraepithelial Lesion or Malignancy (NIL) (496 / 814, 60.93%). A retrospective case review was performed seeking potential morphological cues that might lead to a change in the quality assurance plan, in terms of modifications, for quality control through directed rescreen and / or routing cases directly, for a pathology review.

Materials and Methods: HPV testing was performed using Invader® / Cervista® . Cervista® was approved by the Food and Drug Administration in June 2009, and WSLH certified the method during the same year. WSLH had validated Invader as an Analyte Specific Reagent (ASR) in early 2007. HPV and Pap test data were obtained from the laboratory information system (LIS) that provided not only 2009 data, but also all the available follow-up of the gynecological tests (Pap, HPV, Biopsy / Colposcopy) in 2010 and 2011. Screening criteria was based on several text books and modeled from journal articles by Bollman et al., (2005) and Nijhawan et al., (2010). Third semester students evaluated all cases scoring them by the determined criteria as well as the established abnormal criteria. Instructors adjudicated case discrepancies. All abnormal cases were routed to the pathologist for final evaluation.

Results: Women, 30 years and older, comprised 4665 (84.36%) of the HPV testing results. Using Invader® / Cervista® (Hologic, Madison, WI) high-risk HPV (HRHPV) testing kits, 68.93% (3812 / 5530) reported as not detected, while 14.72% (814 / 5530) detected one of the 14 HRHPV types. General HPV and Pap results in women over 30 years are illustrated in [Table 1]. The retrospective review evaluated the HPV-detected and Pap NIL cases. Screening criteria are described in [Table 2] and include selected cell morphology, inflammation, and infectious agents. The initial review of 319 cases found that a large majority met the multinucleated criteria (40%) as illustrated in [Figure 1]. Of the inflammatory and infectious agents, 40% of the cases also had coccobacilli as seen in [Figure 2].

Conclusions: The next steps in the review process include evaluating a random sample of cases from women of age 30 years and older with NILM Pap and no HPV detection, using the same criteria points. To complete the study, we will also evaluate the sample of cases diagnosed as ASC-US, regardless of the HPV status in the same population demographic. Initial findings show that 40% of the cases reviewed meet the criteria of bi- / multinucleation. Several authors have discussed these soft criteria of HPV. It is also interesting to note the increase in coccobacillus infection with this initial review sample. It is likely that these cases meet closer morphological changes to ASC-US rather than NILM, but do not provide clear diagnostic criteria.


Comparing the positive rates of the qiagen Hybrid Capture II and Hologic Cervista HR tests for high risk human papillomavirus detection in SurePath® cervical cytology specimens

Heather Smith, BS, CT(ASCP), Brenda Sweeney, MS, SCT(ASCP)MB, David Wilbur, MD

Cytopathology, Massachusetts General Hospital, Boston, Massachusetts

Introduction: HPV testing is an important component of the management of equivocal cervical cytology specimens and for the triage of women over the age of 30 years having negative cytological examinations. On the basis of the FDA labeling data showing a Cervista positive rate of 18.5% in the over 30 years negative cytology group, there has been concern in the literature that the Cervista HPV HR assay is too sensitive for use in these roles, and that the positive rates, particularly in the over-30-years population, will be too high compared to the current HC II standard, to yield appropriate clinical results (Kinney et al., Am J Clin Pathol 2010;134:193). In this study we compare the performance of the current standard Hybrid Capture II (HC II) to Cervista HPV HR in SurePath® specimens.

Materials and Methods: HPV test results were collected over a one-year period that used either HC II (six months) or Cervista HPV HR (six months) testing methods. The patient population used for each test was the same and was considered a low-risk screening population. Positive rates with each test were tabulated and the results in the ASC-US triage; and in the over-30-year, cytology-negative populations were compared.

Results: In the first six-month period, HC II testing was performed on 3823 specimens, and in the second six-month period, Cervista testing was performed on 3176 specimens. In the over-30-years negative cytology group a 5% positive HC II rate and a 3% positive Cervista rate were noted. In the ASC-US triage group, HC II yielded a 42% positive rate and Cervista yielded a 35% positive rate. Cases of ASC-H yielded a 71% positive rate with HC II and a 49% positive rate with Cervista. Cases of LSIL tested showed an 86% positive rate with HC II and a 72% positive rate with Cervista.

Conclusions: The data shows consistently lower rates of HPV test positivity in SurePath® samples in all patient groupings and interpretations. Linked with data (Nutter , 2011) showing that HC II with SurePath® specimens may show low-risk virus cross-reactivity leading to false positive HR results, the current data suggests that low-risk virus cross-reactivity may account for at least some of the discrepancies noted. In total, the results do not support the conjecture that the Cervista test is too sensitive for routine clinical use and suggest that the Cervista FDA labeling data represents a population-dependent prevalence effect.


The influence of previous Pap smear history on cervical cytology diagnoses

Andrea Snitchler, DO, FCAP 1 , Jan Silverman, MD 1 , Yulin Liu, MD 1 , Nathaniel Sherwood, DO 1 , Alok Mohanty, MD 1 , Uma Krishnamurti, MD 2 , Kathleen Bryer, BS 2 , Beth Ujevich, MBA, MS, SCT, HT(ASCP) 1

1 Pathology, Allegheny General Hospital, Pittsburgh, Pennsylvania; 2 Pathology, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania

Introduction: The Pap smear cytopathological interpretation may be influenced by each patient's prior history, especially when there is a prior Pap smear diagnosis, with the assumption that the cytological diagnosis may be upgraded in a patient with a prior abnormal Pap smear. The purpose of this study was to determine the influence of previous Pap smear history on cervical cytology diagnoses.

Materials and Methods: One hundred ThinPrep® (Hologic, Marlborough MA) Pap smears were retrospectively reviewed, wherein the original diagnosis was NILM (25%), ASCUS (50%) or LGSIL (25%). Four cytopathologists and two cytopathology Fellows examined the Pap smears with the submitted age and any history of prior Pap smear diagnosis. Then after approximately 14 days, the pathologists re-reviewed the same specimens blinded to case number, age, and previous Pap smear diagnosis. One cytopathologist was only able to review 80 of the blinded slides.

Results: In aggregate, 80 cases (13.8%) were upgraded with the submitted history, that is, NILM to ASCUS or ASCUS to LGSIL. Eighty-eight cases (15.2%) were downgraded with the submitted history. Two cytopathologists showed remarkably low intraobserver variability, with 90% or greater consistency. The other cytopathologists and Fellows showed 47 - 69% intraobserver consistency (31 - 53% variability in diagnosis). The most junior cytopathology Fellow showed the greatest variability. Nine cases (9%) were agreed on by all six evaluators both blinded and unblinded. Compared to the original diagnoses, two of the six cytopathologists tended to underdiagnose when no history was provided. Three of the six cytopathologists overdiagnosed and one showed no difference.

Conclusions: The Pap smear is a screening test with recognized imperfect intraoberserver and interobserver variability. This study demonstrates that there is only a slight (1.4%) difference between the diagnoses being made, if evaluated blinded to demographics and prior history, or with all accompanying information. To better account for test bias the original diagnosis was also compared with the Pap smear interpretation blinded to prior history and again demonstrated little impact on the cytological interpretation. Although there was some interobserver variability, these results substantiated the idea that Pap smear diagnoses were based on cytomorphological findings, with only a minimal impact by demographics and prior history.


Effects on Pap smear screening productivity by implementation of the BD FocalPoint™-guided screener imaging system (GS)

Brenda Sweeney, SCT(ASCP) MB, David Wilbur, MD

Cytopathology, Massachusetts General Hospital, Boston, Massachusetts

Introduction: Over two-thirds of the accredited Cytotechnology training programs have closed since 1976, leaving only 31 active programs in the United States and Puerto Rico. Additionally, only 164 candidates successfully passed the American Society for Clinical Pathology (ASCP) examination in Cytotechnology in the year 2010. In light of staffing shortages and potential changes in the available health care dollars, many laboratories are looking to automation as a partial solution.

Materials and Methods: In order to assess changes in Pap smear screening productivity due to automation we collected average daily screening productivity data from three time periods: 12 months pre-GS implementation, six months post-GS implementation, and seven to eighteen months post-GS implementation. Screening rates for five cytotechnologists were tabulated during each of the three time periods.

Results: [Table 1].

Conclusions: Conclusions: After the GS implementation there was a learning curve that negatively impacted some, but not all technologists. This screening volume downtrend was resolved within six months in all technologists, resulting in a 15.4% overall increase in daily screening volume (range 6.1 - 26.9%). As our laboratory represents a busy academic practice in which technologists perform many other duties outside of Pap smear screening, including HPV testing and an active rapid interpretation FNA service, the daily volumes do not reflect a typical eight-hour screening day. Laboratories that have more concentrated cervical screening environments may find greater levels of productivity resulting from the use of this device.


TelePAPology versus liquid-based ThinPrep® cervical cytology: A comparative study evaluating human papillomavirus testing by Hybrid Capture-2 and in-situ hybridization

Nevene Andraws, MD, Marilyn Davis, CT(ASCP), Susan Dillom, CT(ASCP), Fang Fan, Ossama Tawfik

Pathology and Laboratory Medicine, Kansas University Medical Center, Kansas City, Kansas

Introduction: Digital images are being used for telecytology, automated screening of Pap smears, training and education, as well as, proficiency testing. To date, the impact of digital imaging on routine day-to-day cytology remains far from perfect. Cellblock (CB) preparations from discarded / residual, conventional, and liquid-based GYN samples have been shown to be of diagnostic value. In a pilot study, we have demonstrated the feasibility of utilizing imaging technology to overcome current limitations by digitizing the cytological specimens from CB preparations. The current study was undertaken to evaluate the possibility of performing Human Papillomavirus (HPV) in-situ hybridization (ISH) on CB preparations and compare the results with Hybrid Capture-2 (HC-2) technique.

Materials and Methods: The Cellient system from Hologic (Marlborough, MA) was used to prepare CBs. Fifty-six H and E stained CB slides prepared from residual ThinPrep® (TP) samples were analyzed. These included ASCUS (42), LGSIL (7), normal (4), ASC-H (2), and HGSIL (1) cases. TelePAPology slides were obtained using the Aperio digital imaging system (Vista, CA). Virtual slides were reviewed by three cytopathologists and two cytotechnologists. HC-2 testing (QIAGEN, Inc, Valencia, CA) and HPV ISH testing (iVIEW Blue Detection Kit) (Ventana Medical Systems, Tuscon, AZ) were performed on all samples. Test performance characteristics of TP and TelePAPology samples were compared for diagnostic accuracy and HPV assay performance. Three CB samples / slides were included to reduce the cost and improve efficiency.

Results: TelePAPology virtual slides contained an optimal amount of material from all cases. Compared to TP diagnoses, fewer ASCUS cases were diagnosed by the TelePAPology method (33 vs. 42) and more normal cases were diagnosed (13 vs. 4). The diagnosis remained unchanged for LGSIL and HGSIL cases, except one case that was reclassified as LGSIL from ASCUS. One of the ASC-H cases diagnosed by TP remained as ASC-H by the TelePAPology method and the other was reclassified as ASCUS. A total of 46 of 56 cases were concordant by HC-2 and ISH testing including 24 positive and 22 negative cases. There were 30 positive and 26 negative HC-2 cases compared to 28 positive and 28 negative ISH cases. All discrepant cases were with ASCUS diagnosis. These included four positive by ISH / negative by HC-2 and six positive by HC-2 / negative by ISH. Both HC-2 and ISH reported similar ASCUS to HPV ratio positivity for TP and TelePAPology methods (48% for HC-2 and 45% for ISH).

Conclusions: CB preparations are a valuable source of material for additional ancillary testing such as HPV. In addition, combining the CB technology with digital techniques is a feasible method for widespread adoption, to achieve high quality specimen preparations. We have been successful in performing HPV ISH testing on all cases with an excellent concordance between ISH and HC-2 methods. ISH testing is feasible, cost-effective (three cases / slide), and practical. The concept of TelePAPology is suitable for routine cytology, ISH, and immunohistochemistry testing for HPV and other prognostic markers.


Determining implementation of ASCCP guidelines based on clinical evidence

Marla Taylor, BS, CT(ASCP)IAC, Lubna Sayage-Rabie, MD

Cytopathology, Scott and White Healthcare, Temple, Texas

Introduction: The American Society of Colposcopy and Cervical Pathology (ASCCP) developed practice guidelines for the management of women with an abnormal Pap Test. The guidelines changed the management of adolescent women (≤ 20 years) with a cytological diagnosis of Atypical Squamous Cells of Undetermined Significance (ASC-US) from reflex human papillomavirus (HPV) testing to repeat cytology in 12 months. Also included in the guideline was the recommendation of HPV testing for women ≥ 30 years with a diagnosis of negative for intraepithelial lesion (NIL) on cytology. Due to these changes the actual clinical practice of the guidelines varies among physicians. The aim of this study was to provide physicians with evidence from our patient population to determine if reflex HPV testing of adolescent women should be discontinued and if HPV testing of women ≥ 30 years should be implemented.

Materials and Methods: This study consists of two cohort-specific sub studies defined by cytological diagnosis and HPV testing. One cohort consisted of women ≤ 20 years, with a cytological diagnosis of ASC-US, with or without HPV testing, and biopsy correlation from January 2004 to December 2009. The other cohort consisted of women ≥,30 years with a cytological diagnosis of CIN2+ with biopsy correlation and HPV status for the year 2008. Data for the two cohorts was obtained through a computer search.

Results: There was a total of 1428 ASC-US diagnoses among adolescent women from January 2004 to December 2009; of these 1110 (78%) had HPV testing. High-risk HPV (hr-HPV) was detected in 708 (64%) of the cases by using Digene Hybrid Capture 2. Of these 167 (24%) had a follow-up biopsy. HPV testing was not performed on 318 (22%) of the ASC-US cases. Among those not tested, 89 (28%) had a biopsy. [Table 1] shows the follow-up biopsy correlation of the cytological diagnosis of ASC-US with and without HPV testing among women ≤ 20 years from 2004 to 2009. Between January and December 2008, there was a total of 110 women ≥ 30 years with a diagnosis of CIN 2+. Of these 70 (64%) were confirmed by biopsy. A total of 27,794 women in this age group were screened for cervical cancer in 2008, resulting in a prevalence of 70 / 27,794 or 0.25%. Previous hr-HPV testing was performed on 17(15%) patients, of whom 13 (76%) were positive for hr-HPV with seven (54%) of them having biopsy confirmation of CIN2+. The HPV testing was due to either a diagnosis of ASC-US or ASC-H. The biopsy result of the seven hr-HPV cases are depicted in [Table 2].

Conclusions: The results of the data collected among adolescent women with a cytological diagnosis of ACS-US with and without HPV testing shows no statistically significant difference in the follow-up biopsy diagnosis. This supports the ASCCP recommendation to not perform HPV testing on women 20 years of age or younger. The prevalence of CIN 2+ among women ≥ 30 years for 2008 was 0.25% or 2.5 per 1,000. This is slightly higher than the rate of 1.5 per 1,000 reported among women enrolled at Kaiser Permanente Northwest (Portland, Ore) in 1998. Previous HPV testing was performed on 17 cases, of which 13 were hr-HPV positive with only seven (54%) having a biopsy diagnosis of CIN2+. Even though the HPV testing was the result of an ASC-US or ASC-H diagnosis, this patient population had a higher prevalence of CIN 2+ as compared to other population groups. This would suggest that this institution should implement the ASCCP HPV testing guidelines for this patient population.


The yield of repeat biopsy following a second abnormal papanicolaou test after an initial negative or low-grade biopsy as follow-up for LSIL

Michael Thrall, MD

Department of Pathology, The Methodist Hospital, Houston, Texas

Introduction: The American Society for Colposcopy and Cervical Pathology (ASCCP) currently recommends that most women should undergo colposcopy and biopsy following a Pap test interpreted as LSIL (Wright et al. AJOG 2007;197:246). The exceptions are adolescents (aged < 21) for whom colposcopy is not recommended, and post-menopausal women, for whom colposcopy is one of several options. Many biopsies are negative or find only low-grade dysplasia. On the basis of the Advanced Trauma Life Support (ALTS) trial findings, repeat biopsy is recommended if a follow-up Pap test is abnormal or if high risk HPV is detected (Cox et al. AJOG 2003;188:1406). There is little data about the yield of these second biopsies in clinical practice.

Materials and Methods: The computer database of our institution was searched from the period from 5 / 1 / 2007 to 10 / 30 / 2008, to find women with a Pap test interpreted as LSIL (including LSIL-H). The previous and follow-up Pap test and biopsy results were then recorded for these women. The original slides were not reviewed. The total Pap test volume for this period was 67,223 (80% ThinPrep® and 20% SurePath® ). Biopsy and Pap test follow-up was compiled up to 2 / 28 / 2011.

Results: Over the 18-month period, there were 2329 LSIL Pap tests in our laboratory (excluding a dysplasia clinic). Of these, 1140 were from non-adolescent women with no history of cervical dysplasia or prior abnormal Pap tests; 624 (54.7%) of these women had a first biopsy with the following findings: 221 negative, 351 HPV effect / CIN I, and 52 CIN II-III. Thus, the first biopsy high-grade dysplasia yield was 9.3%. Of the 572 women with no high-grade dysplasia found, 326 (57.0%) had a follow-up Pap test within two years, with the following findings: 126 NILM (HPV- or no HPV), six NILM HPV+, 49 ASC-US, 137 LSIL, and eight HSIL / AGC. Of the 200 women with positive results, 77 (38.5%) underwent repeat biopsy with the following findings: 33 negative, 36 HPV effect / CIN I, and eight, CIN II-III. Thus the second biopsy yield was 10.4% - similar to the first biopsy yield. Among the 137 women with repeat LSIL, five (9.8%) had CIN II-III found in the second biopsy.

Conclusions: Our follow-up data supports the current ASCCP guidelines regarding repeat colposcopy and biopsy for women who do not have high-grade dysplasia found in the first biopsy, but have persistently abnormal Pap tests. The yield of high-grade dysplasia found in second biopsies in this population is similar to the yield of the first biopsies for LSIL.


Use of p16 / ki67 cytology to detect cervical pre-cancer in a US referral population

Nicolas Wentzensen 1 , Joan Walker 2 , Rosemary Zuna 2 , Terence Dunn 2 , Michael Gold 3 , Mark Schiffman 1

1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, Maryland; 2 University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; 3 Vanderbilt University, Nashville, Tennessee

Introduction: Cytology-based screening for and treatment of cervical precancers has led to a substantial reduction of cervical cancer incidence and mortality worldwide. However, Pap cytology has limited sensitivity to detect prevalent cervical precancers. Here, we evaluated the performance of p16 / ki67 cytology to detect CIN2 and greater, in a US referral population.

Materials and Methods: Liquid-based cytology samples were collected from 551 women referred to colposcopy for abnormal cytology screening results. A ThinPrep® slide was produced from the vial and stained using the CINtec plus cytology assay (mtm Laboratories). Pap cytology and HPV genotyping were performed from the same vial. Women with CIN2 or greater identified in colposcopic biopsy were referred for LEEP. This analysis focused on the overall positivity, sensitivity, and specificity of p16 / ki67 cytology, to detect CIN2 or greater.

Results: In total, 136 CIN2+ were detected with colposcopic biopsy; 56.4% of women referred to the clinic were positive for p16 / ki67, while 86.6% were cytology-positive (at an ASCUS cutoff), and 77.7% were positive for carcinogenic HPV DNA. The sensitivity and specificity of p16 / ki67 to detect CIN2 or greater was 87.5% (95%-CI: 80.7 - 92.5%) and 53.8% (95%-CI: 46.3 - 61.2%), respectively. In comparison, the sensitivity and specificity of cytology at an ASCUS cutoff were 95.5% (95%-CI : 86.3 - 100%) and 17.5% (95%-CI: 13.1 - 22.7%); the sensitivity and specificity for carcinogenic HPV testing were 96.3% (95%-CI: 91.6 - 98.8%) and 29.3% (95%-CI: 22.8 - 36.5%).

Conclusions: In this high-risk referral population, p16 / ki67 had good sensitivity and specificity for detection of CIN2 or greater. If p16 / ki67 was used as a triage test, about half of the colposcopies could have been avoided, while most of the CIN2 or greater would have been detected. A detailed analysis of CIN2+ cases missed by p16 / ki67 to rule out non-dysplastic lesions overcalled in histology has been currently conducted.


Reflex HPV testing for ASCUS in a high-risk population

Vladislav Zakharov, MD, Diane Avery, CT(ASCP), Philip Valente, MD

Department of Pathology, University of Texas Health Sciences Center at San Antonio, San Antonio, Texas

Introduction: Reflex HPV testing following ASCUS cannot rule out LSIL. It is well-documented to increase the yield of CIN2+ on biopsy. However, recent guidelines have discouraged its use in women under 30 years of age. This retrospective study evaluates the relative sensitivity of Reflex HPV testing in younger and older patients in a high-risk population.

Materials and Methods: The computer database of the University Hospital in San Antonio, TX, was electronically searched for all ASCUS diagnoses from August 2000 to August 2010. These were further divided by age (Group A < 30 years; Group B ≥ 30 years) and high-risk HPV test status (Digene Hybrid® Capture II).

Results: A total of 6,125 ASCUS diagnoses (2,562 in Group A, 3563 in Group B) were identified. Unequivocal high-risk HPV test results were obtained in 3,036 (1,320 in Group A, 1,716 in Group B). The prevalence of high-risk HPV was 48.9% overall, 65.3% in Group A and 36.2% in Group B. Within a one-year interval, a total of 1,828 (29.8% of all ASCUS cases) had a histological follow-up, of which 908 (49.7%) had high-risk HPV testing with their original diagnoses (384 in Group A and 524 in Group B). A proportion of histological follow-ups differed significantly depending on the high-risk HPV test status. Among women of Group A, who tested negative for high risk HPV, 7.2% (33 out of 458) had a histological follow-up within a one-year interval compared to 40.7% (351 out of 862) in the same age group with a positive result for high-risk HPV. The same trend was observed in Group B , 14.0% (153 out of 1095) high-risk HPV negative versus 59.7% (371 out of 621) with a positive high-risk HPV test. Histological results are summarized in [Table 1]. Of note is that overall, Group A and Group B had similar positive biopsy rates among HPV positive patients (all CIN , 31.6% versus 28.6%; CIN2+ , 10.3% versus 10.8%). Among the small number of HPV negative biopsied patients, positive biopsies were 21.2% (7 out of 33) versus 6.5% (10 out of 153), with all cases in Group A being CIN 1. Five CIN2+ cases in Group B HPV negative patients had biopsies retrieved and reviewed. These were subjected to p16 immunostaining. Only one case was positive for p16. Two were reclassified as immature squamous metaplasia and two had features of CIN 2 despite negative p16 staining [Table 1].

Conclusions: In contrast to the ASCCP guidelines discouraging HPV Reflex testing, our findings suggest that this approach is equally useful in patients under 30 years of age, in a high-risk population. This may be due to a higher rate of HPV persistence in our older population (overall HPV prevalence in ASCUS was 65.3% in Group A, but still 36.2% in Group B). Our CIN 2 biopsies on HPV-negative patients have yielded two histological overcalls of immature squamous metaplasia. Two remaining CIN 2 cases may have been related to undetected low-risk HPV types. In our high-risk population, HPV Reflex testing for ASCUS appears to be useful even in women under 30 years of age.


Analysis of 70,123 cases with negative Pap test and high-risk HPV co-test results in a Large women's hospital

Chengquan Zhao, MD 1 , Xiangbai Chen, MD, PhD 2 , Agnieszka Onisko, PhD 1 , Baoying Weng, MD, PhD 2 , Marshall Austin, MD, PhD 1

1 Pathology, Magee Womens Hospital, UPMC, Pittsburgh, Pennsylvania; 2 Pathology, Conemaugh Memorial Medical Center, Johnstown, Pennsylvania

Introduction: HrHPV-positive rates in women with cytology negative results vary widely in available reports. This variation has been attributed to differences in infection rates, patient ages, rates of HPV-associated cervical abnormalities, intensity of screening, and treatment programs to detect and ablate cervical lesions associated with persistent HPV infections, and false-negative cytology rates attributable to laboratory screening and interpretation.

Materials and Methods: A retrospective computer-based search of the Copath database of Magee Womens Hospital (MWH) of the University of Pittsburgh Medical Center was conducted over a six-year period, from 2005 to 2010, to retrieve data on Pap tests reported as negative for intraepithelial lesion or malignancy (NILM) from women who also tested for hrHPV DNA with the FDA-cleared Hybrid Capture 2 (HC2) method. The HPV-positive and HPV-negative rates for these NILM cases were assessed between younger and older age groups. Beginning in June 2005, the cytology-negative HPV-positive cases were routinely reviewed by a second quality control cytotechnologist and a cytopathologist. Long-term follow-up results of cytology-negative HPV-positive cases were analyzed.

Results: Among a total of 645,541 Pap tests performed over six years, 529,198 (81.98%) were reported as NILM (80.96 - 83.07%, varied yearly). Of these cytology-negative cases, 70,123 of 645,541 (13.25% (4.17 - 20.21%, varied yearly) had hrHPV co-testing. HPV testing of cytology-negative cases markedly increased in 2007. Cytology-negative HPV-positive results comprised of 1.92% (< 30, 7.22%; ≥ 30, 1.73%, P < 0.0001) of 70 and123 NILM cases co-tested for hrHPV. The HPV-positive rate in NILM cases decreased during the study period from 2.9% in 2005 to 1.0% in 2010. Eight hundred and sixty-nine women with NILM Pap and positive HC2 HPV testing results (average age 41.3 years) interpreted from July, 2005 to December, 2009 had repeat cytological or histopathological follow-up results. CIN1 / LSIL and more severe lesions were detected in 211 of 869 cases (24.3%) including 21 (2.4%) CIN2+ with average follow-up of 23.2 months. Kaplan-Meier projections of the cumulative incidence rates for CIN2+ in cytology-negative hrHPV-positive women, after 70 months, were 4.6% (95% CI: 2.4 - 6.8%). More LSIL / CIN1+ was identified with repeat positive HPV results than with repeat negative HPV results (P < 0.001). Since the last year women with negative Pap and positive HC2 HPV testing results underwent reflex HPV16 / 18 genotyping analysis. HPV16 / 18-positive cases accounted for 7.6% (8 / 105) of cytology-negative HC2 HPV positive cases [Table 1].

Conclusions: HPV positive rates in women with NILM Paps were significantly lower among women 30 years and older than among women less than 30 years, probably reflecting the increased transient HPV infection rates, known to occur in younger women. The decreasing HPV-positive rate in cytology negative cases observed in all age groups during the study period probably reflects the progressively increasing scrutiny of slides initially interpreted as negative, which were subjected to additional quality control reviews. As the CIN2+ detection rate was very low in these women and HPV16 / 18 positive cases accounted only for less than 10% of the cytology negative HC2 HPV positive cases, reflex HPV16 / 18 genotyping and triage may be the most cost-effective method for risk stratification of women with NILM Pap and positive HC2 HPV testing.

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Cytology follow-up analysis of the bethesda system - Thyroid diagnostic category follicular lesions of undetermined significance (category III) in African Americans versus non-African Americans

Pamela Archuletta, MD, Raja Gidwani, MD, Opada Alzohaili, MD, Julie Samantray, MD, Jineng Feng, MD, Donping Shi, MD, Lili Geng, MD, Paul Tranchida, MD, Mujtaba Husain, MD, Vinod Shidham, MD, Tamar Giorgadze, MD

Cytopathology, Wayne State University, Detroit, Michigan

Introduction: It has been reported that African Americans (AA) have a higher prevalence of malignancy compared to Non-African Americans (NAA); however, a report by Morris et al. indicates that the rate of thyroid malignancies in AA is half that of NAA. The reports looking at thyroid lesions in AA compared to NAA are few and have conflicting results. Even less is known about the rates of follicular lesions of undetermined significance (FLUS) between these two populations. There are no reports looking at FLUS in this population. The aim of this study is to analyze FLUS in the AA population.

Materials and Methods: We retrospectively reviewed thyroid FNA cytology reports and corresponding surgical pathology reports between January 2005 and January 2011, in cases where the race was known. Two hundred fifty eight patients met our inclusion criteria and were associated with 311 FNA specimens, and 265 surgical pathology specimens. The patients were categorized as AA and NAA. The NAA group included Caucasians (C), Hispanics (H), and Others (O). The rate of FLUS was determined from the total number of thyroid FNAs. Cases predating the latest edition of the bethesda system for reporting thyroid cytology were categorized as FLUS based on microscopic descriptions that paralleled those outlined in the bethesda system for reporting thyroid cytology. All post FLUS diagnostic FNA results were compiled and analyzed. The follow-up surgical pathology diagnosis was used for the final categorization.

Results: Of the 258 patients, 65 were categorized as FLUS and included 36 AA and 27 NAA. The average age for AA was 51 (range 20 - 88) and for NAA was 53 (range 25 - 86) years. The average age of AA diagnosed with FLUS was 48.5 (range 18-80) and NAA was 47.5 (range 24 - 77) years. Similar to the overall population, there were more females than males in both groups. There were more females in the AA versus NAA (85 and 75%, respectively). With respect to the FLUS lesions, there were more females than males (89% for AA and 81% for NAA). The rate of FLUS was similar for AA and NAA (20% and 20.5%, respectively). After a FLUS diagnosis, most patients were followed up with surgical resection as apposed to repeat FNA (91% of AA and 96% of NAA). The time from initial FLUS diagnosis to surgical resection for AA and NAA was six (range 1 - 48.75) months and 2.3 (range 0.25 - 9.57) months, respectively. Three AA (9%) had repeat FNAs averaging 11 (range 1 - 29.75) months after initial FLUS diagnosis, whereas, only one NAA had a repeat FNA 3.75 months after the initial FLUS diagnosis [Table 1]. With respect to the surgical outcomes of FLUS, the rates of malignancy for AA versus NAA were 38% and 56%, respectively. The incidence of papillary thyroid cancer was less in AA versus NAA [Table 2] and [Table 3].

Conclusions: Both groups were more likely to proceed directly to surgical intervention after initial diagnosis of FLUS; however, the time interval between the initial FLUS diagnosis was longer in AA suggesting a delay in surgical intervention compared to NAA. Reasons for this were not elucidated in this study, however, a possible etiology could be a delay in follow-up. Not enough data is present to make any conclusion about the utility of repeat FNA in FLUS lesions in AA versus NAA. AA were less likely to be diagnosed with malignancy than NAA following a FLUS diagnosis. This is an area that warrants further investigation with respect to possible environmental or intrinsic causes.


Insights into false-negative fine needle aspiration of the thyroid: A single institution experience

Brent Bedke, MD, Jordan Reynolds, MD, Jesse Voss, CT(ASCP), Ashley Hansen, CT(ASCP), Brittany Rosas, CT(ASCP), Aziza Nassar, MD, Michael Henry, MD

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Introduction: Fine needle aspiration (FNA) of the thyroid is an integral step in the evaluation of thyroid nodules. Despite high sensitivity even with ultrasound-guided FNA (US FNA), false negatives (FN) occur leading to delayed treatment. We performed a retrospective review of negative thyroid FNAs from 2001 to 2008, which had a follow-up histological diagnosis of carcinoma. In the present study we compare the cytomorphological characteristics of these FNAs with the histological features of the corresponding thyroid resections.

Materials and Methods: All thyroid FNAs with a negative diagnosis from 2001 to 2008 were identified from the computerized data. Cytologica; specimens signed out as 'non-diagnostic, too few follicular cells' were excluded. Available follow-up tissue resection specimens for each case were reviewed to identify FN FNA. Any carcinoma identified in the same thyroid lobe previously aspirated was included. Review of the archived cytology slides was performed by two cytotechnologists, two trainees and one cytopathologist, with a review of histology by the same two trainees and cytopathologist. The FN FNA slides were evaluated for group cellularity, presence of micro / macro follicles, colloid quantity, nuclear hyperchromasia, nuclear groove percentage, intranuclear inclusion quantity, and quantity of lymphocytes. Histology parameters included tumor size, location, and presence of biopsy site change / needle tract within the tumor.

Results: From 2001 to 2008, 11,461 thyroid FNAs were identified, 7225 (63%) were negative of which 37 (0.5%) had histological confirmation of carcinoma. Ten cases could not be retrieved and 27 potential FN cases were obtained, 21 had a histological diagnosis of papillary thyroid carcinoma (PTC), three had a follicular variant of PTC, two had follicular carcinoma, and one had Hurthle cell carcinoma. Median time lapse from FNA to surgical resection was two months (range 0 - 26 months) with median tumor size 0.75 cm (range 0.2 - 9.0 cm). The cytological findings are presented in [Table 1].

Conclusions: Overall, the sampling error was attributed to a majority of cases. False negative thyroid FNAs were associated with minimal lymphocytes, low percentage of grooves, and rare nuclear inclusions. Subgroup analysis revealed a larger number of grooves and intranuclear inclusions in cases with biopsy site change, within the tumor. Attention to detail in the cytological evaluation of these features remains crucial to prevent false negative diagnosis.


Cytohistological correlation of parotid lesions

Feriyl Bhaijee, MD, Israh Akhtar, MD, Mithra Baliga, MD, Rhyne Flowers, MD, Anwer Siddiqi, MD

Pathology, University of Mississippi Medical Center, Jackson, Mississippi

Introduction: Fine needle aspiration (FNA) is often used as a diagnostic modality in the evaluation of palpable parotid lesions. This study correlates the FNA cytology results with the subsequent histopathological final diagnoses.

Materials and Methods: All parotid FNAs performed at a large academic medical center between 2006 and 2010 were reviewed, and patients that underwent a subsequent surgical resection were identified. We obtained the following data for patients: Age, sex, FNA cytology results, and surgical histopathology results.

Results: Over a four-year period, 74 patients underwent a parotid FNA and subsequent surgical resection. Of these, 34 patients had a pleomorphic adenoma, 94% (32 / 34) of which showed cytohistological correlation; 14 had Warthin's tumor, with 79% (11 / 14) correlation; five had squamous cell carcinoma, with 100% correlation; three had mucoepidermoid carcinoma, with 33% (1 / 3) correlation; two had salivary duct carcinoma, with 50% correlation; one had poorly-differentiated adenocarcinoma, without correlation; and the following diagnoses had 100% cytohistological correlation: One adenoid cystic carcinoma, one epithelial-myoepithelial carcinoma, one oncocytoma, one basal cell adenoma, one papillary adenocarcinoma, one medullary carcinoma, three diffuse large B-cell lymphomas, three melanomas, one branchial cleft cyst, one benign lymphoepithelial cyst, and one salivary duct cyst. Overall, the FNA cytology results and histopathology results correlated in 88% (65 / 74) of the patients. Of the nine cases in which there was disparity between FNA cytology results and subsequent histopathology results: one pleomorphic adenoma was preceded by a non-diagnostic (inadequate) smear; one pleomorphic adenoma was called a duct obstruction / retention cyst on FNA; one Warthin's tumor was suggestive of SCC on FNA, another was 'suspicious for malignancy,' and a third was called sialadenitis; one salivary duct carcinoma with squamous differentiation was suggestive of a squamous cell carcinoma on FNA; two mucoepidermoid carcinomas were preceded by benign or non-diagnostic smears; and one poorly-differentiated adenocarcinoma was preceded by a diagnosis of pleomorphic adenoma on FNA.

Conclusions: For parotid lesions, FNA cytology results showed excellent correlation with the subsequent histopathology results. In our setting, a cytological; diagnosis of pleomorphic adenoma, Warthin's tumor, or squamous cell carcinoma was highly predictive of the subsequent histopathological final diagnosis.


Combined cytomorphological and immunophenotypic analysis enhances the fine needle aspiration diagnosis of lymphoproliferative disorders involving the thyroid

Gillian Levy, Alexander Finkelstein, Guoping Cai

Pathology, Yale University School of Medicine, New Haven, Connecticut

Introduction: Fine needle aspiration (FNA) is the choice of diagnostic tool for thyroid lesions. Although cytomorphological analysis alone is sufficient for rendering a diagnosis in most circumstances, the addition of ancillary studies may sometimes become necessary. For example, a variety of lymphoproliferative disorders (LPDs) may have overlapping cytomorphological features with that of Hashimoto / lymphocytic thyroiditis, which is quite a common entity in the thyroid. LPDs seen in the thyroid may represent thyroidal involvement of systemic disease and rarely occur as primary neoplasms. Clinicopathological features that prompt further workups include patient's prior history of LPD, presence of monomorphic lymphocytes, or pleomorphic lymphocytes with increased large cell population and frequent mitoses / apoptotic bodies. In this retrospective study, we aimed to review our experience with the FNA diagnosis of LPDs involving the thyroid.

Materials and Methods: The database of the Department of Pathology was searched for all cases of thyroid FNA with lymphoproliferative disorders in the diagnostic field during the period from January 2008 to March 2011. A total of 16 cases were retrieved, with cytological diagnoses ranging from favor reactive, atypical lymphocytes, suspicious for lymphoma, to malignant lymphoma. Flow cytometric studies were performed in eight cases using the panel of CD20, CD3, CD4, CD8, CD5, CD10, CD23, kappa, and lambda. Follow-ups or prior specimens were available for comparison in eight cases.

Results: The patients included 14 females and two males with ages ranging from 29 to 90 years (mean 53 years). The biopsied lesions involved the right (7 cases), left (4 cases), isthmus (3 cases), and both right and left (2 cases) thyroid. Three patients had a prior history of LPDs including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). Of the eight cases that had flow cytometry, DLBCL was diagnosed in four cases with another case being MCL. Interestingly, the patient who had a prior history of CLL developed DLBCL in the thyroid. The remaining three cases were considered to be Hashimoto / lymphocytic thyroiditis because of negative flow cytometric results. A reactive process was favored in seven out of eight of the cases without flow cytometry. No follow-up was available for the case that was cytologically suspicious for lymphoma.

Conclusions: Involvement of the thyroid by LPDs is a rare occurrence with DLBCL being the most common type. Patient's prior history and recognition of atypical cytomorphological features must help prompt further workups. Flow cytometry is the choice of ancillary study that helps render a definite diagnosis. In selective cases, addition of fluorescence in-situ hybridization for chromosomal translocations may be helpful in further subtyping LPDs.


Reliability of benign non-neoplastic findings on fine needle aspiration of the parotid gland

Richard Cantley, MD 1 , Leonidas Arvanitis, MD 1 , Guy Petruzzelli, MD, PhD 2 , Vijaya Reddy, MD 1 , Paolo Gattuso, MD 1

1 Pathology, Rush University Medical Center, Chicago, Illinois; 2 Otolaryngology, Rush University Medical Center, Chicago, Illinois

Introduction: There is considerable debate over the role of fine needle aspiration (FNA) in the evaluation of parotid gland lesions. Benign findings on FNA can obviate the need for surgical resection. However, the sensitivity of FNA in diagnosing malignant neoplasms of the parotid gland remains an area of controversy. This study aims to assess the accuracy of a benign, non-neoplastic diagnosis on FNA of the parotid gland.

Materials and Methods: A retrospective search of the cytology archives from our institution for a time period of 19 years (January 1991 to January 2010) revealed that a total of 381 patients underwent parotid FNAs. Of these, 211 had benign and non-neoplastic findings, 90 had benign neoplastic findings, 43 had primary malignant findings, three had secondary malignant findings, and 34 (8.9%) had insufficient material for diagnosis. Among the 211 non-neoplastic cases, 37 (17.5%) underwent surgical resection.

Results: Among the patients with surgical follow-up, there were 13 males and 24 females (M : F 0.54 : 1) ranging in age from 32 to 85 years (mean age 58 years). On surgical resection, 33 (89%) had benign findings. Sixteen (43%) had confirmed non-neoplastic pathology. Seventeen (46%) had benign neoplastic pathology, including twelve Warthin's tumors, three pleomorphic adenomas, one lipoma, and one basal cell adenoma. Four patients (11%) had malignant findings on surgical follow-up. Three had a diagnosis of B cell lymphoma, and one had a diagnosis of squamous cell carcinoma. Among the three lymphoma cases, two had a previous diagnosis of lymphoma. The third patient was being worked up for lymphoma and leukemia, including concurrent bone marrow biopsy, based on a strong clinical and laboratory suspicion. The patient with a diagnosis of squamous cell carcinoma had radiological evidence of bony metastasis to the mandible, prompting surgical follow-up, despite benign FNA findings.

In 33 of the 37 cases (89%) with non-neoplastic findings on FNA and subsequent surgical follow-up, resection confirmed the benign FNA findings, although 17 patients (46%) showed benign neoplastic lesions. Warthin's tumor was the most common neoplasm misdiagnosed as non-neoplastic on FNA (12 cases). In the absence of a strong clinical suspicion for malignancy, a finding of non-neoplastic pathology on FNA was a reliable indicator of benignity [Table 1] and [Table 2].

Conclusions: These data suggest that non-neoplastic findings on FNA of the parotid gland could obviate the need for surgical resection when clinical suspicion for malignancy was low, as only four (11%) patients had a diagnosis of malignancy on surgical resection, and all four of these patients had a strong clinical or radiographic evidence of malignancy at the time of FNA.


Thyroid fine needle aspiration cytology: Performance data of malignant and neoplastic cases as identified from the ASCP NonGYN assessment program

Stan Eilers, MD, FASCP 1 , Paula LaPolice, BS, CT(ASCP) 2 , Perkins Mukunyadzi, MD, FASCP 3 , Umesh Kapur, MD, FASCP 4 , Amy Wendel Spiczka, MS, SCT, MB, HTL(ASCP) CM5 , Ajay Shah, MD, FASCP 6 , Husain Saleh, MBA, MD, FASCP 7 , Adebowale Adeniran, MD, FASCP 8 , Amberly Nunez, MD 9 , Indra Balachandran, PhD, SCT(ASCP), CFIAC 10 , Jennifer Clark, BS, SCT(ASCP) CM11 , Larry Lemon, BS, CT(ASCP) 11

1 Pathology, Mercy Medical Center, Cedar Rapids, Iowa; 2 Pathology, Baystate Medical Center, Springfield, Massachusetts; 3 Pathology, Arkansas Pathology Associates, Little Rock, Arkansas; 4 Pathology, Loyola University Medical Center, Maywood, Illinois; 5 Pathology, Mayo Clinic, Scottsdale, Arizona; 6 Pathology, FNA Clinic, Toledo, Ohio; 7 Pathology, Detroit Medical Center, Detroit, Michigan; 8 Pathology, Yale University School of Medicine, New Haven, Connecticut; 9 Pathology, University of Alabama at Birmingham Health System, Birmingham, Alabama; 10 School of Cytotechnology, Albany College of Pharmacy and Health Sciences, Albany, New York; 11 Continuing Professional Development, American Society for Clinical Pathology, Indianapolis, Indiana

Introduction: The ASCP NonGYN Assessment Program is a glass-slide program developed with oversight by the ASCP NonGYN Assessment Committee, and includes Thyroid FNA cases. Participants choose one of the five diagnostic categories. One - Negative / Reactive / Hyperplasia / Developmental; Two - Infectious / Inflammatory Process; Three - Benign Neoplasm; Four - Lesion of Uncertain Biological Potential; and Five - Positive for Malignancy. A secondary specific interpretation is also provided. In thyroid aspirates, the diagnostic category would largely determine the clinical response: Surgical excision, in categories four and five and generally non-surgical follow-up in categories one, two, and three.

Materials and Methods: Data from five cases of thyroid malignancies and neoplasms, with a total of 1558 responses, was reviewed: Papillary carcinoma with 599 responses; Medullary carcinoma with 225 responses; and Anaplastic carcinoma with 163 responses were categorized as positive for malignancy; while follicular neoplasm with 304 responses; and hurthle cell neoplasm with 267 responses were categorized as lesion of uncertain biological potential. responses were placed into groups: Group A represented the correct diagnostic category and correct interpretation (true positives). Group B, which is a summation of the above-mentioned diagnostic categories one, two, and three, represented the incorrect diagnostic category and incorrect interpretation (false negatives). This was the group representing a major error, as it would impact the clinical management of these patients who would generally not undergo treatment by surgical excision. Group C represented the diagnostic category four, lesion of uncertain biological potential, with any incorrect interpretations. Group D represented the diagnostic category five, positive for malignancy, with incorrect interpretations. Group E presented the most common misinterpretation(s) for each case.

Results: Thyroid aspirates that were Positive for Malignancy (Papillary, Medullary, Anaplastic Carcinomas), performed well with false negative (Group B) rates of 5, 7, and 0%, respectively. Group C (Diagnostic category four) and Group D (Diagnostic category five with incorrect interpretation) represented the relatively minor errors that most probably would not significantly alter the eventual treatment and outcome. Thyroid aspirates categorized as lesions of uncertain biological potential and hurthle cell neoplasm, performed in a similar manner to positive for malignancy cases, with a false negative rate of 7% and a correct interpretation rate of 83%. 9.85% of responses of positive for malignancy with incorrect interpretation could lead to a definitive surgical procedure, which may not be indicated. follicular neoplasm, another lesion of uncertain biological potential, showed a false negative rate of 22%, which represented the incorrect diagnostic category and incorrect interpretation (Group B). 17.23% of the responses were in Group D, positive for malignancy with incorrect interpretation, which could lead to a definitive surgical procedure, which may not be indicated [Table 1].

Conclusions: In summary, thyroid aspirates of Papillary carcinoma, Medullary carcinoma, Anaplastic carcinoma, and hurthle cell neoplasm, showed diagnostic sensitivity rates (Group A + Group C) of 92 - 100%, which was similar to the reported rates of around 95%. The follicular neoplasm presented as the most challenging diagnosis, with a 22% false negative rate.


Incidence of microcarcinomas diagnosed by ultrasound-guided fine needle aspiration in the Puerto Rican population: A prospective study

M. Garcia, MD, L. Rivera, FCAP, FASCP, W. Virella, FCAP, FASCP, R. Rodriguez, CT(ASCP), G. Villarmarzo, FCAP

Cytology, Hato Rey Pathology Associates, Inc., San Juan, Puerto Rico

Introduction: Thyroid nodules are a very common clinical finding during palpation, with an estimated prevalence that ranges from 3 to 7%. Because of their high prevalence, a cost-effective strategy for diagnosis and management of thyroid nodules becomes vital. Fine Needle Aspiration (FNA) plays an essential role in the diagnosis of thyroid disease. Ultrasound-guided FNA (US-FNA) is recommended for nodules >10 mm and suggested for nodules < 10 mm, if clinical information or US features are suspicious. Several studies demonstrate no significant difference in cancer risk in patients with nodules < 10 mm, which does not justify an arbitrary cut-off value of 10 mm. However, the behavior of microcarcinomas is controversial: Even as some of them remain indolent, a substantial number show a more aggressive course; therefore, early diagnosis and treatment of small tumors is clinically important. In order to better assess smaller incidental nodules, it is important to review pertinent clinical history as well as sonographic findings . Few studies have been conducted to evaluate the use of US-FNA in thyroid nodules < 10 mm.

Materials and Methods: The aim of the study was to perform a one-year prospective study to evaluate the use of FNA in the diagnosis of microcarcinomas and its variants in a Puerto Rican population and to correlate nodule size and sonographic characteristics with the risk of malignancy. The study includes all the patients seen in our clinics during one year, starting on July 2010, with thyroid nodules less than 10 mm in size. Our five clinics accept patients from all over the island. All USG-FNA were performed by experienced pathologists using a 23-gauge needle attached to a 20 ml syringe and pistol . Each image from the procedure date and biopsy site was evaluated by a radiologist who confirmed the nodule's size and sonographic characteristics.

Results: The preliminary results came from the data obtained from July to December 2010. Out of 280 positive cases, a total of 34 (12%) nodules resulted in incidental microcarcinomas, measuring from 1 mm up to 10 mm, with an average measurement of 4 mm. And out of the 34, 11% resulted in medullary carcinoma, and the remaining 89% were papillary microcarcinomas; 56% of the incidental microcarcinoma nodules were hypoechoic solitary nodules, whereas, nine (24%) of these nodules showed associated microscopic calcifications.

Conclusions: These preliminary results demonstrate the importance of Fine Needle Aspiration in the diagnosis of microcarcinomas and point out the fact that this type of tumor may be missed if only lesions greater than 10 mm are biopsied.


Thyroid fine needle aspiration reporting rates and outcomes pre- and post-bethesda implementation within a combined academic and community hospital system

Aaron Harvey, MD, Debora Smith, CT(ASCP), Dina Mody, MD, Mojgan Amrikachi, MD

Pathology and Laboratory Medicine, The Methodist Hospital, Houston, Texas

Introduction: The current study compares data from our hospital system before and after the 2008 implementation of the Bethesda System for Reporting Thyroid Cytology, to show the effects it has had on the reporting rates and outcomes for thyroid lesions. Our institution had been previously sub-classifying the 'Suspicious for Malignancy' category into the categories recommended by the Bethesda system. However, the 'Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance' (AUS / FLUS) category did not exist in our reporting lexicon prior to the Thyroid Bethesda implementation.

Materials and Methods: A search of the medical record for all thyroid fine needle aspiration biopsies (FNA) was performed for 2002 - 2005 (prior to the Bethesda recommendations) and for 2009 - 2010 (following the Bethesda implementation). The diagnostic outcomes were also reviewed for cases with available follow-up.

Results: From 2002 - 2005, a total of 2958 thyroid FNAs were performed in our hospital system with 67 (2.3%) unsatisfactory, 2514 (85.0%) negative, 261 (8.8%) suspicious for malignancy, and 116 (3.9%) positive for malignancy, upon cytological evaluation. From 2009 - 2010, a total of 2371 thyroid FNAs were performed with 38 (1.6%) unsatisfactory, 2093 (88.3%) negative, 49 (2.1) AUS / FLUS, 107 (4.5%) suspicious for malignancy, and 84 (3.5%) positive for malignancy, upon cytological evaluation [Table 1]. The AUS / FLUS and suspicious thyroid FNAs from 2009 - 2010 with available follow-up showed the following findings [Table 2]. Of the 49 cases of AUS / FLUS on cytological evaluation, 24 (49%) cases had follow-up procedures: Nine had repeat FNAs, 14 had surgical resections, and one had both repeat FNA and surgical resection. Of the nine repeat FNAs, two were AUS / FLUS, one was positive for malignancy, and one was suspicious for follicular neoplasm. Of the 14 surgical resections, three (21.4%) were positive for malignancy (papillary carcinoma) and one was Hurthle cell neoplasm of uncertain malignant potential. The case with both repeat FNA and surgical resection was benign. Of the 64 cases suspicious for follicular neoplasms, 27 had histological material for follow-up and five (18.5%) showed malignancy. Of the 22 cases suspicious for Hurthle cell neoplasm, nine had histological material for follow-up, two (22.2%) showed malignancy, and one was a Hurthle cell neoplasm of uncertain malignant potential. Of the 21 cases suspicious for malignancy, 10 had histological material for follow-up and eight (80%) showed malignancy.

Conclusions: In conclusion, our AUS / FLUS rate of 2.1% is at the lower range of the 'up to 7%' recommendation of the Bethesda guidelines and our 16.7% rate of malignancy of AUS / FLUS with available follow-up is slightly above the 5 - 15% recommendation of the Bethesda guidelines. As the percentage of cases classified as suspicious (follicular neoplasm, Hurthle cell neoplasm, and malignancy) dropped from 8.8% in 2002 - 2005 to 4.5% in 2009 - 2010, we believe the 2.1% of AUS / FLUS cases in 2009 - 2010 would have probably been included within the suspicious category prior to the 2008 Bethesda implementation.


Review of the National cancer institute / Bethesda terminology system for thyroid cytology specimens: The New York university experience, with special focus on the indeterminate category follicular lesion of undetermined significance

Rachel Hudacko, MD 1 , Keith Heller, MD 2 , Kepal Patel, MD 2 , Jennifer Ogilvie, MD 2 , Joan Cangiarella, MD 1 , Aylin Simsir, MD 1

1 Pathology, New York University Medical Center, New York City, New York; 2 Surgery, New York University Medical Center, New York City, New York

Introduction: In 2007, a National Cancer Institute (NCI)-sponsored consensus conference developed a unified 'Bethesda Terminology System (BTS)' for reporting thyroid fine needle aspiration (FNA) specimens. A national source of dilemma has been with the diagnosis, reporting, and treatment of the indeterminate category called 'follicular lesion of undetermined significance (FLUS)'. Current recommendations are that the FLUS diagnostic rate should not be higher than 7%, and that FLUS should be followed up with a repeat FNA in three to six months due to the low incidence of malignancy (5 - 10%). However, follow-up (F / U) studies published since 2007, have reported malignancy rates of up to 48%. We have been using FLUS before the BTS with a special comment favoring the hyperplastic nodule (HN), follicular neoplasm (FN), or the follicular variant of papillary carcinoma (FVPC), when possible, to offer further input for clinical management. In December 2009, we officially reviewed our diagnostic criteria / terminology and adopted the BTS in 2010, as a department in its entirety, with an intention of eliminating further comments for the FLUS category. The purpose of this study is to compare our FLUS diagnostic and malignancy rates with the published data before and after 2010.

Materials and Methods: This study was confined to FLUS cases with surgical F / U divided into the pre-Bethesda (2006 - 2009) and post-Bethesda (2010) periods. The FLUS cases were further categorized into five subtypes as indicated in the original reports: FLUS not otherwise specified (NOS), FLUS favor HN, FLUS favor FN, FLUS favor FVPC, and atypia of undetermined significance (AUS). The surgical F / U diagnoses were recorded. The FLUS diagnostic and malignancy rates were calculated.

Results: In the pre-Bethesda and post-Bethesda periods, there was a total of 6053 and 2261 thyroid FNAs, respectively, both with the same FLUS diagnostic rate of 6.5%. Overall, 223 FLUS cases (41%) were surgically resected. The malignancy rates for each of the FLUS subtypes are shown in [Table 1]. The overall malignancy rate for the FLUS category was 48%. Of the malignant cases, 44% were FVPC, 40% were classic PC, 14% were follicular carcinoma, and 2% were medullary / other carcinoma. After exclusion of the subtypes FLUS favor FN and FLUS favor FVPC, the overall malignancy rate decreased by 8% [Table 2]. The malignancy rates were similar for cases diagnosed in both the pre- and post-Bethesda periods.

Conclusions: Our overall diagnostic rate for FLUS is similar to the published data. Our malignancy rate for FLUS is significantly higher than what is reported by the BTS, but similar to that reported by some institutions. This may partly be due to the inclusion of cases deemed worrisome for FN and FVPC in the FLUS category. Reclassification of some FLUS cases (i.e., favor FN or FVPC) into more appropriate categories as recommended by the BTS (i.e., neoplasm or suspicious for malignancy) helped to decrease the malignancy rate. Therefore, each institution must evaluate their own practice, to determine how their diagnostic criteria / categories compare to the BTS. This is essential for recommending appropriate F / U for FLUS cases. Based on the wide variation of reported FLUS diagnostic and malignancy rates, revision of the FLUS category at a future national consensus conference is warranted.


Fine needle aspiration of thyroid nodules in the pediatric population: A twelve-year cyto-histological correlation experience at long island Jewish medical center

Seema Lale, MD, Nora Morgenstern, MD, Lori Anderson, DO, Chiara Sugrue, MS, SCT(ASCP), Patricia Wasserman, MD, FCAP

Pathology, North Shore Long Island Jewish Health System, Lake Success, New York

Introduction: Diagnostic evaluation of thyroid nodules by fine needle aspiration (FNA) is an acceptable and cost-effective screening test to triage patients for clinical management, based on the rate of malignancy of each diagnostic category. The bethesda system for reporting thyroid cytopathology was published in 2007, by the National Cancer Institute (NCI). Using this classification, we studied our institution's experience in the pediatric population calculating the rate of malignancy for each diagnostic category, and comparing our findings to the overall patient population and that of the literature.

Materials and Methods: There were 13,312 thyroid FNAs performed at our institution between 1998 and 2010. Of these 282 cases were from patients under 19 years of age. We reviewed and reclassified these cases using the new NCI categories, and pursued a cytology-surgical follow-up.

Results: Of the 282 FNA cases, 20.92% (59) were classified as unsatisfactory (U), 48.22% (136) as benign (B), 2.12% (6) as Atypia of undetermined significance (AUS), 14.18% (40) as suspicious for follicular neoplasm (FN), 2.12% (6) suspicious for malignancy (SM), and 12.41% (35) as positive for malignancy (P). The U-category was further subclassified as the non-diagnostic (ND) 12.41% (35) and the cyst (C) 8.51% (24) category. Seventy-four children had a surgical follow-up. The rates of histologically confirmed malignancy were 10% in U (1 / 10), 0% in B (0 / 17), 50% in AUS (2 / 4), 39% in FN (7 / 18), 100% in SM (4 / 4), and 100% in the P (24 / 24) categories, respectively. Among the U category, the malignancy rate was 0% for the ND category and 25% for the C category.

Conclusions: To our knowledge, this is the first study to apply NCI categories to the pediatric population and compare the risk of malignancy in each category in the pediatric population to that of the overall population, as reported in the literature. The rate of malignancy in the U category was only seen in the specimens with cystic-component (25% malignancy rate in cysts compared to 0% in the non-diagnostic category), although only two patients had surgery in this category. The AUS and FN categories had a higher malignancy rate in our pediatric population (50 and 39%, respectively) as compared to that of the overall population reported in the literature (15 and 30%, respectively). Given that the rates of malignancy are higher for cysts and AUS in the pediatric population, the literature recommendation to 'follow-up and repeat' may not apply well to children, where surgery may be warranted instead. As the rate of malignancy is slightly higher for FN, the literature recommendation of diagnostic lobectomy remains sound, unless malignancy can be proven by slide re-review or frozen section, before the lobectomy is final.


Hashimoto thyroiditis: The great obscurer or a mere diagnostic obstacle

Alexander Finkelstein, DO, MD, Gillian Levy, MD, Constantine Theoharis, MD

Pathology, Yale University School of Medicine, New Haven, Connecticut

Introduction: Fine needle aspiration (FNA) is the modality of choice in the diagnostic workup of thyroid nodules. The inflammatory condition hashimoto thyroiditis (HT) is often encountered, and while, in and of itself, HT does not usually present a diagnostic challenge, it is associated with both epithelial and lymphocytic cytological changes that lie within the spectrum of thyroid neoplasms. Furthermore, HT is classically associated with an increased risk of certain thyroid malignancies including papillary thyroid carcinoma (PTC) and non-hodgkin lymphoma (NHL). In light of the wide spectrum of changes and the increased risk of malignancy, HT may be associated with a concurrent neoplastic process that goes unnoticed. We sought to investigate the risk of HT disguising a neoplastic process in a tertiary endocrine referral center, that is , whether the presence of HT on thyroid FNA obscures another pathological condition in the thyroid for which surgical intervention would be warranted.

Materials and Methods: Three hundred and forty-nine cases were retrieved from the Department of Pathology archive representing all cases of thyroid FNA with HT during the five-year period of January 2002 to December 2007. Cytological diagnoses ranging from HT alone, HT with follicular neoplasm (FN), to HT with PTC were represented. Surgical follow-up was available in 39 cases (11%). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated in relation to the histopathological diagnoses.

Results: Twenty-seven (69%) of the 39 operated cases or 8% out of the total 349 HT patients had a cytological diagnosis of either FN or PTC in addition to HT. In the remaining 12 cases (31%), operations were performed for cosmetic reasons. Cases where both cytological and histopathological evaluation exhibited HT with a concurrent neoplastic lesion were designated as true positives (21 cases). Cases where cytological evaluation showed HT and follow-up confirmed HT alone were designated as true negatives (7 cases). Cases diagnosed cytologically as HT with a concurrent neoplasm, but upon resection exhibited HT alone were designated as false positives (6 cases). Finally, cases where cytological evaluation displayed HT only, but where histopathology displayed as both HT and a concurrent neoplasm not detected preoperatively were designated as false negatives (5 cases). These five false negative cases included: Three PTCs, two Follicular Adenomas, and one NHL. Sensitivity in the detection of a neoplasm concurrent with HT was 81% and specificity was 54%. PPV and NPV in our patients were 78 and 58%, respectively.

Conclusions: Neoplasms concurrent with HT, while not exceedingly common, can present a diagnostic challenge. FNA has a relatively high sensitivity and PPV in the detection of these neoplasms (81 and 78%, respectively). However, as shown by the rate of false negatives in this study, the epithelial and lymphocytic cytological changes in HT may obscure the significant concurrent pathology. Therefore, the FNA diagnosis of HT demands some degree of heightened vigilance for the presence of concurrent neoplasms.


Interinstitutional second opinion in thyroid cytology: Should second opinion be mandated prior to definitive surgery if Fine Needle Aspiration was performed elsewhere?

Xiaosong Li, MD 1 , Keithe Heller, MD 2 , Joan Cangiarella, MD 1 , Aylin Simsir, MD 1

1 Pathology, NYU Medical Center, New York, New York; 2 Endocrine Surgery, NYU Medical Center, New York, New York

Introduction: Interinstitutional consultation / second opinion (IC) for review of surgical biopsy slides, prior to definitive surgery, is a routine practice in many institutions, if the original biopsy leading to the definitive surgery has been performed elsewhere. Data on the effect of IC for cytology specimens prior to definitive surgery is somewhat sparse. Studies show major discrepancy rates varying from 7.4 to 9.3% in a variety of gynecological and non-gynecological cytology specimens. Thyroid fine needle aspiration (FNA) is one of the major specimen types leading to a change in diagnosis and patient management upon IC. The Interinstitutional diagnostic concordance rate varies from 40 to 82%. We reviewed our experience with the IC of thyroid FNAs for patients who had the FNA procedure done elsewhere and were referred to NYU for further management.

Materials and Methods: The pathology reports from all IC thyroid FNAs in 2009 and 2010 were retrieved; the original and IC diagnoses were recorded and compared. For the purpose of this study the diagnostic categories were grouped as follows, based on effect on clinical management: (1) Nondiagnostic, (2) benign, (3) atypia of undetermined significance / follicular lesion of undetermined significance (AUS / FLUS), (4) suspicious for follicular neoplasm / follicular neoplasm (susp FN / FN), (5) suspicious / positive for malignancy including papillary carcinoma and other carcinomas. The diagnostic concordance rates were calculated. Surgical follow-up was obtained when possible.

Results: [Table 1] summarizes the original referral and NYU IC FNA diagnoses. The overall diagnostic concordance rate with referral institutions was 74% [Table 2]. The lowest concordance rate was within the AUS / FLUS and susp FN / FN categories. Of the AUS / FLUS cases, 19% were downgraded to benign, 6% were downgraded to unsatisfactory, and 20% were upgraded to susp FN / FN or malignant categories; therefore, within this group, patient management would potentially be altered in 46% of the cases based on the IC review. Of those who underwent surgery, 86% of the AUS / FLUS cases that were downgraded to benign were confirmed to be benign nodules on excision; one of the seven cases was papillary carcinoma. On excision, 86% of the AUS / FLUS cases upgraded to susp for FN / FN or malignant categories were confirmed to be malignant. For the susp FN / FN category, 26% were downgraded to benign, 39% were downgraded to AUS / FLUS, and 3% upgraded to malignant, potentially altering patient management in 68% of the cases. None of the cases downgraded to benign had a surgical follow-up; 67% of the cases downgraded to AUS / FLUS were papillary carcinoma on excision, whereas, 33% were confirmed to be benign. The single case upgraded to malignancy in this group was papillary carcinoma on excision. Malignancy rates on surgical excision are summarized in [Table 3].

Conclusions: (1) The greatest diagnostic concordance with referring institutions is with the unsatisfactory, benign, and susp / positive for malignancy categories. The least diagnostic concordance is with the AUS / FLUS and susp for FN / FN diagnostic categories. (2) The IC review in AUS / FLUS and susp for FN / FN cases can lead to significant changes in management; therefore, IC should be undertaken whenever possible. Based on the surgical outcome results and cytological diagnostic discordance, these lesions continue to pose significant diagnostic and management challenges. (3) The malignancy rates in most diagnostic categories are higher in this study compared to the published Bethesda data. This may be, in part, due to the selection bias, as cases sent for second opinion / surgery may reflect those patients with higher risk of malignancy based on clinical / radiologic findings.


Application of the Hybrid Capture-2 assay to squamous cell carcinomas of the head and neck: A convenient liquid-based approach for the reliable determination of HPV status

Zahra Maleki, MD 1 , Justin Bishop, MD 1 , Alexander Valsamakis, MD, PhD 1 , Xiaofei Chang, MD 2 , Sara Pai, MD 2 , William Westra, MD 1

1 Pathology, The Johns Hopkins Hospital, Baltimore, Maryland; 2 Otolaryngology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: A growing proportion of head and neck squamous cell carcinomas (HNSCCs) are caused by the human papillomavirus (HPV). In light of the unique natural history and prognosis of these HPV-related HNSCCs, routine HPV testing is being incorporated into the diagnostic protocols. Accordingly, there is an escalating demand for an optimal detection strategy that is sensitive and specific, transferrable to the diagnostic laboratory, standardized across laboratories, cost-effective, and amendable to broad application across specimen types including cytological preparations.

Materials and Methods: Cytological preparations (fine needle aspirates and brushes) were obtained from surgically resected HNSCCs and evaluated for the presence of high-risk HPV using the Hybrid Capture 2 assay. HPV analysis was also performed on the corresponding tissue sections using HPV in-situ hybridization and p16 immunohistochemistry. In cases where the immunohistochemical and in-situ hybridization results were discordant, HPV status was determined by real time PCR detection of E6 and E7 expression. HPV status in the tissue sections and corresponding cytological samples were compared. Twenty-four patients who had undergone resection of a primary and / or metastatic HNSCC were selected. During routine surgical pathology dissection of the specimens, the tumor was identified and aspirated or brushed. These FNA specimens were evaluated cytologically (Diff Quick stained air-dried smears) for the presence of tumor cells and overall sample cellularity. Four brushes from the non-neoplastic tonsils and two FNAs from the benign lymph nodes were used as HPV negative controls. All together, 27 cytological preparations from 24 patients were evaluated for high-risk HPV using the Hybrid Capture 2 assay. The HPV status was confirmed in the formalin-fixed and paraffin-embedded tissue sections of the resected HNSCCs. Immunohistochemical analysis targeted the expression of a biomarker of HPV E7 oncoprotein activity, the CDK-inhibitor p16. Tumor blocks were evaluated for the presence of HPV DNA by in-situ hybridization that captured HPV genotypes 16, 18, 33, 35, 45, 51, 52, 56, and 66.

Results: There was a strong agreement for the HPV status across the various methods of HPV analysis. All 13 HNSCCs that were p16 positive by immunohistochemistry and HPV positive by in-situ hybridization (p16+ / HPV+) were HPV positive by Hybrid Capture 2 assay analysis. Conversely, the 10 HNSCCs that were p16- / HPV- were negative by Hybrid Capture 2 assay analysis. The single discordant case (p16+ / HPV-) was found to be HPV positive using the Hybrid Capture 2 assay. Using real-time PCR amplification of the viral oncoprotein E7, the presence of HPV 16 in this case was confirmed at 1.02 copies of HPV 16 / genome equivalent. Based on the benchmark HPV testing of the tissue sections, 14 HNSCCs were classified as HPV positive and 10 as HPV negative. All the corresponding cytological preparations were correctly classified using the Hybrid Capture 2 assay.

Conclusions: The Hybrid Capture 2 strategy, already in widespread use for the detection of high-risk HPV in cervical brushes, is readily transferrable to HNSCCs. Consistent accuracy in cytological preparation suggests its potential application in FNAs from patients who present with lymph node metastases, and may eliminate the need to obtain a tissue, solely for the purpose of HPV testing.


Atypia of undetermined significance: Institutional experience with the Bethesda system

Matthew Olson, MD, Douglas Clark, MD, Yener Erozan, MD, Syed Ali, MD

Department of Pathology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Atypia of undetermined significance (AUS) is one of the least well-understood diagnostic categories within the bethesda system (TBS). As all the eight different scenarios lead to the categorization of AUS, and these range from sample preparation artifact to rare focal nuclear atypia, it follows that the likelihood of malignancy in these nodules would differ. This study is a correlation of the resection specimens from 95 patients with no worse than an AUS diagnosis from a fine needle aspirate (FNA) of the thyroid.

Materials and Methods: The frequencies of TBS categories were calculated from 3,956 thyroid FNAs, interpreted over a 26-month period at a major tertiary care center since the adoption of TBS for reporting thyroid cytopathology. The mmorphological criteria of TBS were applied strictly. The breakdown of TBS diagnoses is given in [Figure 1]. Resection and cyto-histo correlation was performed on 95 cases of AUS.

Results: The rate of false negative diagnoses in our series was 4.8% (11 / 237). Odds ratios and relative risks for this data are listed in [Table 1]. In thyroidectomies performed after the worst diagnosis of AUS (n = 95), 31 (33%) of them had stageable (not incidental microcarcinoma) carcinoma on resection (OR: 10.0, CI: 4.78 - 21.08). Our review disclosed two salient subcategories of AUS: Those with focal nuclear atypia, and those with focal microfollicular features. Of the subset of AUS with nuclear atypia (n = 36), 47% had stageable carcinoma on resection (OR: 18.6, CI: 7.6 - 45.2). When the AUS with nuclear atypia group was separated out, this group demonstrated a significantly higher risk of malignancy that the other forms of AUS (OR: 2.87, CI: 1.18 - 6.99). In contrast, the microfollicular features did not differ significantly from the other forms of AUS (OR: 1.61, CI: 0.57 - 4.54).

Conclusions: Although nuclear atypia and microfollicular features are both morphological subsets of AUS, in terms of clinical significance, nuclear atypia correlates more with the risk of stageable carcinoma on resection. These findings support the conclusion that AUS is a heterogeneous yet clinically valid diagnosis, with a significantly higher risk for stageable carcinoma on resection. Additionally, AUS with nuclear atypia is independently correlated with a higher risk for stageable carcinoma on resection.


Atypia of undetermined significance in thyroid fine needle aspiration predicts the absence of features associated with aggressive behavior for papillary thyroid carcinoma

Paul VanderLaan, MD, PhD, Jeffrey Krane, MD, PhD

Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Introduction: The vast majority of malignancies identified by thyroid fine needle aspiration (FNA) are papillary thyroid carcinoma (PTC), a cancer with an excellent overall prognosis. We had previously noted a high percentage of follicular variants among the PTCs identified following an AUS diagnosis. Here, we set out to determine if other features of PTC, particularly those associated with a more aggressive clinical behavior, correlate with the preceding cytological diagnosis.

Materials and Methods: Thyroid FNAs performed at our hospital from January 2005 through May 2010 were correlated with subsequent histopathological findings. FNAs were evaluated by staff cytopathologists using a six-tiered Bethesda-like reporting system. The FNA diagnoses that were evaluated in this study were malignant (POS), suspicious for malignancy (SUS), and atypia of undetermined significance (AUS). Only the diagnosis for the targeted nodule was considered. Nodules diagnosed as PTCs were classified as being high-risk subtypes (tumors having tall cells, columnar, and / or diffuse sclerosing features), follicular variants, conventional variants, or other subtypes not in the high-risk subcategory. The following clinical parameters were also assessed for PTCs: Tumor size, the AJCC TNM stage, multifocality, lymphovascular invasion, extrathyroidal extension, and recurrence / metastasis after surgery.

Results: For this study period, 168 thyroid nodules were resected following a single or repeat diagnosis on FNA of AUS; 202 nodules for a SUS FNA, and 192 thyroid nodules for a POS FNA. Malignancy was identified in 56 (33%) of the AUS cases, including six minimally invasive follicular carcinomas (only one with lymphovascular invasion) and one lymphoma (following a diagnosis of atypical lymphoid cells). PTC was confirmed in 49 (29%) of the AUS FNAs, 163 (81%) of the SUS FNAs, and 183 (95%) of the POS FNAs. The mean size of the resected PTCs did not differ between the FNA-diagnosis groups (AUS: 2.0 ± 1.2 cm, SUS: 2.1 ± 1.4 cm, POS: 1.9 ± 1.1 cm), nor did the likelihood of a multifocal disease (AUS and SUS: 55%; POS: 63%). However, the PTCs following an AUS FNA were less likely to have properties associated with aggressive behavior. No aggressive variants of PTC were identified with the AUS category. AUS-associated PTCs were more likely to be a lower AJCC T stage, to be a lower AJCC N stage (all AUS-associated PTCs were N0) and to lack lymphovascular invasion or extrathyroidal extension [Table 1]. All T3 PTCs associated with AUS were > 4 cm, with one also having extrathyroidal extension. Only two PTCs had distant metastasis at the time of surgery, and 16 patients later went on to develop recurrence and / or distant lymph node metastasis after surgery; all these cases had a POS FNA diagnosis preceding surgery.

Conclusions: This study indicates that the probabilistic-based Bethesda system for reporting thyroid cytopathology not only conveys the risk that a thyroid nodule is malignant, but also predicts the presence of pathological risk factors associated with aggressive biological behavior when the malignancy is PTC. AUS diagnoses identify low-risk PTCs, most of which are follicular variants. These findings suggest that a more conservative clinical approach for nodules diagnosed repeatedly as AUS, particularly those of small size, may represent a reasonable alternative to immediate surgery.


Can cytomorphology further stratify thyroid fine needle aspirates diagnosed as follicular lesion of undetermined significance?

Ann Walts, MD, Catherine Bresee, MS, Shikha Bose, MD

Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California

Introduction: Fine needle aspiration (FNA) provides a cost-effective method for the triage of thyroid nodules, but inconsistent terminology and low levels of diagnostic agreement have been problematic. The bethesda system for reporting thyroid cytopathology (TBS-T) was created to standardize the criteria and terminology for thyroid FNA reporting into six categories, each with an assigned 'risk of malignancy'. Follicular lesion of undetermined significance (FLUS), with an assigned 5 - 10% risk of malignancy, is the most frequent abnormal diagnosis rendered in thyroid FNAs. TBS-T recommends that the FLUS not exceed 7% of thyroid FNA diagnoses; however, this percentage varies widely across cytopathologists and laboratories. In our laboratory, FLUS comprises of 10% of the thyroid FNA diagnoses. Stratification of FLUS could benefit patient management. This retrospective study of cases diagnosed as FLUS was designed to determine if cytomorphology could predict carcinoma (CA) in the subsequent thyroidectomy.

Materials and Methods: Eighty-three consecutive cases diagnosed as FLUS on FNA and subsequently resected were retrieved from our files. The patients ranged from 27 to 77 years in age; 76% were female. The final diagnostic groups (benign vs. malignant) were compared for age, gender, interval between FNA and resection, and the presence of cell sheets, microfollicles, nuclear grooves / irregular nuclear membranes (NG), nuclear overlap / crowding (NC), nuclear enlargement (NE), nuclear pseudoinclusions (NI), bland / compact chromatin, Hurthle change, lymphocytes, and colloid as described in the cytology reports. The confounding effects of subjective terms (e.g., rare, few, some, focal) were eliminated by scoring each feature on a binary scale (present or absent). Statistical analyses were performed using the SAS software.

Results: Histological diagnoses of the aspirated thyroid nodules (median 1.8 cm) included 58 benign, 21 CAs (10 follicular variants of papillary CA (FVPTC), five CAs with mixed papillary / follicular patterns, three classical papillary CAs, two minimally invasive follicular CAs, one anaplastic CA), and four neoplasms of undetermined malignant potential. For statistical analysis, these four cases were included in the malignant group. Comparison of the FNAs from the benign and malignant cases showed significant differences (P < 0.5) in the frequencies of the four features studied - NG, NC, NI, and Hurthle change - between the two groups [Table 1]. When each predictor was considered independently using unadjusted analysis with logistic regression, NG and NC emerged as significant predictors of CA. Ten (66.7%) of the 15 cases with NG and 15 (62.5%) of the 24 cases with NC were malignant on excision. The presence of NC showed a strong positive correlation with the presence of NG, NI, and NE (Spearman rank correlations). In a forward step-wise selected, adjusted, logistic regression analysis, an increased risk of malignancy on excision was only associated with NC. Thyroid malignancy was present in 17 (61%) of the 28 cases whose FNAs exhibited ≥ 1 of the three significant cytological features [Table 2].

Conclusions: The follicular variant of papillary thyroid carcinoma (FVPTC) is the most frequent CA identified in thyroidectomies following an FNA diagnosis of FLUS.

  • The presence of NG, NI, and / or NC in FNAs diagnosed as FLUS was associated with malignancy in 61% of the subsequent thyroidectomies. These FNAS were best diagnosed as suspicious for malignancy to which TBS-T assigned the risk of malignancy as 50 - 75%.
  • Large prospective studies are needed to confirm our findings.


Web-based laboratory provider outreach tool to ease transition to the Bethesda system for reporting thyroid cytopathology

Scott Whitworth, MD

Department of Pathology, Walter Reed National Military Medical Center, Bethesda, Maryland

Introduction: The 2010 bethesda system for reporting thyroid cytopathology provides a framework for uniform interpretation of thyroid fine needle aspiration cytology. For some laboratories, adoption of this new reporting framework may require a significant adjustment in terminology and report format. This may create confusion for providers and uncertainty regarding treatment recommendations in some instances. The Bethesda System for reporting thyroid cytopathology allows flexibility in the reporting framework for each laboratory to tailor its interpretations and format, to meet the needs of its providers. This flexibility may lead to modified terminology and long explanatory commenting in individual aspiration reports. A prototype web application was developed to enhance the adoption of the bethesda system for reporting thyroid cytopathology, standardize interpretation commenting, and improve the laboratory-clinician outreach.

Materials and Methods: A web-based application was written in the Python programming language against the Django web-development framework, using open-source technology and modern programming practices, including source control and unit-testing. Application data was stored in SQLite. The application presented the providers with a list of diagnostic categories and specific diagnoses used by the laboratory. Pages for the selected diagnosis included laboratory-specific explanatory comments and recommendations for clinical management. External links included reference abstracts on Pubmed.gov, diagnosis-specific image sets in the online atlas of the bethesda system for reporting thyroid cytopathology, and management recommendations in the american thyroid association professional guidelines. the application is deployed on an inexpensive virtual private server using an open-source web server stack consisting of an Ubuntu linux operating system and Nginx web server.

Results: A functional demonstration is available at http//www.scottwhitworth.com/thyroid/. The source code is available under a free software license and is hosted on a popular code sharing and collaboration site where it is under active development. Current development efforts are focused on user-facing design and experience improvement. Application utilization and provider satisfaction studies are planned.

Conclusions: Adoption of the bethesda system for reporting thyroid cytopathology may cause a significant adjustment in former reporting terminology and create confusion for providers. This tool provides a low-cost solution for laboratory-clinician outreach, and reduces the need for lengthy explanatory comments in individual thyroid fine needle aspiration reports. The introduction of powerful web development frameworks for popular scripting languages and the open-source climate of today's internet, reduces the cost and expertise barriers to produce laboratory web tools.


Do smears contribute to the diagnosis of thyroid fine needle aspiration preparations?

Heather Wilgenbusch, CT(ASCP), MP(ASCP), Gail Mueller, MS, CT(ASCP), DLM, Nola Munasifi, MD, Margaret Neal, MD

Ketchum Wood and Burgert, Pathology Associates, Tallahassee, Florida

Introduction: Three types of cytological specimen preparations (two types of liquid-based preparations and prepared smears) are commonly used to diagnose thyroid fine needle aspiration (T-FNA). At present, there is lack of consensus as to which of these preparations is most influential in the diagnosis of FNAs or whether considering the three methods in conjunction might have a synergistic effect on the diagnosis. For example, the two liquid-based preparations (LBPs) provide an additional diagnostic tool with complementary morphological features. The standard procedure in our laboratory is to prepare one ThinPrep® (Hologic) and one SurePath® (BD) from the needle rinse in addition to reviewing two to eight smears (air-dried, Romanowsky stain and fixed, Papanicalou stain) provided by the clinician. The purpose of this study is to measure how closely the diagnoses based on LBPs reviewed alone correspond to our original diagnoses based on a combination of LBPs and smears.

Materials and Methods: Seven cytotechnologists and seven pathologists reviewed 272 archived liquid-based slides from 136 T-FNA cases received sequentially over a two-month period. These review diagnoses were based on liquid-based preparations alone and were compared to the original archived diagnoses, which were based on a review of liquid-based slides and clinician-prepared smears. The original diagnosis served as a reference point because it contained a composite of cytological material and more complete clinical information. The review diagnosis was blind to the original diagnosis and limited patient history was provided along with the ultrasound report, when available. The two LBPs were diagnosed separately, but were not blinded to each other. Diagnoses were based upon the bethesda system for reporting thyroid cytopathology. Comparison of the original diagnosis and review diagnosis was evaluated according to a scoring grid based on one- and two-step discrepancies.

Results: Comparison of the diagnoses revealed that 79.8% of the review diagnoses made using only LBPs corresponded exactly with the original diagnoses made on smears and LBPs, and an additional 17.8% of the diagnoses made on these LBPs reflected the original diagnosis within one step of the diagnostic category. All unsatisfactory cases in the original diagnoses were correctly identified, while 1.7% of the cases were two-step discrepancies based on the original diagnosis (six by cytotechnologists and three by pathologists) and 0.7% of the cases showed > 2-step discrepancies (four by cytotechnologists). A histological follow-up was available for only two cases.

Conclusions: LBPs reproduced the diagnoses of FNAs when compared with LBPs and submitted smears in 98% of the cases within one step in this study of 136 sequential T-FNAs. This study supports the previous findings that the use of liquid-based preparations alone provides sufficient material for the evaluation of FNAs. The inclusion of prepared smears added another dimension for review, with minor resultant differences in the interpretation, in 2% of our cases. LBPs offered advantages over smear preparations of a more standard preparation, ease of additional preparations for stains or molecular studies, and a clearer cellular detail, with no apparent disadvantage in diagnostic material, in this study.

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Use of the Cellient™ automated cell block system to process small surgical biopsy specimens that may not survive traditional processing

Dawn Underwood, MS, CT(ASCP), Alisha Cotman, Joseph Begany, PA(ASCP), Brad Skilton, PA(ASCP), Linda McDonald, HT(ASCP), EM(EMSA), Jennifer Brainard, MD

Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio

Introduction: Small biopsy specimens with minimal visible tissue are a challenge to process. At our institution, these samples are particularly common in gynecological pathology. Samples ≤ 1 mm in one dimension or those that consist predominantly of mucus are vulnerable to loss during processing. In our experience, approximately one-third of these cases are insufficient for diagnosis. The Cellient™ automated cell block system is a novel method for creating cell blocks from cytology samples, using controlled vacuum to deposit material onto a filter, followed by infiltration of the sample with reagents and paraffin. Cellular material is concentrated to maximize cell yield in an automated and more consistent manner when compared to traditional cell block methods. We decided to see if the use of the Cellient™ System to process small gynecological biopsies would facilitate diagnosis and result in fewer unsatisfactory samples.

Materials and Methods: Small biopsies that may not survive processing are defined as samples that measure ≤ 1 mm in one dimension or those that consist predominantly of mucus, with scant visible tissue. Traditional processing involves filtering formalin-fixed samples into a nylon mesh biopsy bag, followed by a transfer of the contents of the bag to a histowrap paper. The histowrap paper is placed into a cassette for further processing and paraffin embedding. For Cellient™ cell block processing, the formalin-fixed biopsy samples were washed twice with 20 ml of CytoLyt and placed in a ThinPrep® vial containing PreservCyt solution. The samples, without visible tissue fragments, were directly transferred from the vial into the cassette / filter assembly by the instrument. If tissue fragments were visible, they were manually loaded by the cytopreparatory personnel into the cassette / filter assembly. The sample was augmented by aspiration of additional cellular material from the ThinPrep® vial. The instrument processed the sample and infused paraffin to produce a cell block. The blocks were cut and stained with H and E. At least two tissue sections were present on each of the two levels. All slides in the study were reviewed by one gynecological surgical pathologist.

Results: In 2010, the data for 422 gynecological surgical biopsies with minimal tissue were processed, using the traditional methods that were available. A diagnosis was rendered in 279 (66%) cases. The remaining 143 (34%) were insufficient for evaluation. Cellient™ cell block processing was introduced during a one-month study period, in April 2011. During this time, 135 samples were processed, including 111 endocervical curettings (ECC), 19 endometrial biopsies (EMB), two endometrial curettings, and three cervical biopsies (CXBX). Twenty-six cases (19%) processed using the Cellient™ System were insufficient for evaluation (18 ECC, 6 EMB, and 2 CXBX). Nineteen cases were manually processed and all were diagnostic. Dysplastic squamous cells were identified in seven ECC (three CIN 2, two CIN 1, and two squamous dysplasias that could not be further classified). All endometrial and cervical biopsy diagnoses were benign.

Conclusions: Rates of unsatisfactory gynecological biopsies decreased from 34% with traditional processing to 19% using the Cellient™ System, during the study period. The Cellient™ method concentrates the cellular material in the center of the microscopic slide, facilitating microscopic evaluation. Close communication between surgical pathology, histology, and cytology is necessary for optimal specimen handling.


Constructing a twenty-first century cytology laboratory

Janie Roberson, SCT(ASCP), Allison Wrenn, CT(ASCP), John Poole, AIA, Andrew Jaeger, Isam Eltoum, MD, MBA

Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Introduction: Constructing or renovating a laboratory can be both challenging and rewarding. UAB Cytology (UAB CY), recently undertook a project to relocate from a building constructed in 1928 to a new space. UAB CY is part of an academic center serving two Cytotechnology programs, two Fellowships, and support a staff active in research and publication. Our objectives were to provide a safe, aesthetically pleasing space, and gain efficiencies through Lean processes.

Materials and Methods: The phases of any laboratory design project are planning, schematic design (SD), design development (DD), construction documents (CD) and Construction. Laboratory personnel are most critical in the planning phase. During this time stakeholders, relationships, budget, square footage, and equipment are identified. Equipment lists, including what will be relocated, newly purchased, and projected for future growth ensure that utilities are matched to the need. A chemical inventory is essential for providing adequate storage space. Regulatory and safety requirements are discussed determining that the wet laboratory space be designated as Bio safety level 2 (BSL2). Tours and high level process flow diagrams help architects and engineers understand the daily laboratory work. Future needs are addressed through a questionnaire, which identifies the potential areas of growth and technological change. Throughout the project, decisions are driven by the data from the planning phase. During the SD phase, objective information from the first phase is used by architects and planners to create a general floor plan. This is the basis of a series of meetings to brainstorm and suggest modifications. DD brings more detail to the plans with engineering, casework, equipment specifics, and finishes. Design changes must be completed at this phase. The next phase, CD, takes the project from the laboratory purview into a purely technical mode. Construction documents are used by the contractor for the bidding process and ultimately the construction phase.

Results: The project fitted out a total of 9,000 square feet; 4,000 laboratories and 5,000 office / support. The laboratory space included areas for prep, CT screening, sign out, and imaging. The adjacent space housed faculty offices and conferencing facilities. Transportation time was reduced (waste removal) by a Pneumatic Tube System, specimen drop window to the prep laboratory, and a pass through window to the screening area. The open screening and prep areas allowed visual management control. Efficiencies were gained by ergonomically placing the CT Manual and Imaging microscopes and computers in close proximity, also facilitating a paperless workflow for additional savings. Logistically, closer proximity to Surgical Pathology maximized the natural synergies between the areas.

Conclusions: Laboratory construction should be a systematic process based on sound principles for safety, high quality testing, and finance. Our detailed planning and design process could be a model for others undertaking similar projects.


Trends in practice before and after implementation of EUS-FNA: A disruptive innovation effect

Evans Alston, MD, Janie Roberson, CT(ASCP), Isam Eltoum, MD, MBA

Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Introduction: The CAP Futurescape Forum has introduced the concept of disruptive innovation as a means to assess the impact of technology on pathology practice. Little has been reported on changes in the volume and types of pathology specimens, spectrum of pancreatic disease, and the accuracy of cytologic diagnosis, before and after implementation of EUS-FNA. In this study, we assessed these parameters before and after implementation of EUS-FNA, in a large academic center. We also attempted to determine if the impact of EUS-FNA cytology on replacing tissue biopsy in the diagnosis of pancreatic disease fulfils the Christensen's criteria of disruptive innovation.

Materials and Methods: Electronic records of 20 years of pancreatic specimens, both cytological and histological evaluations (10 years before and 10 years after implementation of EUS-FNA) were reviewed. For these two periods, the following parameters were compared: The total and annual average of each and every type of pancreatic specimen, diagnostic categories, and accuracy of cytological diagnoses, using histology as the gold standard. The chi-square test was used as appropriate with the difference considered significant at P < 0.05. Disruptiveness of the cytology relevant to biopsy was assessed by comparing the utilization trend and the accuracy of diagnosis over time.

Results: Over 20 years, there was a gradual increase of all pancreatic specimens [Figure 1]: The mean annual volume (SD) of cytological specimens was 24 (11) before versus 231(10) after implementation (9 non-EUS-FNA, 222 EUS-FNA), and that of histological specimens was 97 (42) before versus 377 (148) after implementation. The average annual percent of cases managed following cytology alone was 19% (10) before versus 51% (8) after implementation. In contrast, those managed after histology alone was 56% before versus 23% after implementation. Non-EUS FNA decreased from 36% to 1% of the total cytological pancreatic specimens. Out of all the pancreatic surgical pathology specimens, the total number of needle biopsies decreased from 7 to 1%; and the other biopsy types from 29 to 9%. Whipple procedures increased from 19 to 49%, while partial resections remained the same: 44 versus 41%. More definitive diagnoses such as categorization of neoplastic and cystic diseases were observed after implementation. A significant reduction in unsatisfactory (7 vs. 1%), atypical (16 vs. 4%), and suspicious (16 vs. 3%) cytological diagnostic category rates was noted. The accuracy of the cytological diagnosis significantly improved; for ductal carcinoma, the sensitivity (C.I.) and specificity (C.I.) were 55% (38 - 70%) and 78% (58 - 89%) before versus 88% (84 - 91%) and 96% (93 - 98%) after, respectively. The diagnostic odds ratio (C.I) for a positive cytology was 4.0 (1 - 13) compared to 200 (94 - 422) before and after implementation, respectively.

Conclusions: EUS-FNA has greatly improved the accuracy of cytological diagnosis, reduced the number of indeterminate diagnoses, and largely replaced the need for tissue biopsy. Given the cost and simplicity as compared to tissue biopsy, this trend represents a disruptive innovation effect.


Does cell block quality need to be sacrificed for turn-around time? The impact of rapid microwave processing on cytomorphological and immunohistochemical characteristics

Rebecca Heintzelman, MD, Hillary Kimbrell, MD, Fernando Garcia, MD, Xiaoli Chen, MD

Department of Pathology, Drexel University College of Medicine, Philadelphia, Pennsylvania

Introduction: Processing cell blocks with traditional tissue processors can delay the turn-around time (TAT) of cytology cases. Microwave tissue processors can decrease the processing time to less than an hour, resulting in better patient care, although there are no studies evaluating the appropriate time of fixation and the effect on cellular morphology. We have undertaken this study to evaluate whether processing cell blocks on a microwave processor adversely affects cytomorphology or immunoreactivity.

Materials and Methods: Sixty-four effusion fluid specimens were collected consecutively and divided evenly into two blocks, one processed on the traditional processor and the other using the microwave. Each block was assigned to a formalin fixation time of 1 - 2 hours, 2 - 8 hours, 8 - 24 hours, or 24 - 72 hours. Hematoxylin and Eosin (H and E) stained sections were prepared from each block and randomized. Each slide was blindly scored on a scale of 0 - 3, with regarf to the nuclear, cytoplasmic, and cell membrane details of the mesothelial cells by three reviewers. Each reviewer was also asked to predict the processing method that was used. MOC31, calretinin, and cytokeratin 7 (CK7) immunostains were performed on 39 paired cases, using the Ventana Benchmark system. Each slide randomly contained both microwave and traditional sections from the same fluid. The reviewers blindly compared the extent and intensity of the staining (1 - 3 scale) for each section. The results were averaged among the three reviewers and compared across the two processing methods and more specifically within the different formalin fixation time periods, using the Chi Square method.

Results: The average nuclear, cytoplasmic, cell membrane, and total cytomorphological scores demonstrated no significant differences between the processing methods and the fixation times in any of the categories (P > 0.6) [Table 1]. Reviewers correctly identified the processing method and the majority of the time, ranging from 60 to 73%. Notably, the mesothelial cells in the microwave blocks demonstrated a fine nuclear chromatin pattern more often than a hyperchromatic one (52% vs. 18%); the reverse held true for traditionally processed blocks (45% vs. 32%) [Figure 1]. Retrospectively, this was noted to be a helpful feature utilized by the reviewers when predicting the fixation method. There was also concordance among the reviewers with regard to the extent and intensity of immunohistochemical staining between the two processing methods. Immunostaining intensity for CK7 was consistently 3+, regardless of the processing method and formalin fixation time. MOC-31 and calretinin staining intensity results are demonstrated in [Figure 2] and [Figure 3].

Conclusions: 1) Overall, the total cytomorphological scores are similar, regardless of the processing method or formalin fixation time. 2) Microwave processing and formalin fixation time do not affect the immunohistochemical staining extent for CK7, MOC31, and calretinin. Staining intensity for CK7 and MOC-31 is unaffected and calretinin shows an 89% concordance rate between the two processing methods. 3) Microwave processed cell blocks tend to have mesothelial cells with finer chromatin instead of a hyperchromatic pattern, regardless of the fixation time. 4) Rapid-microwave processing can replace traditional processing methods as a viable option to make 'same-day processing' a reality for cytology and improve patient care.


Prospective use of glacial acetic acid in a grossly bloody Pap test: Reduction in hologic ThinPrep® rate of unsatisfactory specimens

Vickie Johncox, CT(ASCP), Vijayalakshmi Padmanabhan, MBBS, MD

Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire

Introduction: Presence of blood, lubricant, mucous or inflammation can contribute to an unsatisfactory (unsat) Pap test (PT). When we recently changed vendors for Pap tests from BD SurePath® to Hologic ThinPrep® , one of our prime concerns was the unsat rate. The unsat rate for ThinPrep® has been reported at 1.2%; while with BD SurePath® our rate was 0.51%. Scant squamous cellularity in the presence of abundant fresh blood has been reported with ThinPrep® specimens. It has been postulated that excess blood in ThinPrep® samples may cause obstruction of membrane filters competing for the capture of epithelial cells producing a scantily cellular smear. Reprocessing with glacial acetic acid (GAA) for an unsat PT, a process that takes time, has been approved for improving adequacy. The aim of this study was to study the prospective use of GAA in grossly bloody specimens and incorporate this in the flow of the laboratory.

Materials and Methods: All grossly bloody samples accessioned in the laboratory between 8 March, 2011 and 28 April, 2011, were processed using Hologic's ThinPrep® Pap Test protocol and Hologic's reprocessing protocol using 10% glacial acetic acid, without and with the addition of GAA. Both slides were reviewed by the cytotechnologists who signed out the normal cases, while the pathologist signed out the abnormal cases. All the cases were then reviewed by the pathologist and the slides were assessed for cellularity, cellular detail, presence of obscuring blood and abnormality. The overall unsat rate during the study period and the cause for an unsat PT, and the unsat rate for the previous 12 months were determined. An attempt was made to correlate them with the HPV test results (Invader assay performed without addition of GAA) and the histological follow-up. In addition, five known HPV positive and five known HPV negative cases were treated with GAA and reassessed using the Invader assay.

Results: Eighty cases were grossly bloody and were processed as two slides, with and without GAA, during the study period. There were 71 NILM, one other, three ASCUS, one ASCH, one AGUS, three LSIL, and no HSIL. One case was HPV positive and two were HPV negative. The unsat rate between May 2010 and April 2011, since the change in PT platform, had increased to 0.91%. Many of these were attributable to the use of a lubricant. Over the last six months the unsat rate was 0.67%. During the study period, the unsat rate reduced to 0.32%. When the two preparations were compared uniformly, all the slides with GAA showed a marked increase in cellularity, absence of obscuring blood, and improved cellular details.

Conclusions: GAA is useful in improving the cellularity of the bloody specimens. As it is considered 'off-label' to perform a GAA prep without first obtaining an UNSAT result using the Hologic's ThinPrep® Pap test protocol, it was determined that a GAA preparation was equivalent to Hologic's standard Pap Test protocol in this study. Although some of these specimens without GAA may not have been strictly considered unsat, addition of GAA vastly increased the cellularity, improved the cellular detail, and reduced the obscuring blood. Specimens with abundant blood could potentially be from patients with glandular lesions and may benefit from this process and manual screening instead of image-guided screening where they may be missed. Using GAA on grossly bloody specimens saves time upfront and can easily be incorporated into the work flow of the laboratory.

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Role of cytology in classifying lung adenocarcinoma and its various subtypes: A five-year retrospective review with emphasis on cytomorphology

Israh Akhtar, MD, Anwer Siddiqi, MD, Rhyne Flowers, MD, Ric Bowlin, SCT, Mithra Baliga, MD

Department of Cytopathology, University of Mississippi Medical Center, Jackson, Mississippi

Introduction: Cytological techniques are of utmost importance in the diagnoses of lung cancer, which is the leading cause of cancer deaths in the United States for both men and women. Adenocarcinoma, arising from the bronchial mucosal glands, is the most frequent non-small cell cancer, representing 35 - 40% of all primary lung cancers. The pulmonologists, radiologists, and internists in conjunction with the cytopathologists are the key players in establishing the diagnoses and subtyping these lesions.

Materials and Methods: A retrospective study was conducted, wherein we reviewed the cytology database for adenocarcinomas of lung for the last five years (September 2005 - August 2010). The specimens included bronchial washes, bronchial brushes, bronchoalveolar lavage, transbronchial fine needle aspiration biopsy (FNAB), endobronchial ultrasound-guided FNAB (EBUS), pleural fluid cytology, image-guided transthoracic FNAB, core biopsy, and core imprints.

Results: A total of 146 cases of adenocarcinomas were diagnosed by various cytological methods. The cases included both primary lung as well as metastatic adenocarcinomas, including 74 male and 72 female patients, with an age range of 32 to 87 years. Lung adenocarcinoma in this study was observed most commonly between the fifth and seventh decades, with an increased incidence in the sixth decade (36.9%). Conventional adenocarcinoma was the most commonly diagnosed subtype (n = 112, i.e., 76.7%). The remaining cases included three bronchoalveolar carcinomas (BAC), mucinous subtype (2.05%), two BAC non-mucinous subtypes (1.3%), three adenocarcinomas with papillary configuration and psammoma bodies (2.05%), one signet ring cell type adenocarcinoma (0.68%), and one mucinous adenocarcinoma (0.68%). Twenty-four cases (16.4%) were metastatic adenocarcinomas from various body sites; colon (n = 10), breast (n = 8), kidney (n = 5), and endometrium (n = 1). The diagnosis of metastatic tumors was confirmed by a clinical workup and immunohistochemistry. On the basis of cytomorphology the conventional subtype, was graded into well, moderate, and poorly differentiated, in most cases.

Conclusions: Primary pulmonary adenocarcinomas may display a wide variety of cytomorphological patterns, which can be difficult to recognize in a limited sample. Familiarity with these unusual patterns, in conjunction with ancillary studies, such as immunohistochemistry, is the frontier for diagnosing and prognosticating subtypes of lung adenocarcinomas. The cytological mode of diagnoses has not only proven to be cost-effective and rapid, but has reduced the morbidity resulting from open biopsies.


Variability in the diagnostic yield of endobronchial ultrasound-guided transbronchial fine needle aspiration biopsy

Dustin Woods, MD, Charles Beavers, MD, Sunati Sahoo, MD

Department of Pathology and Laboratory Medicine, University of Louisville Health Sciences Center, Louisville, Kentucky

Introduction: EBUS-FNA is a minimally invasive procedure that has been increasingly used by clinicians for the diagnosis and staging of lung carcinoma. Over the past four years, the frequency of EBUS-FNA has been increasing at our institution. The purpose of this study was to examine the utility and diagnostic yield of this procedure.

Materials and Methods: A computer database search was performed at the University of Louisville Hospital for malignancy on EBUS-FNA cases, between January 2006 and October 2010. A total of 124 cases were retrieved and reviewed for FNA sites and diagnosis.

Results: FNA material from 156 sites was obtained on 124 patients using EBUS-FNA, at the University of Louisville, from 2006 to 2010. The total number of FNA sites were as follows: Lung masses 63, subcarinal nodes 46, paratracheal nodes 27, and peribronchial nodes 20. Of 156 FNAs, 40 were interpreted as non-diagnostic (25.6%). The non-diagnostic rate for lung masses was 17.5% (11 / 63). The non-diagnostic rates for lymph node sites were as follows: Peribronchial 25.0% (5 / 20), subcarinal 32.6% (15 / 46), and paratracheal 33.3% (9 / 27). The number of EBUS-FNAs and the overall non-diagnostic rate from 2006 to 2010 were as follows: 2006, 3 / 10 cases (30%); 2007, 3 / 13 cases (23%); 2008, 13 / 39 cases (33.3%); 2009, 16 / 64 cases (25%); and 2010, 5 / 30 cases (16.7%).

Conclusions: The diagnostic yield of EBUS-FNA was improved significantly as the operator gained more experience. At our institution, the yield of diagnostic material from lung masses was higher than that of lymph nodes.


Rapid onsite evaluation of endobronchial ultrasound-guided fine needle aspiration: Correlation between adequacy assessment and final diagnosis in patients with bronchogenic carcinoma

Kathryn Dyhdalo, MD 1 , Dawn Underwood, MS, CT(ASCP) 1 , Julie Shorie, CT(ASCP) 1 , Christine Booth, MD 1 , Peter Mazzone, MD 2 , Jennifer Brainard, MD 1

1 Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio; 2 Pulmonary and Critical Care Medicine, Cleveland Clinic, Cleveland, Ohio

Introduction: Endobronchial Ultrasound-Guided Fine Needle Aspiration (EBUS FNA) is a non-invasive method for diagnosis and staging of lung carcinoma, which utilizes real-time optical and ultrasound images to guide the biopsy procedure. Rapid onsite evaluation (ROSE) of FNA samples has been shown to increase the diagnostic yield. There are relatively few reports in the literature that specifically address discordant results between on site adequacy assessments and the final diagnoses. We decided to study our EBUS samples and investigate cases with discrepancies between rapid and final diagnoses in patients with bronchogenic carcinoma.

Materials and Methods: We reviewed all EBUS FNAs over a two-year period (January 2009 to December 2010). In all cases, the slides used for ROSE were stained with diff quik stain (DQ). Pap stained smears, ThinPreps® , and / or cell blocks were reviewed before a final diagnosis was rendered. We compared onsite adequacy assessments with the final diagnoses. If the interpretation at the time of ROSE agreed with the final diagnosis, the aspirate was classified as concordant. If the initial interpretation differed significantly from the final diagnosis, based on a review of all available material, then the case was classified as discordant. All available slides from discordant aspirates were reviewed and re-screened. The reason for the discordant result was categorized as either sampling or an interpretive / screening error at the time of ROSE. In discordant cases due to sampling, the location of the diagnostic material was noted.

Results: There were 340 EBUS procedures performed in 335 patients (168 men, 167 women, median age 65 years) with 575 sites sampled during the study period. A diagnostic discrepancy between ROSE and final diagnosis occurred in 138 (24%) total aspirates, including 65 (11%) aspirates from 53 patients with bronchogenic carcinoma. The remaining aspirates were concordant, including all cases called positive for carcinoma at the time of ROSE. The distribution of diagnoses in discrepant cases is summarized in [Table 1]. Of the 65 discrepant cases, 52 (80%) were positive for carcinoma on final review. All slides were available for re-screening in 60 discordant aspirates, including 47 with a final positive diagnosis. Re-screening of DQ stained slides in cases with a final positive diagnosis showed either no tumor cells or insufficient tumor cells for diagnosis in 28 (60%) cases. The location of the diagnostic cells in these discrepant cases due to sampling is summarized in [Table 2]. The remaining 19 (40%) cases were classified as interpretive or screening errors at the time of ROSE. The majority of these errors occurred in aspirates called atypical or atypical suspicious, which upon re-screening were considered diagnostic of carcinoma (16 aspirates, 84%).

Conclusions: In our study, EBUS adequacy assessments and final diagnoses were concordant in 89% of the aspirates from patients with bronchogenic carcinoma. All aspirates with a positive interpretation at ROSE were concordant. in the discordant cases, all aspirates with 'atypical cells suspicious for malignancy' and 86% of aspirates with 'atypical cells' on ROSE had a final diagnosis of carcinoma. The majority of discordant cases with a positive final diagnosis were due to sampling (60%). In cases lacking tumor on DQ slides, the tumor was most commonly identified in the ThinPrep® . In cases with insufficient tumor cells for diagnosis on DQ, both the ThinPrep® slide and Pap stained smears were helpful diagnostically.


Multinucleated histiocytes in Fine Needle Aspirations of pulmonary hamartomas: A new morphological finding

Cherie Paquette, MD 1 , Gladwyn Leiman, MD1 , Pam Michelow, MD 2 , Sara Brownschidle, MD 1

1 Cytopathology, Fletcher Allen Health Care and University of Vermont, Burlington, Vermont; 2 Cytopathology, NHLS, University of the Witwatersrand, Johannesburg, South Africa

Introduction: Pulmonary hamartomas (PH) are among the most frequently encountered benign neoplasms of the lung. Commonly presenting as solitary lung nodules, peripheral hamartomas outnumber endobronchial variants by 9 : 1. The radiological diagnosis is specific in most cases; no further diagnostic intervention may occur, the PH being left in-situ under surveillance or resected, as dictated clinically. In cases with inconclusive radiology, fine needle aspiration (FNA) may be undertaken. The cytodiagnostic features of PH are well-described, resting primarily on recognition of the mesenchymal elements (cartilage and fibromyxoid matrix) admixed with benign bronchial cells and pneumocytes. In a recent case with otherwise characteristic features of a PH, numerous giant multinucleated histiocytes (MNHs) were observed. The literature review yielded no mention of this finding. This prompted a retrospective review of cases previously diagnosed as PH, specifically to determine whether the cytological presentation of PH may include MNHs.

Materials and Methods: Archived FNA slides diagnosed as PH by the above-described criteria were retrieved from the files of two institutions and rescreened to confirm the original cytodiagnoses, to ensure that no other pathology (particularly granulomatous) was missed, and they were specifically assessed for MNHs. Inclusion criteria required that MNHs be at least 3x the size of pneumocytes, display 3+ separately identifiable nuclei, and contain no carbon particles. We documented the number of MNHs per slide with the highest count: Findings were grouped as 1 - 4 (rare), 5 - 9 (moderate numbers) or greater than 10 (numerous) MNHs. The MNHs were further subclassified by size: 20 - 60 (regular) or greater than 60 microns (giant). The selected cases were subjected to immunochemical stains to confirm the histiocytic phenotype.

Results: Aspirates of 31 FNA-diagnosed PH from two institutions were retrieved. All cases were confirmed as hamartomas on review; there was no instance in which other pathology, particularly granulomatous, had been overlooked. Of the total of 31 cases, 30 (65%) were found to contain MNHs. MNHs were few in eight cases, moderate in a further eight cases, and numerous in the remaining four cases (quantified as defined earlier); the average number of MNHs per case was eight. The average MNH size was approximately 40 to 50 microns. However, in approximately 25% of the cases with MNHs, giant MNHs (greater than 60 microns) were a prominent and readily identifiable component on the slides reviewed [Figure 1]. Immunochemical stains confirmed that the cells were of the histiocytic type [Figure 2].

Conclusions: Reliable criteria for FNA diagnosis of PH have been well-described. To our knowledge, MNHs have not been mentioned as part of the spectrum of FNA morphology of these lesions. This study indeed shows that in the majority of cases, MNHs are either rare or of regular size, with insufficient prominence to be noted as conspicuous. Nevertheless, when specifically sought, MNHs were found in 65% of the 31 FNAs from PH. In approximately 25% of the positive cases, the MNHs may appear sufficiently large or numerous to impact the diagnosis. In cases with classic stromal representation, the diagnosis of PH would not be in doubt; however, in suboptimally aspirated nodules, the finding of giant MNHs might lead to confusion with granulomatous disease. This study identifies and confirms MNHs as within the range of features seen in FNAs from PH.


The performance of ThinPrep® respiratory cytology in the diagnosis of pulmonary small cell carcinoma

Cheng Wang, MD 1 , Qiuli Duan, MSc 2 , Margaret Kelly, MBChB, PhD, FCPath(SA), FRCPC 1 , Maire Duggan, MSc 1

1 Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada; 2 Clinical Trials Unit, Tom Baker Cancer Center, Calgary, Alberta, Canada

Introduction: Calgary Laboratory Services (CLS) is the sole provider of respiratory cytology to nearly 1.3 M residents in the Calgary Health Region (CHR) and the cytology, since 1996, is prepared using the ThinPrep® method. This provided an opportunity to carry out the largest study on the sensitivity, specificity, and the positive (PPV) and negative (NPV) predictive values of ThinPrep® prepared bronchial wash and brush specimens in the diagnosis of pulmonary small cell carcinoma (SCCa).

Materials and Methods: All patients in the CHR with a diagnosis of SCCa between 2000 and 2004 were identified from the provincial cancer registry. The laboratory information system (LIS) was searched for all respiratory cytology performed in that interval. Bronchial wash and brush specimens of SCCa patients and those negative for lung cancer of any other type were identified. Cytology diagnoses of SCCa or suspicious for SCCa were considered test positive and all other cytology results were test negative. Unsatisfactory results were excluded. True positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) were classified using the cancer registration as the gold standard.

Results: Of the 309 patients registered as SCCa, 199 (64.4%) had respiratory cytology. There were 129 brush and 170 wash specimens. There were 1,762 respiratory cytology (409 brush and 1,283 wash) specimens on 1,122 patients who were negative for lung cancer. Nearly 50% of the 538 brush and 1,453 wash specimens were diagnosed by five pathologists who did not have prior experience with ThinPrep® cytology. Among the 527 satisfactory brush (1, 440 wash) specimens, there were 78 (90) TP, two (14) FP, 48 (79) FN, and 399 (1,257) TN results [Table 1]. Differences in sensitivity and specificity between brush and wash specimens were not significant, but differences in PPV and NPV were (P = 0.009 and 0.0006, respectively). When the results were stratified by the year of diagnosis, the bronchial brush sensitivity increased from 51.0% in 2000 - 2001 to 68.8% in 2002 - 2004, and the difference was significant (P = 0.04)

Conclusions: ThinPrep® prepared bronchial wash and brush cytology has moderate sensitivity, excellent specificity, PPV and NPV, in the diagnosis of SCCa. The bronchial brush is more sensitive than the wash, and has the potential to increase further with the pathologists' experience.


Use of mutation-specific antibodies to detect epidermal growth factor receptor status in small biopsy and cytology specimens of lung adenocarcinoma

Adnan Hasanovic, MD, Andre Moreira, MD, PhD

Pathology, Memorial Sloan-Kettering Cancer Center, New York

Introduction: The epidermal growth factor receptor (EGFR) mutation status is the best predictor of response to tyrosine kinase inhibitors in adenocarcinoma of the lung. Approximately 70% of lung cancers are diagnosed in advanced stages, where small biopsies and cytological specimens are the only source of material for both diagnosis and mutation testing. In recent times, antibodies that can detect mutant EGFR protein have been introduced. We have evaluated the detection of the EGFR mutant protein by immunohistochemistry in small biopsy and cytology specimens of lung adenocarcinoma.

Materials and Methods: Forty-six cases of adenocarcinoma were analyzed (28 cytology cell blocks, 10 lung core biopsies, and 8 bone biopsies). Antibodies against the exon 19 deletion (15 bp deletion) and the L858R mutation were used under optimized conditions (Cell Signaling Technology). Stains were scored as negative (weak and focal staining in < 10% of tumor cells), 1+ (faint staining in > 10% of tumor cells), 2+ (moderate staining intensity), and 3+ (strong and diffuse staining). All the cases were analyzed by the standard molecular methods to detect EGFR mutations.

Results: Deletions in exon 19 were detected in 22% (4% cytology specimens, 30% core biopsies, and 67% bone biopsies) and L858R mutation in 11% (11% cytology specimens, 10% core biopsies, and 11% bone biopsies) of the cases. Using a cutoff of 2+ as positive, the positive predictive value (PPV) for exon 19 deletion and L858R mutation was 100%, as no false positive cases were detected. The negative predictive values (NPV) were 83 and 92%, respectively, which reflected the rate of false negative results. When a staining score of 1+ was considered positive, the PPV for exon 19 deletion and for L858R mutation was 76 and 86%, respectively. The drop in PPV was a result of the increased number of false positive cases.

Conclusions: Immunostains for specific mutant EGFR show a good correlation with mutation analysis and can be used as a screening method to identify patients for tyrosine kinase inhibition therapy. Cases with weak positivity (1+) should be considered equivocal due to the risk of false positive results. This methodology is potentially useful in areas where molecular analysis is not available and for use in small biopsies when the material is too scant for molecular analysis or in bone biopsies where the decalcification process interferes with the DNA quality.


Fine needle aspiration as a predictor of squamous cell carcinoma and adenocarcinoma subtypes of non-small cell lung carcinoma

Seema Lale, MD, Nora Morgenstern, MD, Daniel Soto, CT(ASCP), Chiara Sugrue, MS, SCT(ASCP), Patricia Wasserman, MD, FCAP

Pathology, North Shore Long Island Jewish Health System, Lake Success, New York

Introduction: Recent changes in therapeutic modalities, such as, targeted therapy with epidermal growth factor receptor (EGFR) inhibitors, have increased the need for a sub-classification of non-small cell carcinomas on both biopsy and cytology specimens. Recent literature has demonstrated the reliability of cytological diagnosis of lung cancer by fine needle aspiration (FNA). The majority of lung cancers are not resectable at the time of presentation. The diagnosis and treatment decisions are made with increasing use of transbronchial and endobronchial ultrasound-guided needle (EBUS) aspirations. The aim of this study is to measure the accuracy of cytology in distinguishing squamous cell carcinoma (SCC) and adenocarcinoma (ADC) of the lung when compared to tissue diagnosis, and to assess the contribution of immunocytochemistry to this differential.

Materials and Methods: We retrospectively reviewed 613 cases of lung FNA from 2005 to 2010. Of these, 123 patients had FNA diagnosis of ADC, SCC, and Non-small cell lung carcinoma not otherwise specified (NSCLC - NOS), with confirmatory tissue diagnosis. Small cell carcinomas, neuroendocrine tumors, and metastatic carcinomas were excluded from this study. The immunocytochemical stains for TTF-1, p63, and CK5 / 6 were performed on 62 cases. The cytology results were correlated with the histological diagnosis.

Results: Forty-six percent (57 / 123) of the surgical specimens were diagnosed as ADC, 39% (48 / 123) as SCC, and 15% (18 / 123) as NSCLC-NOS. A specific cytological diagnosis was rendered in 81% (100 / 123) of the cases. The cytology cases were accurately classified based only on cytomorphological features in 75% (43 / 57) of ADC, 83% (40 / 48) of SCC, and 100% of (18 / 18) NSCLC-NOS. In 17 cases the initial cytology was NSCLC, but immunocytochemistry permitted sub-classification into adenocarcinoma in (11 / 57) 19% and squamous carcinomas in (6 / 48) 12%. Based on cytomorphology alone, three SCCs by cytology had subsequent tissue diagnosis of ADC, confirmed by immunohistochemistry. Conversely, two ADCs by cytology had subsequent tissue diagnosis of SCC confirmed by immunohistochemistry.

Conclusions: The distinction between adenocarcinoma and squamous carcinoma was accurate based on cytomorphology alone in 75 and 83% of the cases, respectively. Immunocytochemistry was needed in 19 and 12% of the cases. Cytomorphology alone was inaccurate in (3 / 57) 5% of the ADC and in (2 / 48) 4% of the SCC. These findings highlight the importance of immunocytochemical confirmation on both cytology and histology in cases of poorly differentiated ADC and non-keratinizing SCC. This study also supports the cytology-based sub-classification of NSCLC with immunocytochemical aid for diagnosis and treatment decisions.


Subclassification of NSCLC / carcinoma on 240 FNA specimens: Can p63 and napsin A be used as a minimal immunohistochemical panel in addition to TTF?

Grzegorz Gurda, MD, PhD 1 , Lei Zhang, MD 2 , Syed Ali, MD 1 , Frederic Askin, MD 1 , Edward Gabrielson, MD 1 , Qing Li, MD, PhD 1

1 Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland; 2 Pathology, The University of Hawaii, Honolulu, Hawaii

Introduction: Over 60% of non-small cell lung cancer (NSCLC) patients present with a locally advanced or metastatic (stage III or IV) disease at the time of diagnosis. Current treatments for NSCLC have advanced to the point at which further sub-classification of NSCLC into squamous cell carcinoma (SQC) and non-SQC, such as adenocarcinoma (ADC) and others, is necessary. Fine needle aspiration (FNA) cytology plays a critical role in the diagnosis and staging of lung cancer patients; the distinction between SQC and ADC, however, may be difficult on FNA specimens, due to the scant material. Although several studies have investigated the utility of immunohistiochemical (IHC) markers in the sub-classification of NSCLC, most are based on surgical or biopsy material and often with a limited number of cases. The study of using a minimal panel of markers for classification of cytological samples is still suboptimal. Our previous study has shown that Napsin A and TTF form a useful panel for identifying lung ADC. In this study, we have retrospectively assessed the clinical utility of p63 in the sub-classification of NSCLC into SQC and ADC on FNA specimens, in addition to Napsin A and TTF.

Materials and Methods: A computer search of a major teaching hospital identified 240 FNA cytological cases of 'NSCLC or carcinoma with IHC studies' over a seven-year period (2004 - March 2011). The cases consisted of lung primaries (n = 74), metastases of a lung primary (n = 61), as well as other malignancies of non-lung primary (n = 105). Among 240 cases, 125 cases (52%) had a surgical follow-up.

Results: The p63 staining of 135 lung cancers is summarized in [Table 1]. In SQC, p63 was positive in 87% (40 / 46) and negative in 13% (6 / 46) of the cases, while, in non-SQC, p63 was positive in 21% (19 / 89) and negative in 79% (70 / 89) of the cases. Particularly, p63 was positive in 19% (8 / 43) and negative in 81% (35 / 43) of the lung ADC cases. The overall sensitivity and specificity of p63 were 87 and 78%, respectively. The addition of Napsin A in 2008 reduced the unclassified NSCLC from 20% (5 / 25 cases) to 9% (10 / 108 cases), with an increased diagnostic rate of ADC. The combination of p63 and Napsin A showed a better sensitivity and specificity of 94 and 88%, respectively. The staining pattern of p63 in 105 non-lung carcinomas is summarized in [Table 2]; and it reveals a sensitivity and specificity of 91% and 77%, respectively, for SQC, as urothelial carcinoma was not included. The correlation between the cytological FNA specimen and surgical pathology was 95%.

Conclusions: Our data demonstrated that p63 and Napsin A can be efficiently used in the sub-classification of NSCLC in addition to TTF. Both false positive and negative cases were identified in our study. The combination of p63 and Napsin A showed a better sensitivity and specificity than p63 alone. Therefore, from a practical standpoint, p63, Napsin A, and TTF may be used as a minimal panel of IHC markers.


Retrospective review of bronchial brushing specimens from peripheral lung nodules utilizing morphology and fluorescence in-situ hybridization characteristics

Trynda Oberg, MS, CT(ASCP), Jesse Voss, CT(ASCP), Justin Cassett, Mark Vande Haar, Jordan Reynolds, MD, Kevin Halling, MD, PhD, Amy Clayton, MD, Michael Henry, MD

Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Introduction: The sensitivity of routine cytology is suboptimal due to sampling difficulties of peripheral lung nodules, and fluorescence in-situ hybridization (FISH) assays have been shown to increase the detection of these lesions. Since 2006, our laboratory has offered a FISH reflex assay for the detection of lung cancer using residual bronchial brushing (BB) cytology specimens. The goal of this study is to determine the value of FISH for detecting peripheral lung neoplasms with equivocal BB cases and to evaluate the potential cytological criteria indicative of carcinoma in these specimens.

Materials and Methods: The medical records were searched for equivocal (atypical or suspicious) BB specimens from peripheral lung nodules that had reflex FISH testing and clinical follow-up information. FISH was called positive when ≥ 5 hypertetrasomy (polysomy) cells (≥ 3 signals for ≥ 2 probes, with at least one of the probes showing five signals) were present. The gold-standard was determined by using the pathological and / or radiological evidence of a lung neoplasm. The BB slides were re-screened by the cytotechology students and a cytopathology Fellow without knowledge of the clinical outcome, and each case was evaluated for numerous cytological criteria in the most abnormal appearing cells on the slides. Positive (n = 5) and negative (n = 5) control cases were reviewed, but not included for data analysis.

Results: From June 2006 through January 2011, 37 BB samples from 36 patients met the study criteria for inclusion. Patient demographics included 17 females and 19 males with a mean age of 71.6 years (range 49 - 87 years). Lung malignancy was found in 10 of 21 (48%) atypical cytology specimens and 14 of 16 (88%) suspicious cytology specimens. Cancer subtypes included: Adenocarcinoma (n = 8), typical carcinoid (n = 1), non-small cell carcinoma (n = 3), squamous cell carcinoma (SCC) (n = 8), and small cell carcinoma (SCLC) (n = 4). Polysomy FISH results were present in 13 specimens (n = 7 atypical, n = 6 suspicious) and all were found to have cancer on follow-up. Conversely, 46% (11 / 24) of the specimens diagnosed as negative by FISH were found to have lung cancer. Hyperchromasia in the majority of abnormal cells was identified in significantly more patients with cancer (63 vs. 15%; p = 0.006). Absent nucleoli were found significantly more often in patients with cancer (25 vs. 0%; p = 0.049) and we observed that these six cases were SCC (n = 5) and SCLC (n = 1). Hypochromasia was identified in the majority of abnormal cells in patients without cancer (77 vs. 33%; p = 0.011). Notably, all five cases (100%) with gland-like architecture and 11 / 14 (79%) with architecture of single abnormal cells, had cancer. Interestingly, 12 / 18 cases (67%) with abnormal single cells containing cilia / terminal bars present were found to have cancer on follow-up. All four cases (100%) with cells present, with nuclei four to five times the normal size, had cancer.

Conclusions: In our small pilot study, in the BB specimens diagnosed as equivocal, we did not find clear-cut cytological criteria indicative of cancer; however several appeared to be helpful. The numerous types of cancers might partially explain why the distinct cytological features were lacking. All patients with a polysomy FISH result had a lung neoplasm; FISH might be a useful tool in determining which patients with a peripheral lung nodule and an equivocal cytology result were most likely to have cancer on follow-up.


Utility of cell block preparations in endobronchial ultrasound-guided transbronchial-needle aspirates

Arbaz Samad, MD 1 , Charanjeet Singh, MD 1 , David Tasso, MD 1 , Tetyana Mettler, MD 1 , Rafael Andrade, MD 2 , Stefan Pambuccian, MD 1

1 Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota; 2 Surgery, University of Minnesota, Minneapolis, Minnesota

Introduction: Endobronchial ultrasound-guided transbronchial-needle aspirates (EBUS-TBNA) is a novel, minimally invasive technique used for staging of patients with lung cancer and the diagnosis of accessible mediastinal lymphadenopathies or masses. The aim of this study was to determine the diagnostic utility of Cell block preparations (CBP) in addition to smears in the EBUS-TBNA samples.

Materials and Methods: We recorded specimen adequacy, cytological diagnoses, adequacy of CBP, and the use of immunoperoxidase stains (IPS) and special stains (SS), FISH, and molecular studies on CBP, in the EBUS-TBNA performed between January, 2008 and April, 2011. EBUS-TBNA specimens were procured by thoracic surgeons in the operating room. Preparation of slides, triage of the specimens, and rapid onsite evaluation (ROSE) was performed by a cytopathologist. After air-dried and alcohol-fixed smears were made, the residual specimen was placed in 10% buffered formalin for CBP. When ROSE was suspicious for lymphoma or infection, part of the residual material was submitted for flow cytometry or culture. IPS or SS were ordered immediately based on the ROSE impression; additional stains were ordered as needed. Cell blocks were prepared by the HistoGel method and sections were stained with H and E.

Results: During the study period, 244 patients (M = 131 / F = 113) aged 15 to 89 (mean 61) years, underwent EBUS-TBNA. One to five (mean 2) nodal and / or pulmonary sites were sampled for a total of 480 individual specimens. In 88 specimens (18.3%) the EBUS-TBNA result was 'inadequate' based on the lack of sufficient lymphoid cells in the absence of granulomas or neoplastic cells. Of the remaining 392 specimens that were considered to be diagnostic, 40 (10.2%) had inadequate CBP cellularity. In these cases, the diagnosis was based on smears alone, and it was 'malignant' in 17 cases and 'negative for malignancy' in 23 (granulomas, n = 14 and reactive lymph node, n = 9). The remaining 352 specimens had adequate CBP. [Table 1] shows the differences in diagnosis between the two groups in the 'non-negative' cases. Of the 73 cases of nonsmall-cell lung carcinomas (NSCLC) (adenocarcinomas (ADCA), n = 44, squamous cell carcinoma (SqCC), n = 25, and NSCLC, NOS, n = 4), 48 (66%) were diagnosed based on their characteristic morphology on smears and CBP and comparison with the previous histology (when available), while in the remaining 25 cases (34%), a panel of IPS (CK5 / 6, CK7, TTF1, and p63) was used, resulting in a diagnosis of ADCA in 19 and SqCC in two. Four cases could not be classified as ADCA or SqCC and were diagnosed as NSCLC, NOS. Of the cases with adequate CBP, 30 patients had granulomas: AFB / GMS stains were successfully performed in all cases and were negative in all but three cases, which showed fungal organisms consistent with Histoplasma. Molecular studies for EGFR, and in some cases also for KRAS and EML4-ALK were performed in 22 adenocarcinomas; 20 were successful and showed EGFR mutations in two. In one case of metastatic Ewing sarcoma, FISH was positive for the EWSR1 translocation.

Conclusions: Whenever possible, CBP should be made from EBUS-TBNA material to perform IPS, SS, and molecular studies because they allow:

  1. frequent, specific, and definitive diagnoses (no 'atypical' diagnoses)
  2. of NSCLC and their nodal metastases to allow accurate therapeutic decisions
  3. of infectious agents in the majority of cases with granulomas
  4. of molecular studies for EGFR, KRAS, and EML4-ALK in cases of adenocarcinoma, allowing personalized treatment options


Evaluation of the effectiveness of CT-FNA cytology in stage I lung cancer

Melissa Paoni, CT(ASCP) 1 , Mary-Beth Beasley, MD 1 , David Burstein, MD 1 , Matthew Cham, MD 2 , David Yankelevitz, MD 2 , Maoxin Wu, MD, PhD 1

1 Anatomic Pathology, The Mount Sinai School of Medicine, New York, New York; 2 Radiology Associates, The Mount Sinai School of Medicine, New York, New York

Introduction: Lung carcinoma accounts for the most cancer-caused deaths in both men and women. CT-guided fine needle aspiration (CT-FNA) biopsy cytology of small lung lesions has become crucial in the early diagnosis and management of lung carcinomas. The objective of this study is to evaluate the effectiveness of CT-FNA cytology in diagnosing stage I lung cancer (< 3 cm).

Materials and Methods: In the period from 16 February 2010 to 29 March, 2011, 312 CT-FNA lung biopsies were performed at the Mount Sinai School of Medicine. There was 138 cases with recorded tumor sizes of 3.0 cm or less, based on CT. These cases were cytologically classified as 'Malignant, Suspicious for Malignancy, Neoplastic, Atypical, and Benign.' Statistical analysis was performed for the cases with surgical follow-up (SFU) using surgical diagnosis as the gold standard.

Results: Among the 138 cases [Table 1], there were 86 (62%) malignant, 14 (10%) suspicious, 5 (4%) neoplastic, 18 (13%) atypical, and 15 (11%) benign. There were 45 cases that had surgical correlations including 29 (64%) malignant, six (13%) suspicious, two (4%) neoplastic, six (13%) atypical, and two (4%) benign cases. As shown in [Table 2], all 39 cases within malignant, suspicious, neoplastic, and benign groups show virtual agreement with the surgical diagnoses and thus achieve 100% sensitivity, 100% specificity, 100% positive predictive value (PPV), 100% negative predictive value (NPV), and 100% accuracy. The rest of the six atypical cases [Table 3] showed three (50%) carcinomas and three (50%) non-neoplastic lesions.

Conclusions: The results suggest that CT-FNA cytology is a sensitive and specific diagnostic modality for stage I lung cancer with great positive and negative predictive values and great accuracy in the majority of cases except a small percentage of atypical cases.


The accurate subtyping of lung carcinoma on cytology specimens: A cytomorphological and immunohistochemical comparison

Scott Lauer, MD, Momin Siddiqui, MD, Cynthia Cohen, MD, Michelle Reid, MBBS

Pathology, Emory University School of Medicine, Atlanta, Georgia

Introduction: Accurate subtyping of non-small cell lung carcinoma into adenocarcinoma (AD) and squamous cell carcinoma (SQC) has become critical because of the emerging histology-specific therapies. However, while drugs such as Bevacizumab (Avastin) are effective in the treatment of lung AD, the same drug may cause life-threatening pulmonary hemorrhage in patients with SQC of the lung. Therefore, accurate cytological distinction between SQC and AD is paramount. However, it can be especially difficult for cytopathologists to make this distinction in limited cytology specimens, particularly when the tumors are poorly differentiated. Several immunohistochemical (IHC) stains aid in the distinction of these two subtypes, but with variable results. We compared the accuracy of cytomorphological and IHC diagnosis of lung AD and SQC on fine needle aspiration (FNA) to the gold standard histological (biopsy or resection) specimen.

Materials and Methods: A total of 43 resected or biopsied lung AD (24), SQC (13), sarcomatoid (SAR) (1) and poorly differentiated carcinomas (PDC) (5), all with previous FNA material, were identified. These 43 FNA specimens were submitted for IHC staining with two AD markers (TTF1 and MOC31) and two SQC markers (p63 and cytokeratin 5 [CK5]). IHC staining was considered positive for SQC if both SQC markers were positive. IHC staining was considered positive for AD if at least TTF1 (a lung AD marker) was positive. In addition, the specific cytomorphological features of AD (gland formation, cytoplasmic mucin, and SQC keratin, intercellular bridges) were sought in the cytology material. Based on the cytomorphology and IHC analysis a cytological diagnosis of AD or SQC was rendered and compared to the gold standard histological diagnosis.

Results: The results of the cytomorphological and IHC analysis of 42 of the 43 cytology cases (excluding the single SAR case) are summarized in [Table 1]. The histologically diagnosed SAR carcinoma was also called SAR carcinoma on cytology and was negative for all four IHC stains. After completing the cytomorphological and IHC analysis of all 43 cytology specimens, 14 cases were still unclassifiable as AD or SQC, either by cytomorphology or by IHC analysis. The final resolution of these 14 cases is shown in [Table 2].

Conclusions: When compared to histology, accurate cytologic subtyping of lung carcinomas can be accomplished on FNA material using both cytomorphological and immunohistochemical methods. Immunohistochemistry was surprisingly only slightly superior to cytomorphology in the subtyping of lung AD and SQC. In cases where one method was unable to classify the tumor the other method played a complementary role and resolved the diagnosis in 71% of the cases. Of the five cases that were diagnosed histologically as poorly differentiated carcinoma, cytomorphology or immunohistochemical staining of the cytology material was able to definitely classify four of these as AD or SQC. The combination of cytomorphological and immunohistochemical analysis helped to accurately subtype the majority of lung carcinomas on cytology specimens, including tumors being classified as poorly differentiated carcinoma on histology. In some cases the cytomorphological and immunohistochemical analysis of the cytological material may be superior to histology. Despite the advent of the recent immunohistochemical stains, a prudent cytological practice should combine cytomorphological and immunohistochemical analysis for the accurate classification of these tumors.


Diagnostic accuracy of routine Cytology, cell block preparation and fluorescence in-situ hybridization for the detection of neuroendocrine tumors in bronchial brush specimens

Jordan Reynolds, MD, Jesse Voss, CT(ASCP), Shannon Brankley, CT(ASCP), Jill Caudill, SCT(ASCP), Michael Henry, MD, Amy Clayton, MD, Kevin Halling, MD, Aziza Nassar, MD

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Introduction: Bronchial brush (BB) cytology carries a low sensitivity for detecting neuroendocrine carcinoma (NEC) and typical carcinoid (TC) tumors of the lung. The aim of this study is to investigate the detection of TCs by routine cytology on BB, as well as ancillary testing, including cell block (CB) preparation and fluorescence in-situ hybridization (FISH), using the LaVysion™ probe set.

Materials and Methods: Lung biopsy or resection specimens containing the term 'neuroendocrine' or 'carcinoid' in the diagnosis were searched, from 2008 to 2011. Residual BB specimens retained in the (PreservCyt Hologic, Inc. Bedford, MA) media were retrieved and a ThinPrep® slide (Hologic) was prepared for FISH analysis utilizing probes to pericentromeric regions of 6p11 and the locus-specific regions of 7p12 (EGFR), 8q24 (MYC) (Abbott Molecular Inc., Des Plaines, IL), and 5p15. Slides were screened for chromosomal gains or losses by a technologist. A CB with H and E and IHC for chromogranin and synaptophysin were prepared from the remaining BB specimen. A cytopathologist and a fellow screened the BB slides for tumor cells and atypical features. Patient demographic and clinical follow-up information was collected from the medical records.

Results: From 2008 to 2011, 187 cases (mean age 75) had the diagnosis of NEC or TC. Sixteen had residual BB cytological material within one year of the diagnosis and were used to make 16 FISH and 15 cell blocks for additional analyses [Table 1].

Nine tumors had a central location and seven were peripheral. Most TCs were < 2 cm in size (< 1 cm, n = 6; 1 - 2 cm, n = 6; > 4 cm, n = 4). Three of three primary NEC (1SCLC, 2TC) that were positive by FISH were centrally located with sizes of 3 cm, 1.8 cm, and 2 cm. The SCLC showed polysomy (86% abnormal cells), while the two TCs showed a gain of 7p12 (15% abnormal cells) and a gain of 5q15 (72% abnormal cells). Two of three TC cases positive by FISH (1 SCLC and 1 TC) also had CBs positive for chromogranin and synaptophysin. Cytology diagnosed one case as positive for malignancy (SCLC), one as suspicious for adenocarcinoma, and 14 as negative for malignancy. After a retrospective rescreening of all cases, only one additional TC was diagnosed on BB and it demonstrated medium-sized, bland nuclei, vague rosetting, and evenly distributed chromatin with small nucleoli. This case was positive by FISH and CB. Thirteen of fifteen CBs were negative, showing macrophages and bronchial cells, indicating lack of tumor sampling. For the detection of all NECs by BB sampling, the sensitivities were 6.25% for cytology, 12.5% for CB, and 31.3% for FISH.

Conclusions: TCs are difficult to detect by BB cytology, This is most probably due to lack of tumor cells present in the specimen. Performing CB on all cases detected only one additional TC, while FISH detected three additional cases. Further studies are needed to explore the role of FISH and cytology for the detection of clinically apparent NEC centrally located tumors using bronchial brush specimens.


Clinical characteristic of pediatric population undergoing bronchoalveolar lavage for lipid laden macrophage index

Alice Coogan, MD, Joyce Johnson, MD, Catherine Smith, MD

Pathology, Vanderbilt University Medical Center, Nashville, Tennessee

Introduction: Chronic respiratory symptoms lead to consideration of aspiration in pediatric patients. The lipid-laden macrophage index (LLMI) semi-quantitatively evaluates alveolar macrophage lipid content in bronchoalveolar lavage (BAL) specimens and is used for evaluating chronic aspiration. The majority of LLMI studies are negative. The clinical setting(s) in which this test is most useful has not been established.

Materials and Methods: One hundred sequential pediatric (< 18 years) BAL specimens, from September 2008 to October 2009, were reviewed. Charts were reviewed for age, presenting symptom(s), and underlying disease(s). The Ding modification of the Colombo scale was applied, with a score of 5 considered positive. Patients were categorized into nine clinical groups: Cystic fibrosis (CF), transplant (including solid organ and bone marrow), recurrent pneumonia, airway obstruction, cardiac abnormalities, congenital pulmonary abnormalities, prematurity / failure to thrive (FTT), chronic cough otherwise healthy, and reactive airway disease (RAD) / asthma.

Results: Twenty-three of the 100 studies were positive. The average age of the positive group was 4.3 years; the average of the negative group was 5.8 years [Table 1].

Conclusions: Most BAL samples were from patients with CF or recurrent pneumonias. The average age of positive patients was younger than that of the negative ones. This held true for eight of the nine clinical groups, the exception being chronic cough only. The highest percent of positive studies was in the prematurity / FTT group, followed by RAD / asthma, and recurrent pneumonias. None of the children with congenital pulmonary abnormalities had a positive study. CF patients were unlikely to have a positive test. LLMI may be best suited for identifying increased pulmonary lipid in younger children and those with prematurity / FTT, RAD / asthma, and recurrent pneumonias, and in older, otherwise healthy children with chronic cough.


Cell block examination is critical for sarcoidosis diagnosis by transbronchial ultrasound-guided mediastinal lymph node fine needle aspiration

He Wang, MD, PhD 1 , Akshatha Rao, BS 2 , Anthony Lafranco, MD 2 , Anil Vachani, MD 2 , Andrew Haas, MD, PhD 2 , Prabodh Gupta, MD 1

1 Pathology, Hospital of University of Pennsylvania, Philadelphia, Pennsylvania; 2 Pulmonary Medicine, Hospital of University of Pennsylvania, Philadelphia, Pennsylvania

Introduction: Sarcoidosis, a multiorgan disease, presents with intrathoracic lymphadenopathy in more than 85% of the patients. Transbronchial lung biopsy (TBBx) has been the standard approach for the diagnosis of pulmonary sarcoidosis; however, recent studies suggest that endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is safer, with a superior diagnostic yield. Our center is one of the first to adopt EBUS-TBNA in sarcoidosis diagnosis in North America, with onsite Diff-Quick smear interpretation by cytopathologists, with variable degrees of training and experience, as well as cell block preparation, for all cases.

Materials and Methods: In this study, we report our experience with EBUS-TBNA in a total of 61 consecutive patients (female: 33, 54.1%) from 2008 to 2010, with clinical suspicion of sarcoidosis. They all underwent EBUS-TBNA using an Olympus EBUS-TBNA endoscope. One to three mediastinal lymph nodes (LN) were sampled (max. 5 passes / site) using a 21- or 22-gauge needle. The last one to two FNA specimens / site were collected in Normosol for additional studies including cell block preparations, they were not examined on site.

Results: A definitive diagnosis of sarcoidosis, based on the presence of non-necrotizing granulomas identified by either EBUS-TBNA or TBBx examination, as well as clinical indication, was made in 46 patients (73.7%, 45 patients by aspiration or biopsy, 1 patient by other clinical information); an alternative diagnosis (pneumonia, carcinoma, and lymphoma) was established in 11 patients (18%); the last four patients remained clinically suspicious for sarcoidosis after our diagnostic procedures. Of the 46 sarcoidosis patients, 37 patients were diagnosed with EBUS-FNA (80.4%), 10 out of the 37 patients (27.0%) were diagnosed (7 patients) or suspected (3 patients) onsite. Of the 37 patients diagnosed by EBUS-TBNA, LN sampling was done in one (17 patients, 17 LNs), two (14 patients, 28 LNs) or three (6 patients, 18 LNs) locations. Granulomas were observed in 17 / 17 LNs, 20 / 28 LNs, and 13 / 18 LNs, respectively. In the 46 sarcoidosis patients, the diagnostic yield of LNs at different locations varied dramatically: 50% in R4, 67.9% in the subcarinal, 80% in R11, and 100% in the R12 sites. A total of 34 patients underwent both EBUS-FNA and TBBx, the diagnoses from the two approaches were identical in 22 patients (64.7%); TBBx independently identified sarcoidosis in eight patients, whereas, EBUS-TBNA independently identified sarcoidosis in four patients.

Conclusions: Our study indicates that cell block preparation is valuable for EBUS-FNA diagnosis of sarcoidosis. On site interpretation appears to be experience- and preparation-dependent. EBUS-TBNA and TBBx are effective and complimentary tools for mediastinal LN biopsy. In this study, concomitant bronchoalveloar lavage or bronchial washing were not useful in establishing the finial diagnosis. Laterality (right side), size of the lymphadenopathy, and cost effectiveness of EBUS-TBNA are areas of interest for further studies.

 » Lymph Node / Hematopoietic Top


The cytomorphological and clinicopathological spectrum of pleuropulmonary extranodal marginal zone lymphoma

Hyang Mi Ko, MD, PhD, Gilda da Cunha Santos, MD, PhD, FRCPC, Scott Boerner, MD, FRCPC, Denis Bailey, MD, PhD, FRCPC, William Geddie, MD, FRCPC

Laboratory Medicine and Pathobiology, University of Toronto, University Health Network, Toronto, Ontario, Canada

Introduction: Extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) is the most common primary lung lymphoma and may also be seen in the pleura. Fine needle aspirates (FNA) and bronchoalveolar lavage (BAL) samples are frequently the first materials presented for the diagnosis of pleuropulmonary EMZL (PP EMZL). Histopathological findings have been well-described, but the spectrum of cytomorphological findings in FNA and BAL has not been fully explored. We investigated the utility of cytomorphological examination in a combination with ancillary tests in the specific diagnosis of PP EMZL, as defined by the 2008 revised World Health Organization (WHO) classification.

Materials and Methods: Cases of PP EMZL diagnosed cytologically from 2003 to 2010 were retrieved. All Romanowsky and Papanicolaou stained slides were reviewed by three cytopathologists. Immunophenotyping, interphase FISH, molecular assays, and imaging data were collated from the original reports. Clinical information was obtained from electronic patient records. Concurrent surgical biopsies were also retrieved.

Results: Eleven transthoracic FNA (10 lung, 1 pleura) and one BAL from five male and seven female patients with a median age of 66 years were identified. Two patients had a history of pulmonary lymphoma and one had a history of peripheral blood lymphoproliferative disorder. Six cases were incidentally discovered on radiologic examination (5 respiratory symptoms and 1 chest pain). The radiologic lesions varied from small ground glass opacities (GGO) to mass-like consolidations, which included unilateral solitary (5) and unilateral or bilateral multiple lesions (7). The radiologic differential diagnosis included lymphoma, bronchioloalveolar carcinoma (BAC), and infection in 11 cases. In two cases an inflammatory process was initially favored. Prior chronic infection (tuberculosis) was documented in only one case (FNA pleura). All samples were composed predominantly of small centrocyte-like cells. Three cases showed follicular dendritic cell meshworks and marked plasma cell differentiation. All cases showed occasional lymphoid tangles with entrapped epithelial cells, but no classical lymphoepithelial lesions. Multinucleated giant cells were present in seven cases of which two showed granulomas. Granulomas prompted submission of the sample for culture in one case. Immunophenotyping by laser scanning cytometry (10 cases) and immunohistochemistry (2 cases) confirmed B cell clonality and CD5-, CD10- immunophenotype typical of MZL in all cases. PCR showed B cell clonality in three samples (2 FNA, 1 BAL). FISH confirmed a MALT1 translocation in four out of eight cases tested, and trisomy 3 in two out of two cases. Concurrent surgical biopsies were diagnosed independently as EMZL in seven cases. Clinical management was undertaken only on the basis of the combined cytomorphological and molecular findings in five cases.

Conclusions: Recognition of a lymphoid infiltrate composed predominantly of small centrocyte-like cells and occasional lymphoid tangles with entrapped epithelial cells at rapid onsite assessment of transthoracic FNA or lymphocytosis in a BAL, in the context of a mass lesion or GGO on imaging, should prompt cell surface marker immunophenotyping, even if granulomas are identified. Specific diagnosis of PP EMZL could also be confirmed by demonstration of MALT1 translocations or trisomy 3 by FISH.


Extramedullary hematopoiesis: Cytomorphological, histological, and radiological findings in 12 cases

Nemanja Rodic, MD, PhD, Zahra Maleki, MD

Pathology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Extramedullary Hematopoiesis (EMH) is a tumor-like mass commonly seen in the liver, spleen, and lymph nodes. EMH is a physiologica; response to chronic anemia, observed in various hematological disorders such as myelofibrosis. Unanticipatted EMH is a very rare diagnosis that typically presents as a solitary mass of undetermined significance. As such, knowledge of the cytopathological characteristics as well as the clinical and radiological correlates of EMH is paramount. We share the salient clinical and radiological findings, as also the diagnostic cytopathological features of EMH in 12 cases, diagnosed with fine needle aspiration (FNA), in an effort to accustom the cytopathological community with this fascinating, albeit rare, entity.

Materials and Methods: Twelve cases of EMH were retrospectively retrieved from the cytopathology archives of a teaching hospital over the past 22 years. The cytological material was obtained by ultrasound-guided FNA. Smears were stained with Diff-Quik® (air-dried smears) and Papanicolaou stains (alcohol-fixed smears). Radiological images, medical records, and the available histology (12 cases) were reviewed.

Results: A total of 13 EMH cytopathological cases were seen. The median age at diagnosis was 63 years (40 - 88 years) and there was no gender bias (male-to-female ratio, 1 : 1.15). The presenting signs and symptoms varied widely, from incidental radiographic findings to right hemiparesis, chronic back pain, lower extremity pain, bloating, pleural effusion, and a lung mass. Likewise, presumptive clinical diagnoses in five of the thirteen cases were benign, while five of thirteen were considered malignant prior to the diagnostic FNA. The most common anatomic site for EMH was the liver (n = 4), followed by the presacral soft tissue (n = 3), and the pleura. Even as most EMH nodules were singular, few presented as up to three radiographically distinct nodules. An average EMH nodule measured 2.8 cm, whereas, EMH liver nodules were larger and measured 4.3 cm on an average (P = 0.0043). Cytological smears revealed trilineage hematopoeitic cells including megakaryocytes. The cytological findings were correlated with the histological findings in available cases. No immunohistochemical stains were performed.

Conclusions: Extramedullary hematopoiesis most commonly presents as a mass lesion, which can mimic a neoplasm radiologically. Megakaryocytes may cause diagnostic pitfalls as a poorly differentiated carcinoma or a high-grade sarcoma. EMH might also be misinterpreted as bone marrow contamination. FNA may provide accurate diagnosis and may prevent more aggressive procedures.


Fine needle aspiration / core biopsy of retroperitoneal lesions: Diagnostic advantages and pitfalls

Manisha Mishra, MD, Yurong Wheeler, MD

Pathology, East Tennessee State University, Johnson City, Tennessee

Introduction: Fine needle aspiration (FNA) / core biopsy appears to be an attractive method for diagnosing lesions at surgically inaccessible or difficult-to-access regions of the body, including the retroperitoneum. However, the role of FNA in the diagnosis of these lesions, especially lymphoma, is still debatable. The small sample size, triage, and representativeness issues as well as lack of tissue architecture contribute to the challenges of interpretation of these cases. We did a retrospective analysis of FNA / core biopsy cases of retroperitoneal lesions at our institute over a seven-year period. The diagnostic advantages and pitfalls are discussed.

Materials and Methods: Sixty-nine cases of FNA / core biopsy cases were retrieved from the archive of a teaching hospital (from the year 2004 to 2011). The FNA material and touch prep specimen from the core biopsy were prepared mostly by the Diff-Quik® stain method for onsite evaluations. On most occasions, a needle core biopsy was obtained for H and E stain and other ancillary studies. All slides and ancillary studies were reviewed and clinical correlation was performed.

Results: Of the 69 cases, 34 were male and 35 were female (male : female = 0.97 : 1), and the age ranged from 30 to 81 years (average = 62.2 years). The initial FNA / core biopsy diagnoses were separated into three arbitrary classes: lymphoproliferative lesions (n = 27), metastases (n = 25), and miscellaneous (n = 17) classes. The lymphoproliferative lesions included Hodgkin lymphoma (n = 2), non-Hodgkin lymphoma (n = 14), atypical lymphoproliferation (n = 5), and benign lymphoid hyperplasia (n = 6). In the fourteen non-Hodgkin lymphoma cases, nine were diffuse large B-cell lymphoma, three were follicular lymphoma, one was small lymphocytic lymphoma, and one was unclassified. The primary sites for the metastases were the female reproductive tract (n = 7), gastrointestinal (n = 6), genitourinary (n = 3), and other primaries (n = 9).The miscellaneous group included primary neoplasms, like myelolipoma, sarcoma and germ cell tumor, as well as non-neoplastic entities like fibrosis. The metastatic lesions, especially those with a known primary, were relatively easy to diagnose, with the use of immunohistochemical studies. The lymphoid neoplasms were challenging to diagnose, due to the small tissue size and sample representativeness issues. Morphologically, the neoplastic lymphocytes were present as monotonous dyshesive small or large cells with a peripheral membrane of blue hue. In our experience, with core biopsy, a majority of the lymphoproliferative lesions could be subclassified (81%) with the correlation of clinicoradiological findings and ancillary studies, such as a battery of immunohistochemical stains, flow cytometry, cytogenetics or molecular studies. About 19% of the cases were placed in the atypical category mainly due to sampling issues.

Conclusions: FNA / core biopsy provides a less invasive and minimally traumatic diagnostic method for retroperitoneal lesions. It is an accurate diagnostic tool in patients with metastatic tumors. However, when dealing with a lymphoproliferative disorder, it is important to exercise caution in using this method, especially in view of the limitations of the procedure in sampling the lesion adequately and obtaining the representative material. Therefore, correlation with clinical information and ancillary studies is very important.


Sub-classification of lymphoproliferative disorders in effusions, a ten-year experience (2001 - 2010)

Leung Tong, MD 1 , Hyang Ko, MD 2 , Mauro Saieg, PhD, MD 2 , Scott Boerner, MD 2 , William Geddie, MD 2 , Gilda da Cunha Santos, PhD, MD 2

1 Departments of Laboratory Medicine and Pathobiology, University of Toronto, 2 Laboratory Medicine Program, University Health Network, Toronto, Ontario, Canada

Introduction: The incidence of a lymphoproliferative disorder involving body cavities is well-documented. However, with the new WHO classification and advances in personalized therapies, there is an increasing need for lymphoma sub-classification and standardization on the use of ancillary techniques, such as, immunophenotyping (IP) and molecular tests in cytological samples. The aims of the present study are to evaluate the effectiveness of our laboratory in the diagnosis and sub-classification of lymphoma in serous effusions and to assess the impact of the contribution of ancillary studies to achieve a more specific diagnosis.

Materials and Methods: Reports from the cytological fluid specimens were retrospectively collected from 2001 to 2010. Cases with a diagnosis of lymphoproliferative disorder or were suspicious for lymphoma were included in the study. The following clinicopathological characteristics were extracted from the pathology report or electronic medical record: Age at the time of the diagnosis, gender, previous clinical history of lymphoma, site, gross volume, and final diagnosis. The use of ancillary tests including IP, either flow cytometry or laser scanning cytometry, immunohistochemistry (IHC), and molecular tests, which included PCR for B-cell or T-cell monoclonality, PCR for HHV-8, and FISH for translocations were also recorded.

Results: One hundred and sixty-eight cytological specimens from 110 patients were included in the study. Their mean age was 58.7 (ranged 19 - 92) years. Seventy-two patients were male and 38 female. Sixty-five patients (59%) had a previous clinical history of lymphoma. The specimen site included 133 pleural, 30 peritoneal, and five pericardial fluids. The specimens' mean volume was 238 ml with a minimum of 2 ml to a maximum of 1 liter. There were 129 cases (77%) with a morphological diagnosis of lymphoma, while 39 cases (23%) were diagnosed as suspicious for lymphoma. The distribution of lymphoproliferative disorder diagnoses is shown in [Table 1]. Overall, 106 cases were submitted for ancillary tests, while 62 were not submitted for any ancillary tests. IP was performed in 96 cases and immunohistochemistry in 22 cases. Molecular tests were completed in 20 cases. Among the lymphoma cases, 83 out of 129 cases (64%) underwent IP. Sixteen out of 129 (12%) lymphoma cases were also submitted for molecular tests, of which five showed B-cell or T-cell monoclonality by PCR, three cases showed MYC rearrangement, two cases showed IGH / BCL2 translocation, and six cases had negative results. Ancillary tests helped to establish a specific lymphoproliferative disorder diagnosis in 54 out of the 106 cases (51%) submitted for ancillary tests. Among the suspicious cases, 13 out of 39 cases (33%) underwent IP, of which three showed clonality, one showed a non-clonal B-cell population, six cases showed reactive T lymphocytes and three were non-contributory due to degeneration or non-specific binding.

Conclusions: The use of immunophenotyping and molecular tests in fluid samples has proven to refine the diagnosis of lymphoproliferative disorders and can be performed in a substantial number of cases, as the mean volume of these samples is sufficient to run multiple analyses. In the effusion samples, a specific lymphoma sub-type can be achieved in over 50% of the cases, by using a combination of cytomorphology and ancillary techniques.

 » Pancreas / Liver Top


Diagnostic accuracy of EUS-FNA for pancreatic adenocarcinoma in solid pancreatic lesions: A meta-analysis

Shantel Hebert-Magee, MD 1 , Faisal Mukhtar, MD 1 , Mohamad Eloubedi, MD, MPH 2 , Isam Eltoum, MD, MBA 1

1 Departments of Pathology, 2 Gastroenterology, University of Alabama at Birmingham, Birmingham, Alabama

Introduction: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is an established diagnostic modality that reports high success in accurately diagnosing pancreatic adenocarcinoma. However, most EUS-FNA studies are traditionally restricted to single institutional data regarding diagnostic accuracy, frequently biased by combining clinical follow-up and surgical pathology as the 'collective' gold standards. To date, a meta-analysis has not been performed to critically evaluate the diagnostic accuracy of the EUS-FNA of pancreatic ductal carcinoma and determine the sources of heterogeneity among different studies. A meta-analysis was performed to determine if the variability of the gold standard and other sources of heterogeneity significantly impact diagnostic accuracy, following the cochrane collaboration guidelines.

Materials and Methods: We systematically searched MEDLINE and SCORPUS to identify the potential studies published between 1994 and 2011. The selection criteria were limited to studies consisting of consecutive patients who underwent EUS-FNA for suspicious solid pancreatic lesions and supported the construction of a 2 x 2 contingency table for analysis. Two authors independently extracted data and entered it into a Microsoft Excel spread sheet, with the following parameters: Year, number of cases, TP, TN, FP, FN, presence of cytopathologist, sample adequacy, and the reference standard used. Computational analysis of the data was performed with the Cochrane Diagnostic Accuracy protocol using the Review Manager (RevMan5) and Statistical Analysis Software (SAS / STAT). RevMan uses a modified quality assessment tool for diagnostic accuracy studies, named QUADAS, to assess the quality of each study. Analysis and data presentation were performed as suggested by the Cochrane Diagnostic Accuracy. Covariates (assessing the presence / absence of a cytopathologist and the reference standard used) were evaluated for variations, utilizing the comparative ROC model and Forest plot summaries.

Results: We identified 783 (465 MEDLINE, 218 SCORPUS) published articles. Only 41 studies met the inclusion / exclusion criteria and included 3492 patients with 2119 pancreatic adenocarcinomas. The sensitivity ranged from 0.33 to 1.00, the specificity from 0.88 to 1.00. The lower sensitivity was consistent with the lack of onsite cytopathologist evaluation or sampling error. The overall diagnostic accuracy was found to be as follows: 0.91(CI : 0.87 - 0.94) for sensitivity and 1.00 (CI:0.99 - 1.00) for specificity. The gold standard was histology (n = 4), clinical course (n = 3), and histology and / or clinical (n = 34). Onsite evaluation by the cytopathologist was present (n = 18), sometimes (n = 4), not indicated (n = 9), and not present (n = 10). By receiver operating characteristic (ROC) curve, EUS-FNA performed with the assistance of a cytopathologist was greater than that of the assessment performed without onsite assistance. Results of the summary receiver operating characteristic (SROC) curve analysis, evaluating the reference standard, suggested lower diagnostic accuracy when the reference standard was the histological 'gold standard'.

Conclusions: Our study shows that evaluating diagnostic accuracy in EUS-FNA of the pancreas using clinical follow-up or combined clinical and histological follow-up introduced a bias that falsely elevated the diagnostic accuracy. Our met-analysis also supports the previously published literature, that EUS-FNA evaluation for pancreatic adenocarcinoma is highly accurate and better when rapid onsite evaluation (ROSE) is performed.


Autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis): Cytomorphological characteristics and clinical correlates

Brittany Holmes, MD 1 , Ralph Hruban, MD 1 , Christopher Wolfgang, MD, PhD 2 , Syed Ali, MD 1

1 Department of Pathology, The Johns Hopkins Hospital, Baltimore, Maryland; 2 Department of Surgery, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Formerly known as lymphoplasmacytic sclerosing pancreatitis (LSP), autoimmune pancreatitis (AIP) is an inflammatory, IgG4-associated condition that is primarily treated by non-surgical methods. However, the clinical and radiological features of AIP overlap with the more prevalent and often fatal pancreatic ductal adenocarcinoma, thus creating diagnostic confusion between the two entities on fine needle aspiration (FNA).

Materials and Methods: The pathology archives from a large tertiary care center were searched for a period of 26 years (1985 - 2011) for all surgical pancreatic specimens diagnosed as AIP or LSP, in patients with a prior FNA of the pancreas. FNA material was obtained by endoscopic ultrasound-guided FNA, and direct smears were prepared and stained with Diff-Quick as well as wet fixed for Papanicolaou staining. The surgical and cytopathological data were correlated.

Results: A total of 20 FNAs from 17 patients were identified. There were 12 males and five females (M : F, 2.4 : 1) with an age range of 29 to 76 years (mean 60 years). Imaging studies demonstrated a pancreatic mass or ill-defined density suspicious for malignancy in 65% (n = 11) of the patients with a size range of 2.1 to 5.2 cm (mean 3.5 cm, n = 4) and peripancreatic lymphadenopathy in 12% (n = 2). Of the 20 aspirates, 5% (n = 1) were diagnosed as adenocarcinoma, 5% (n = 1) were suggestive of a mucinous cystic neoplasm, 50% (n = 10) were atypical, 25% (n = 5) were benign, and 15% (n = 3) were scant or non-diagnostic. Of the 10 aspirates diagnosed as atypical, 70% (n = 7) were described as having rare or scattered atypical ductal cells, while 10% (n = 1) were markedly atypical, 10% (n = 1) were suspicious for malignancy, and 10% (n = 1) could not exclude a neuroendocrine neoplasm. Pancreatic intraepithelial neoplasia (PanIN) was present in the surgical specimens corresponding to 58% (n = 7) of the 12 aspirates diagnosed as atypical or neoplastic. Beginning in 2008, immunohistochemical labeling for IgG4 was performed, with nine out of ten demonstrating increased numbers of positive plasma cells.

Conclusions: On FNA, AIP more often leads to an 'atypical' cytopathological interpretation due to the presence of significant epithelial atypia. Thus, caution is warranted to avoid overdiagnosis on FNA, even when a mass lesion is reported by the imaging studies. Performing IgG4 immunohistochemistry on the cytologic cell block material may increase the diagnostic accuracy of AIP on fine needle aspiration.


A rare cytomorphological feature in endocrine neoplasms of the pancreas

Gillian Levy, MD 1 , Alexander Finkelstein, DO, MD 1 , Guoping Cai, MD 1 , David Chhieng, MD 1 , Uzma Siddiqui, MD 2 , Harry Aslanian, MD 2 , Malini Harigopal, MD 1

1 Pathology, Yale University School of Medicine, New Haven, Connecticut; 2 Medicine and Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut

Introduction: Endocrine neoplasms of the pancreas (PENs) are relatively uncommon, accounting for 1 - 2% of all pancreatic neoplasms. A diagnosis can be rendered cytologically for PENs with characteristic cytomorphological features. However, PENs may infrequently display unusual features, such as, oncocytic changes, mucin accumulation, intracytoplasmic inclusions, and clear cell or vacuolated cytoplasm. These morphological variations may pose a diagnostic challenge. Here we report three cases of PENs that had extensive cytoplasmic vacuolization, in which the differential diagnosis may include pancreatic adenocarcinoma, metastatic clear cell renal cell carcinoma, and adrenocortical carcinoma.

Materials and Methods: Three cases of PENs were diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) at our institution, between 2007 and 2010. The biopsy was performed in the endoscopy suite and a cytopathologist was available for a rapid onsite evaluation. The smears prepared from the aspirates were stained with Diff-Quik® or Papanicolaou techniques for cytological evaluation. The additional aspirate material was fixed in CytoRich Red and processed for cell-block. Immunohistochemical studies were performed on the cell-block sections using a panel of antibodies including chromogranin, synaptophysin, CD56, AE1 / AE3, vimentin, CD10, inhibin, and Melan A. The final diagnosis was rendered based on cytomorphological and immunophenotypic features.

Results: The patients included one male and two females, aged 41, 46, and 61, respectively. The tumors were all located in the body / tail of the pancreas with sizes of 1.9 cm, 3.5 cm, and 14 cm each. One patient received a distal pancreatectomy. while the other two were inoperable due to documented liver metastasis or large tumor size with local invasion. Overall, the aspirates were moderately cellular and consisted of single and dyscohesive clusters of relatively uniform neoplastic cells. Extensive cytoplasmic vacuolization was seen in the tumor cells in all cases. The endocrine nature of neoplastic cells was confirmed by the positivity for chromogranin, synaptophysin, or CD56.

Conclusions: Extensive cytoplasmic vacuolization can rarely be seen as a cytomorphological feature in PENs, which can become a diagnostic pitfall for pancreatic adenocarcinoma and other metastatic tumors. Recognition of this rare occurrence with adjunct immunohistochemical studies will help render a correct diagnosis.


Small cell carcinoma of the pancreas: Primary or metastatic?

Faisal Mukhtar, MD, Shantel Hebert-Magee, MD, Isam Eltoum, MD, MBA

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Introduction: Primary small cell carcinoma (SCC) of the pancreas is extremely rare, with approximately 35 cases reported in the literature. The aim of this study is to review a large center experience with SCC and if possible determine whether the origin of the tumor is primary or metastatic.

Materials and Methods: This a retrospective correlation study in which we reviewed electronic medical records, cytology, and histology reports of all endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) of the pancreas between 2000 and 2010, trying to find cases with a diagnosis of small cell carcinoma of the pancreas. We reviewed the history, imaging, and pathologica; findings to determine whether the lesion was primary or metastatic. A case was considered metastatic when the patient was known to have had a mass elsewhere, with immunostains supporting the origin of the tumor.

Results: Based on the search through medical records, we identified 2445 pancreatic FNAs during the study period from 2000 to 2010. One hundred and thirty-four cases were endocrine neoplasms with eight (6%) patients identified with small cell carcinoma (poorly differentiated endocrine carcinoma). Four of the eight patients were male, and four of the patients were known smokers, with one patient being a passive smoker. Median age at diagnosis was 62 years (range 39 - 81 years). Two of the patients were African American and six were Caucasians. The patients presented with vague abdominal pain (n = 3), jaundice (n = 2), and were asymptomatic (n = 3). The tumor size ranged from 3.0 cm to 8.0 cm. Four patients had a tumor in the head of the pancreas, one in the body of the pancreas, and the rest had tumors that were peripancreatic. Five patients had primary lung carcinoma, one had a primary cancer in the cervix, and the other two had primary lesions that could not be identified, making the prevalence of SCC 1.5% for all endocrine neoplasms. In all patients, the cytological features and endocrine markers were diagnostic of small cell carcinoma. With the exception of one patient, all patients died of the disease with survival ranging from 5 to 32 months (median 11 months) after the diagnosis.

Conclusions: SCC whether primary or metastatic to pancreas carries a grave prognosis. Primary pancreatic SCC is extremely rare. The majority of the SCC of pancreas is metastatic, therefore, a search for the origin is required, particularly given the recent reports, which show a comparatively good response of primary SCC to surgical resection.


Second opinion in pancreatic cytopathology

Armanda Tatsas, MD 1 , Joseph Herman, MD, MSc 2 , Ralph Hruban, MD 1 , Daniel Laheru, MD 3 , Christopher Wolfgang, MD, PhD 4 , Elliot Fishman, MD 5 , Syed Ali, MD 1

1 Departments of Pathology, 2 Radiation Oncology and Molecular Radiation Sciences, 3 Oncology, 4 Surgery, 5 Radiology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Fine needle aspiration (FNA) of the pancreas and biliary brushing (BB) are commonly used techniques to preoperatively evaluate pancreaticobiliary disease. Patients with pancreatic and biliary tract neoplasms are often treated with radical surgery, or referred directly for chemo- and / or radiation therapy. Given the importance of accurate interpretation of the cytological specimens in these patients, we sought to examine the rate of diagnostic disagreement in cases of pancreatic FNA and BB referred to our institution for confirmation of diagnosis.

Materials and Methods: A retrospective search of the cytopathology archives at a large tertiary care center revealed 405 pancreatic FNA (88.7%) and BB (11.3%) cases submitted to our institution for confirmation of diagnosis from January 1, 2009 to December 31, 2010. A scanned copy of the diagnostic report from the originating institution was examined and that diagnosis was compared to the second opinion diagnosis rendered at our institution. Cases with incomplete or missing scanned diagnostic reports were excluded from the study. Each case was categorized as 'no diagnostic disagreement,' 'minor diagnostic disagreement,' or 'major diagnostic disagreement.' Minor or major disagreements were established based on a 1 or 2+ step discrepancy, respectively, on a scale that classified diagnoses as 'benign, atypical, suspicious for malignancy / neoplasm, and malignant / neoplasm.' Cases that did not fit readily into this classification scheme were assessed on an individual basis.

Results: In 318 (78.5%) cases, the second opinion diagnosis correlated closely with the original diagnosis, indicating no diagnostic disagreement. Minor diagnostic disagreements were found in 76 cases (18.8%). Major diagnostic disagreements were found in 11 cases (2.7%), nine of which had follow-up surgical pathology or cytopathology specimens. The second opinion diagnosis was supported in six of the nine cases with follow-up specimens. The most serious error detected was one case diagnosed as 'benign, non-specific changes,' at the originating institution, with a second opinion diagnosis of 'adenocarcinoma.' The lesion was resected and found to be a poorly differentiated infiltrating adenocarcinoma, thus the patient was spared a repeat procedure at our institution prior to definitive treatment.

Conclusions: The rate of major diagnostic disagreements in pancreatic FNA and BB samples in our study is 2.7%. Although this rate is slightly lower than the reported rate of major discrepancies in all cytopathologies, this still represents a significant discrepancy rate, given the serious clinical implications of these diagnoses. Our study supports the importance of mandatory second opinion review in pancreaticobiliary cytopathology as part of the ongoing efforts to improve patient safety and quality of care.

 » Quality Assurance Top


FNA rapid onsite assessment of adequacy: The experience of an academic institution

Nariman Gobara, MS, MBChB, Li Liang, MD, Nora Morgenstern, MD, Patricia Wasserman, MD, Chiara Sugrue, MS, SCT(ASCP)

Cytopathology Division, North Shore LIJ Health System, Lake Success, New York

Introduction: Cytotechnologists and cytopathology Fellows often perform rapid onsite assessment (ROSA) of a fine needle aspirate (FNA) to provide immediate feedback regarding the adequacy of the material and to triage the specimens for additional ancillary studies. The current study was designed to assess the concordance of onsite adequacy assessment of FNA as correlated with the final cytology diagnosis.

Materials and Methods: A retrospective search of all FNA ROSA between 1 January 2009 and 31 December 2010 yielded a total of 1,520 cases. The FNA ROSA was correlated with final cytology diagnosis and classified as either 'concordant' or 'discordant'. An 'adequate' FNA ROSA was considered 'concordant' if a definitive final cytology diagnosis was reached and 'discordant' if no definitive final diagnosis was reached or was reached based on limited material. An 'inadequate' FNA ROSA was considered 'concordant' if no definitive final diagnosis was reached and 'discordant' if a definitive final diagnosis was possible. Special consideration was given to inherently hypocellular lesions (e.g., cysts, colloid nodules, abscesses, etc.) as well as to diagnoses limited by the nature of the lesion rather than the sample (e.g., follicular neoplasm of the thyroid).

Results: Out of the total of 1,520 FNA ROSA analyzed, 1,230 cases were considered adequate and 290 cases were considered inadequate. Out of the 1,230 adequate FNA ROSA, 1,180 (96%) were considered concordant with final diagnosis, for a discordance rate of 4%. Out of the 290 inadequate FNA ROSA, 275 (95%) were considered concordant with final diagnosis, for a discordance rate of 5%. FNA of the salivary glands yielded the highest discordance rates (6%), while liver FNA showed the lowest discordance rates (1%). Two hundred and twenty cases of FNA ROSA were performed without image-guidance and showed a 5% discordance rate, a value similar to that of image-guided FNA ROSA. Finally, the number of passes for the discordant cases was analyzed and it revealed that 23% of the discordant cases had less than two passes, 64% had three to five passes, and 13% had more than six passes.

Conclusions: Cytotechnologist and cytopatholology Fellows are, overall, highly competent in providing rapid onsite assessment of adequacy. Salivary gland FNA showed the highest discordance rate, while liver FNAs showed the lowest. The number of passes and location of the lesions did not significantly influence the discordance rates. The focus review and study of these discordant cases may improve ROSA accuracy. A maximum discrepancy ROSA rate of 5% could be used as a quality assurance benchmark in FNA cytology.


Additional levels are not an effective way to reconcile discrepancies between reported cytological and histological diagnoses in cervicovaginal biopsies

Julie Kunkel, MD, Hannah Krigman, MD

Pathology and Immunology, Washington University, St. Louis, Missouri

Introduction: Colposcopic cervical biopsies for reported cytological abnormalities do not always show intraepithelial neoplasia on the three levels routinely performed at our institution. We examined the utility of the three additional tissue levels in biopsies for detection of epithelial lesions, because the current ASCCP guidelines treat biopsy-confirmed epithelial abnormalities more aggressively. We examined the discordance rate for Paps and biopsies with and without additional levels. Endometrial lesions were not included.

Materials and Methods: The Pathology database from January 2009 to July 2010 was searched for all patients with abnormal cervicovaginal cytology diagnoses and any surgical pathology within three months of Pap smears. All cases with concurrent cervicovaginal smears and biopsies were recorded. The paired samples were categorized as concordant (195 cases), discordant (200), or atypical on Pap ((ASCUS (145) or AGUS (83), ASC-H (35)). Concordant biopsies and paps were within one degree of the diagnosis.

Results: Eighteen concordant cases (9%), 14 atypical cases (7%), and 19 discordant cases (10%) were further evaluated by additional levels. The eighteen concordant cases were nine low-grade squamous intraepithelial lesions (LSIL), eight high-grade in squamous intraepithelial lesions (HSIL), and one case of invasive carcinoma, arising from the HSIL. In the concordant cases, most levels were obtained to exclude higher grade lesions. Only in two of the concordant cases (1% of sampled cases) did the additional levels change the diagnosis. The atypical cases with levels examined were ASC-H (3) or ASCUS positive for HPV (11). The discordant cases had diagnoses of HSIL (7) and LSIL (11). Of the ASC-H cases, one had denuded stroma, one was CIN-1, and one was benign. No diagnoses changed after the levels were obtained. In case of the ASCUS cases, six were benign even after six levels. Two ASCUS cases were diagnosed as CIN1 after six levels; while three cases were CIN2-3. Of the LSIL Pap cases, 10 were benign after extensive levels. One case changed from benign to CIN2 after six levels. In one HSIL and one LSIL case, the reorientation and levels found the CIN that was present on other biopsy fragments, so no additional data were obtained.

Conclusions: At our institution, additional levels did not provide significant information in the majority of cases, regardless of the category. For this reason, additional levels were not considered as a cost-effective or time-effective mean for reconciling differences in the diagnoses. Resampling, re-examination, and follow-up provide more effective means of reconciliation. Roughly a third of the biopsies are concordant with the diagnosis rendered on cervicovaginal cytology. Additonal levels were examined on a few of these to exclude higher grade lesions; diagnoses did not change significantly. A third of the biopsies were discordant. A third of the cases represented biopsies after an atypical Pap (ASCUS or AGUS). Of those 50 cervicovaginal biopsies, examination of additional levels changed the diagnosis in each of the three cases with Pap diagnosis of LSIL. In two of those cases, a diagnosis of CIN1 was made and in the other CIN 2, after the levels. This series suggests that additional levels are not an effective way to reconcile discordance between Pap smear and biopsy in these patients, who will already be followed for abnormal Pap.


Cytology-histology correlations in gynecological pathology, trends and results

Debora Smith, CT(ASCP), Mary Schwartz, MD, Subhendu Chakraborty, MS, Todd Fairley, CT(ASCP), Dina Mody, MD

Pathology, The Methodist Hospital, Houston, Texas

Introduction: Cytohistological correlation of high-grade squamous intraepithelial lesions and malignant cases is mandated for gynecological cytology under the Clinical Laboratory Improvement Amendments of 1988. However, details as to how this is done and timelines are not specified. The results of and statistics derived from these correlations are mandated to be a part of the laboratory's quality improvement data and are reviewed by accrediting agencies as part of the cytology laboratory inspections. We reviewed our laboratory's cytohistological correlations over a ten-year period. The case mix includes Pap tests and biopsies obtained in private practice ob-gyn offices, charity clinics including a dysplasia clinic, and gynecological oncologists' offices.

Materials and Methods: Data derived from gynecological cytohistological correlations performed at our institution between 2000 and 2009 was reviewed for trends. All cervical and vaginal biopsies with previous abnormal cytology in the HSIL / carcinoma and adenocarcinoma in-situ categories were included. Between 2000 and September 2004, the biopsies were interpreted by the general surgical pathology service. Beginning in Fall 2004, that practice changed and all gynecological cytology as well as all cervical and vaginal biopsies with previous abnormal cytology in our files were interpreted by pathologists on the gynecological (gyn) cytology service. The pathologists on the gyn cytology service were all Board-certified cytopathologists who practiced both gynecological cytology and surgical pathology. Not all surgical pathologists who signed out cervical and vaginal biopsies during 2000 to 2004, signed out gynecological cytology or had subspeciality expertise in gyn pathology. During that same time period, gynecological cytology was signed out by anatomical pathologists who signed out cytology and not all had subspeciality expertise in cytopathology. The Pap tests included ThinPrep® and SurePath® , both manually screened and imager screened. The data was viewed in aggregate for 2000 - 2009 and a comparison of the earlier years (2000 - 2003) to the later years (2005 - 2009) was also performed. Data from 2004 was excluded from the comparison as it was the transition year.

Results: The most frequent cause of noncorrelation between the Pap test and biopsy was a colposcopic biopsy sampling variance followed by cytology interpretive variance. When comparing the earlier time period with the later period, the number and percent of cases due to cytological and histological interpretive variances dropped and this difference was statistically significant [Table 1]. Technical issues related to tissues were resolved in the later period by deeper levels, reorientation of blocks or immunohistochemical staining for biomarkers. While technical issues were eliminated in the later time period, this difference did not reach statistical significance with a P value of 0.058 on Fisher's exact chi square test.

Conclusions: Gynecological-cytohistological correlation is a quality indicator and the results of this correlation are critical for optimal patient management. This study demonstrates the benefit to patient care by changes in pathology practice where real-time correlation by the same group of individuals with subspecialty expertise can decrease interpretive variances and variances due to tissue-related technical issues.


More frequent use of p16 / Ki67 on cervical biopsy specimens improves cervical cytohistological agreement rates

Tetyana Mettler, MD 1 , Charanjeet Singh, MD 1 , Arbaz Samad, MD 1 , Kay Savik, MS 2 , Samy Amrouche, CT(ASCP) 3 , Jana Holler, CT(ASCP) 3 , Bharat Thyagarajan, MD 1 , Evin Gulbahce, MD 1 , Stefan Pambuccian, MD 1

1 Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota; 2 School of Nursing, University of Minnesota, Minneapolis, Minnesota; 3 Cytology Laboratory, University of Minnesota Medical Center, Fairview, Minneapolis, Minnesota

Introduction: Cervical cytohistological correlations are routinely performed in cytology laboratories as a measure of quality assurance. However, the 'gold standard' against which the cytological (Pap test) diagnosis is judged, the histological (biopsy) diagnosis is itself marred by subjectivity. Studies have shown high inter-pathologist variability of cervical biopsy diagnoses and that p16 and Ki67 stains performed on cervical biopsies improved the diagnostic agreement between pathologists and their diagnostic accuracy. The aim of this study was to determine the impact of different pathologists' frequency of use of p16 / Ki67 stains on cervical biopsies on cervical cytohistological correlations.

Materials and Methods: We identified all cervical biopsies on which p16 / Ki67 stains were performed (or not) in our institution from 1 January, 2005 to 30 September, 2010. The following data were recorded for each biopsy: (1) patient age, (2) preceding Pap test, and when available, HPV result obtained within six months of the biopsy, (3) the most severe grade of dysplasia on biopsy, dichotomized as CIN0 / 1 or CIN2 / 3+ and (4) biopsy pathologist. Minor and major cytohistological discrepancy rates (One- and respectively two-step differences between Pap test and biopsy result) were determined for each biopsy. The frequency of use of p16 / Ki67 in cervical biopsies was calculated for each pathologist. The pathologists were then classified as 'high users' of p16 / Ki67 when their frequency of use of p16 / Ki67 was above the unweighted mean of all pathologists, or 'low users' when it was below. Immunostains for p16INK4a (clone E6H4™, CINtec® ) and Ki67 (clone 30-9, Ventana) were performed on Ventana NexES.

Results: During the study period, 23 pathologists interpreted cervical biopsies from 11,850 women aged 14 to 83 (mean 33 ± 11.8) years. Prior Pap test results showed NILM (2,302, 19.4%), ASC-US (3,161, 26.7%), LSIL (3,458, 29.2%), ASC-H (837, 7.1%), LSIL-H (710, 6.0%), AGC (367, 3.1%), and HSIL+ (1015, 8.6%). The biopsy diagnoses included 2,240 (18.9%) CIN2 / 3+. p16 / Ki67 staining was used in 1201 / 11850 (10.14%) cases. p16 / Ki67 stains were ordered for biopsies following Pap tests diagnosed as NILM (8.08%), ASC-US (31.39%), LSIL (26.23%), ASC-H (12.57%), LSIL-H (7.33%), HSIL+ (12.03%). p16 was used the most when the prior Pap test result was ASC-H (18.04%), followed by HSIL (14.39%), LSIL-H (12.39%), ASC-US (11.93%), and least when the prior Pap test diagnosis was LSIL (9.11%) or NILM (4.21%). The 23 pathologists included in the study diagnosed 59 - 1811 (mean 518 ± 543) cervical biopsies and used p16 / Ki67 in 0 - 21.31% of these biopsies (unweighted mean 7.83%). Individual pathologists diagnosed 6 - 413 (mean 97 ± 115) cases as CIN 2 / 3+, which represented 9.49 - 24.12% of their cervical biopsy diagnoses. The 11 'high users' of p16 / Ki67 had a lower rate of major cytohistological disagreements than the 12 'low-users' (1059 / 8391, 12.62% vs. 516 / 2943, 14.92%, P < 0.0001). 'High users' of p16 / Ki67 also had a lower rate of biopsy diagnoses of less than CIN2 / 3 in women with prior Pap test diagnoses of ASC-H, AGC or HSIL+ (1146 / 8391, 13.66% vs. 570 / 3459, 16.48%, P < 0.0001).

Conclusions: Our results show a high variability of use of p16 / Ki67 in cervical biopsies by pathologists practicing within the same institution. We also found that more frequent use of p16 / Ki67 by biopsy pathologists improved cervical cytohistological correlation rates, by lowering the rates of major discrepancies.


Does immunohistochemical subtyping of non-small cell lung carcinoma in fine needle aspiration play a similar role as in surgical resection tissue?

Brooke Koltz, Zachary Sletten, Donna Russell, Thomas Bonfiglio, Haodong Xu, Zhongren Zhou

Pathology and Laboratory Medicine, University of Rochester, Rochester, New York

Introduction: Personalized therapies require subtyping of non-small cell lung carcinomas (NSCLCs) into adenocarcinoma and squamous cell carcinoma. A four antibody (p63, CK5 / 6, TTF-1, and napsin A) immunohistochemical (IHC) panel is widely applied for subtyping NSCLSs in fine needle aspiration (FNA), biopsy, and surgical resection tissue (SRT). However, the IHC study is unreliable in some FNA cell blocks. A quality control study in matched lung FNA and surgical resected lung cancer has rarely been done.

Materials and Methods: Three hundred and ninety-eight lung resections were performed at our institution during 2008 - 2011, of which 24 cases had the diagnosis of adenocarcinoma or squamous carcinoma with matched FNA and satisfactory cell blocks. The immunohistochemical studies for TTF-1, napsin A, cytokeratin 5 / 6 and p63 were used for both SRT and FNA cell blocks in 12 cases (five adenocarcinomas and seven squamous carcinomas). The results were evaluated independently by three pathologists.

Results: Six (86%) of seven squamous cell carcinomas in SRT showed both p63 and CK5 / 6 positive IHC staining. One case was positive for CK5 / 6, but negative for p63 stain. The identical IHC staining pattern was observed in the matched FNA cases. Four (80%) of five adenocarcinoma cases in SRT exhibited both TTF-1 and napsin A positive IHC staining. One case only showed positive TTF-1. All five FNA cases showed both TTF-1 and napsin A positive IHC staining. Two (40%) of the five adenocarcinoma cases in SRT showed a focal p63 IHC stain, which was not identified in all the five adenocarcinoma FNA cases.

Conclusions: The IHC studies on FNA cell blocks and SRT showed similar staining patterns. The IHC subtyping of non-small cell lung carcinomas in the FNA specimen played an important role similar to that in the SRT specimen. Focal p63 positivity found in a portion of surgically resected adenocarcinomas, but not in matched FNA indicated that the p63 positive cells were not sampled by FNA.

 » Soft Tissue / Bone Top


Is fine needle aspiration cytology a sensitive diagnostic tool for granular cell tumors? A cytohistological review with emphasis on pitfalls

Susan Haley, MD, Vicki Schnadig, MD, Ranjana Nawgiri, MD

Pathology, The University of Texas Medical Branch, Galveston, Texas

Introduction: Granular cell tumors (GCT) are relatively uncommon neoplasms thought to be of neural origin. Although they may appear as cellular smears of cohesive and single cells with bland nuclei and abundant granular cytoplasm, the cytological appearance on fine needle aspiration (FNA) is variable. The differential diagnoses are many and include infections, benign, and malignant neoplasms. The polygonal cells and granular cytoplasm can be mistaken for necrosis and histiocytes; therefore, atypical infections, granulomatous inflammation, and histiocytic tumors are considered in the differential diagnoses. In the breast, GCT may resemble fibrocystic changes, including apocrine metaplasia; and in thyroid, cells can resemble oncocytic Hürthle cells. CGT presenting as a fragile bare nuclei with prominent nucleoli may also resemble renal cell carcinoma, alveolar soft parts sarcoma, or melanoma.

Materials and Methods: All cases of suspected GCT on cytology within the previous ten years were identified, and nine cases were retrieved. Three patients had been lost to follow-up. The medical records of the remaining six patients were then reviewed for histopathological and clinical findings.

Results: Two painful neck masses in a 65-year-old woman and 29-year-old man were confirmed by histology as GCT. A thyroid nodule in a 40-year-old woman was later diagnosed as Hürthle cell adenoma; a lung mass in a 44-year-old man was ruled as poorly-differentiated carcinoma; and a breast mass in a 78-year-old woman was ruled as a fibrocystic change. One case of suspected malignant GCT was later diagnosed as unclassified sarcoma.

Conclusions: Granular cell tumor is a rarely encountered entity on FNA preparations and may closely mimic other conditions, and the cases presented highlight the often unusual cytology findings seen in entities that resemble GCT. Awareness of this entity and its differential diagnoses, along with careful clinical correlation is critical in the accurate interpretation of the findings.


Giant cell tumor of tendon sheath: Cytomorphological, histological, and radiological findings in fourteen cases

Cheng-Ying Ho, MD, PhD, Zahra Maleki, MD

Pathology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Giant cell tumor of tendon sheath (GCTTS), also known as pigmented nodular tenosynovitis and fibrous xanthoma of synovium, is a common soft tissue lesion. The lesion occurs in young adults with a peak incidence in the age group of 40 to 50 year olds. It most commonly affects small joints of hands and is known to be associated with degenerative joint disease. It presents as a firm, multilobular, slow-growing, nontender mass located adjacent to the synovium of the tendon sheath. Radiological features include a well-defined, round, soft tissue mass, eccentric to or enveloping the tendon sheath, with or without osseous erosion. Bone involvement is considered to be more aggressive. Complete excision is the treatment of choice. The incidence of local recurrence is high, ranging from 4 - 45%. Herein, we describe the cytomorphological features of GCTTS and the usefulness of ultrasound-guided FNA as an initial diagnostic modality.

Materials and Methods: Fourteen cases of GCTTS were retrospectively retrieved from the cytopathology archives of a teaching hospital. The cytological material was obtained by ultrasound-guided FNA. Smears were stained with Diff-Quik® and Papanicolaou stains. Radiological images, medical records, ancillary studies, and available histology (12 cases) were reviewed.

Results: The 14 cases selected included three males and 11 females ranging in age from 11 to 63 years (mean = 32 years). GCTTS involved the lower extremity in 11 cases and upper extremity in three cases. The site of involvement included knee (n = 3), toe (n = 3), ankle (n = 2), leg (n = 2), foot, wrist, palm, and finger (one case each). Small joint involvement was seen in five cases. The entire 14 cases presented with a unifocal lesion. The majority (8 cases) of the lesions were immobile. Bone involvement was seen in three cases. The tumor size ranged from 1.5 cm to 9.5 cm (Mean = 3.6 cm). Three cases had bone involvement. One case had subcutaneous involvement. Six cases had a painful presentation. Two cases had a known history of trauma. Only one case was a recurrence. Microscopic examination revealed hypercellular smears with abundant polygonal, round or spindle stromal cells. Multinucleated giant cells, hemosiderin-laden macrophages, and foamy histiocytes were noted. The stromal cells lacked pleomorphism and displayed occasional nuclear grooves or intranuclear inclusions. Mitosis was absent. The stromal cells were immunoreactive for CD68 and negative for cytokeratin, CD34, and desmin. Cytomorphological findings were correlated with the histological findings in all 12 cases. Histological sections demonstrated a round-to-oval mass surrounded by a thin layer of fibroconnective tissue. The mass consisted of sheets of stromal cells varying in morphology from small oval shaped with minimal cytoplasm to large polygonal with abundant vacuolated cytoplasm. Numerous multinucleated giant cells were seen, the majority of which were in clusters. Extracellular hemosiderin deposition was most prominent at the periphery of the stromal cells. Occasional hemosiderin-laden macrophages were noted. Mitotic figures were rare.

Conclusions: Our study demonstrates that ultrasound-guided FNA is a useful method to obtain diagnostic material for GCTTS. Awareness of the cytomorphological features of GCTTS and the typical clinical setting will permit an accurate diagnosis and eliminate the need for preoperative biopsy.


Fine needle aspiration of epithelial tumors and tumor-like lesions of the skin: A single institute experience

Manisha Mishra, MD 1 , Surendra Verma, MD 2 , George Youngberg, MD 1 , Neelaiah Siddaraju, MD 2 , Tamar Giorgadze, MD 3

1 Pathology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; 2 Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India; 3 Pathology, Wayne State University, Detroit, Michigan

Introduction: Although fine needle aspiration (FNA is widely practiced as a diagnostic method for superficial and deep lesions, it has not enjoyed as much popularity for the diagnosis of skin nodules. Its advantages include low cost, simplicity of technique, and minimal trauma. In spite of its obvious advantages in its use for the diagnosis of skin lesions, few large studies have been published about the role of FNA as a primary method of diagnosing epithelial lesions presenting as skin nodules. We encountered 47 cases at a tertiary care institute in India over a 22-month period.

Materials and Methods: All patients presenting with non-inflammatory skin nodules between August 2003 and June 2005 underwent FNA before incisional / excisional biopsy. A diagnosis was rendered on FNA and the forty-seven cases that received a subsequent histological diagnosis of an epithelial lesion were included in this study.

Results: Cyto-histo correlation was performed and a statistical analysis was done [Table 1], [Table 2] and [Table 3]. Sensitivity was 78.9%, specificity was 96.2%, and diagnostic accuracy was 88.8% for FNA as a primary diagnostic modality for the diagnosis of epithelial skin lesions.

Conclusions: FNA is a highly specific and moderately sensitive method of diagnosis for epithelial lesions of the skin. It has a high diagnostic accuracy for such lesions and is very safe and cost-effective as a diagnostic technique.


Role of fine needle aspiration as a primary diagnostic method for mesenchymal tumors and tumor-like skin nodules: Twenty-two months' experience at a single institute

Manisha Mishra, MD 1 , Surendra Verma, MD 2 , George Youngberg, MD 1 , Neelaiah Siddaraju, MD 2 , Tamar Giorgadze, MD 3

1 Pathology, East Tennessee State University, Johnson City, Tennessee; 2 Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India; 3 Pathology, Wayne State University, Detroit, Michigan

Introduction: Fine needle aspiration (FNA) is a widely used diagnostic procedure in the current medical milieu. It is inexpensive, simple to perform, and is a relatively less invasive diagnostic modality. However, few large studies have been done to evaluate the role of FNA as a primary method of diagnosing mesenchymal lesions presenting as skin nodules. We intend to present our study of 49 cases of skin nodules, which we encountered during a 22-month period at a tertiary care institute in India.

Materials and Methods: All patients presenting with skin nodules between August 2003 and June 2005 underwent FNA before incisional / excisional biopsy. Inflammatory lesions were excluded from the study. A cytological diagnosis was rendered. Only forty-nine cases in which a subsequent histological diagnosis was available and revealed a mesenchymal lesion were included in this study.

Results: The results of our study are outlined in [Table 1], [Table 2] and [Table 3]. Statistical analysis showed that the sensitivity was 93.3%, specificity was 90.9%, and diagnostic accuracy was 91.7% when FNA was used as a primary diagnostic modality for the diagnosis of mesenchymal skin lesions.

Conclusions: Our study showed high diagnostic accuracy of FNA as a primary diagnostic modality in the diagnosis of mesenchymal lesions of the skin. The sensitivity was higher than the specificity for diagnosing mesenchymal lesions, but both values were above 90%, which made FNA a very valuable diagnostic procedure for such lesions, because it was also an inexpensive and minimally invasive diagnostic technique.


Utility of fluorescence in-situ hybridization in bone and soft tissue cytopathology: A seven-year experience at an academic medical center

Fay Jia, PhD, Walid Khalbuss, MD, PhD, Sara Monaco, MD, Urvashi Surti, PhD, Kathleen Cieply, BSc, Liron Pantanowitz, MD

Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Introduction: Fluorescence in-situ hybridization (FISH) is a sensitive and highly specific method that can be used for diagnostic purposes, as well as for the evaluation of predictive biomarkers for therapeutic decision-making. There has accordingly been a greater demand to perform FISH tests on cytology specimens. The aim of this study is to report our experience by utilizing FISH in a large series of non-gynecological cytology samples.

Materials and Methods: A retrospective study was performed evaluating all bone and soft tissue cytopathology cases in which ancillary FISH testing was performed over a seven-year period. In all cases the final cytology diagnosis, FISH test results, and other ancillary studies (immunocytochemistry, flow cytometry) were recorded. The additive value of FISH for each case was determined. Data were captured and analyzed using descriptive statistics (Excel).

Results: In 128 cases FISH was ordered as an ancillary test. FISH was performed for four diagnostic categories, including 51 hematolymphoid cases (40%), 44 soft tissue tumors (34%), 30 metastatic carinomas (24%), and three miscellaneous cases (mesothelioma, neuroendocrine carcinoma, benign bone lesion). FISH was unsuccessful in six cases (5%) due to insufficient material procured from five soft tissue neoplasms and one metastatic breast carcinoma. For lymphomas, FISH was helpful in 46 (90%) cases used to evaluate clonality (IgH), specific translocations (MYC, BCL1, BCL2, BCL6, MALT1), and ALK rearrangement. In five cases (10%) diagnostic of lymphoma (1 Hodgkin, 4 non-Hodgkin), FISH was not deemed additive as IgH rearrangement was inconclusive (n = 1) or negative (n = 4). Among soft tissue tumor cases, FISH proved helpful in the diagnosis of 41 cases (93%), specifically for liposarcomas (CHOP, FUS, MDM2), alveolar rhabdomyosarcoma (FKHR), synovial sarcoma (SYT), and EWSR1 positive sarcomas. FISH in one case of recurrent synovial sarcoma (SYT negative) and two cases of PNET (EWSR1 negative) was not additive. In all adenocarcinoma cases, FISH provided Her2 / neu (28 breast carcinomas) and EGFR (two lung tumors) results.

Conclusions: These data show that at our institution FISH can be successfully performed on the majority (95%) of bone and soft tissue cytology specimens. FISH is especially helpful in diagnosing lymphomas and soft tissue sarcomas, as well as in evaluating therapeutic biomarkers (like Her2 / neu and EGFR) in cytology samples containing breast or lung adenocarcinoma.


Nodular fasciitis: Cytomorphological findings and diagnostic issues on fine needle aspiration

Gang Zheng, MD, PhD, Matthew Olson, MD, Syed Ali, MD

Pathology, The Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Nodular fasciitis (NF) is a common benign mimicker of sarcoma on fine needle aspiration (FNA). The cytomorphological characteristics have rarely been described in published series in the literature. We retrospectively analyzed 13 cases of NF from a single institution with elaboration of the morphological features and differential diagnosis.

Materials and Methods: A retrospective search of the pathology archives of a large tertiary care center identified 13 cases of NF in a 12-year period (1999 - 2011). Of these, eight cases had tissue confirmation (biopsy and / or resection). Material was obtained by ultrasound-guided FNA and direct smears were stained with Diff Quik® and Papanicolaou stains.

Results: The patients ranged in age from 7 to 63 years (median: 38) with a M : F ratio of 1 : 1.7. The cytopathological diagnoses included 'NF' (9) and 'Spindle cell proliferation or neoplasm, NOS' (3). One case was deemed non-diagnostic. The lesions ranged in size from 2 to 5.5 cm (mean: 3.5 cm). These were located in the lower extremity (37.5%), upper extremity (25%), face (12.5%), parotid (12.5%), and trunk (12.5%). The patients presented with pain in 63% of the cases. No prior history of injury or trauma was available in any of the cases. Salient cytomorphological features included hypercellularity with mostly single cells arranged haphazardly, 'tissue culture appearance', few tissue fragments with cells embedded in the myxoid matrix, variable amount of loose myxoid stroma, bipolar spindled cells with oval nuclei, and pale blue wispy cytoplasm. Four cases showed few multinucleated giant cells and large ganglion-like cells. Focal significant atypia (pleomorphism, high N / C ratios, macronucleoli) was present in half of the cases. Mitosis and necrosis were absent. Immunostaining with smooth muscle actin performed in few cases was diffusely positive.

Conclusions: The cytomorphological characteristics of NF often overlap with a number of benign and malignant spindle cell neoplasms, resulting in a broad differential diagnosis on FNA. In the right clinicoradiological context, the presence of cytomorphological features, as mentioned earlier, may support the diagnosis of NF. Immunostaining with muscle cell actin may help narrow the list of differential diagnosis. A conservative diagnostic approach is recommended when cytological atypia is encountered in NF, to avoid false positive diagnoses.

 » Special / New Techniques Top




Discordances in immunoperoxidase stains performed in evaluation of carcinomas: A comparison between results on cytological and surgical specimens

Nyasha Bullock, MD, Mary Fiel-Gan, MD, Richard Cartun, PhD, Srinivas Mandavilli, MD

Pathology and Laboratory Medicine, Hartford Hospital, Hartford, Connecticut

Introduction: Immunoperoxidase (IPOX) stains are established ancillary methods for the evaluation of carcinomas on cytological specimens. IPOX stains are optimally performed on formalin-fixed, paraffin-embedded (FFPE) tissues, including cell blocks. However, FFPE tissue / cell blocks are not available in all cases and in such instances IPOX stains could be applied directly on alcohol-fixed cytological smears (AFCS). There is limited literature evaluating the reliability of the results of IPOX on AFCS. The aim of this study was to compare the results of IPOX stains performed on AFCS and FFPE tissue using the same cohort of cases.

Materials and Methods: Five hundred and eighty two tumors (primary and metastatic carcinomas from a variety of body sites) were retrieved from the pathology files over a two-year period (2008 - 2009) and 226 of those cases had a tissue follow-up. There were 57 cases in which IPOX stains were performed in both AFCS and surgical specimens. IPOX concordance (results reported as positive or negative in both specimens) or discordance (positive in one and negative in the other specimen) were tabulated. IPOX was performed on AFCS and FFPE using polymer-based HRP / DAB detection with careful evaluation of controls.

Results: There was 100% concordance in the following antibodies (numbers in parentheses represent # of cases): CK-7 (7), CK34BE12 (4), Calcitonin (4), Calretinin (3), Thyroglobulin (3), CA125 (2), CEA-m (2), CD117 (2), PLAP (1), Mammaglobin (1), PSA (1), CD34 (1), CD31 (1), PSAP (1), and WT1 (1). There was 100% discordance in the following antibodies: CD10 (1), EMA (1), MOC31 (1), Beta catenin (1); 50% discordance in the following antibodies: Hep Par1 (2), RCC (2); and 33% or less discordance with TTF-1 (13), CK cocktail (12), Synaptophysin (11), Chromogranin (11), ER (8), CK-20 (7), CDX2 (4), p63 (4), S-100 (4), and PR (3). The discordance was overwhelmingly represented in cases with IPOX negative on AFCS and IPOX positive FFPE material. There were only four cases (ER, p63, TTF-1 and RCC - all one each) that were IPOX positive on AFCS and negative on FFPE material.

Conclusions: ( 1) ICC stains can be successfully performed on AFCS when FFPE is not available, with 100% concordance seen in several commonly utilized antibodies. (2) Although this is a relatively limited study, the results suggest that there can be discordances in the IPOX results between AFCS and FFPE material and the vast majority of such discrepancies represent a likely 'false negative' IPOX result on AFCS. (3) Immunoperoxidase results have to be carefully interpreted on AFCS and evaluated in the individual clinical context.


Molecular testing in cytopathology

Frances Cate, MD, Doha Itani, MD, Cindy Vnencak-Jones, PhD, Joyce Johnson, MD, Alice Coogan, MD

Pathology, Vanderbilt University Medical Center, Nashville, Tennessee

Introduction: Molecular testing has widely become the standard of care for lung cancer and melanoma. Cytological samples are frequently the diagnostic material obtained due to decreased morbidity of the procedure, as compared to more invasive procedures. In July, 2010, molecular assays for melanoma and lung cancer became available at Vanderbilt University Medical Center (VUMC). This is a report of our laboratory's experience using cytology specimens for these analyses.

Materials and Methods: All tumors were tested in the Molecular Diagnostics Laboratory at VUMC using the ABI PRISM® SNaPshot® Multiplex Kit and a laboratory developed test, with appropriate positive and negative controls. The assay included testing for KRAS, BRAF, NRAS, PIK3CA, MEK1, AKT1, PTEN, and EGFR mutations in primary lung tumors and BRAF, NRAS, GNA11, GNAQ, KIT, and CTNNB1 mutations in melanomas. Routine molecular testing of cytology specimens was not implemented, but rather was performed only on request by the treating physician. The specimens were tested using a cell block or a spun-down fresh cell pellet. On cell blocks, the amount of submitted tissue was measured in millimeters. A percentage of the tumor cells present was determined by a pathologist, to establish the adequacy of the sample and judge whether it was possible to enrich for tumor. Our procedure was to enrich for tumor as much as possible. Samples having 10% or more tumor cells were considered adequate for testing, based on the sensitivity data established in the molecular diagnostics laboratory. For tissues that could not be enriched, the percentage of tumor cells was noted and paraffin sections were obtained. As for cases that could be enriched, unstained slides were obtained and macro-dissection of the enriched tumor areas was conducted, to acquire material for molecular testing. An H and E stained slide was prepared after tissue sectioning, to ensure that the tumor was consistently present in the intervening slides tested. Fresh tissue pellets were also accepted for testing provided a cytospin obtained from the same material met the adequacy criteria.

Results: From July 1, 2010 to February 28, 2011, 24 cytology specimens were referred for testing. Four had less than 10% tumor cells and were considered inadequate. One sample was referred to the laboratory in error and in one case, testing was canceled by the treating physician. The 18 remaining specimens included 15 lung cancers and three melanomas. Two of the lung cancers were tested with pellets of fresh tissue. The cell blocks on the remaining 16 samples had a range of 3 mm to 15 mm of tissue that was macro-dissected. Fifteen of these had greater than 10% tumor cells on the initial and final H and E slides. One sample of a primary lung tumor had 10% tumor cells on the initial H and E and 3% on the final H and E; however, the sample was tested at the request of the physician and a mutation was detected. Ten out fifteen lung tumors were positive for genetic mutations: Seven KRAS, two EGFR, and one MEK1. Nine mutations were identified from the cell block samples and one from a pellet sample. Two out of three melanomas were positive for NRAS mutations.

Conclusions: We conclude that the DNA yield from cytology specimens is adequate for molecular mutation analysis. Material submitted for testing should be enriched for tumor as much as possible in order to increase the confidence in targeted tumor testing. Specimens without a cell block can have reliable studies performed from a pellet of fresh cells.


A comparative study of thyroid ultrasound-guided fine needle aspiration biopsies performed by cytopathologists versus others

YoonSun Choi, CT(ASCP), David Burstein, MD, Hua Chen, MD, PhD, Hui Liu, MD, Sambasivarao Sunkara, SCT(ASCP), Arnold Szporn, MD, David Zhang, MD, PhD, Maoxin Wu, MD, PhD

Anatomic Pathology, Mount Sinai Medical Center, New York, New York

Introduction: In the past, the majority of thyroid fine needle aspirations (FNA) were done by palpation. Today there is an increased number of FNA biopsies performed routinely using ultrasound guidance (USG). The objective of this study was to evaluate the effectiveness of thyroid USG-FNA performed by pathologists compared with others, including endocrinologists and radiologists, from the institution.

Materials and Methods: Thyroid USG-FNA cases submitted during the period of September 1, 2010 to April 6, 2011 were reviewed. There were 134 performed by cytopathologists and 368 performed by others. The Bethesda System (BS) for thyroid cytology was applied to classify each group. A surgical follow-up study was also recorded. Statistical analysis for cases with surgical follow-up (SFU) was performed by using surgical diagnosis as a gold standard.

Results: As shown in [Table 1], among the 134 cases performed by cytopathologists, there were four (3%) non-diagnostic / unsatisfactory (BS 1), 89 (66%) benign (BS 2), 11 (8%) atypical / follicular lesions of undetermined significance (BS 3), nine (7%) follicular neoplasms (FN) / suspicious for FN (BS 4), seven (5%) suspicious for malignancy (BS 5), and 14 (11%) malignant (BS 6), and there were 27 (20%) cases with SFU. In comparison, the 368 cases performed by others showed 76 (21%) BS 1, 238 (65%) BS 2, 26 (7%) BS 3, 10 (3%) BS4, nine (2.5%) BS 5, and nine (2.5%) BS6, and there were 26 (7%) cases with SFU. The statistical results based on SFU are presented in [Table 2] and [Table 3].

Conclusions: USG-FNA performed by pathologists with the onsite evaluated group shows less unsatisfactory cases (3% vs. 21%), higher rate of SFU (20% vs. 7%), and better sensitivity (87% vs. 70%), specificity (90% vs. 88%), and PPV (93% vs. 78%) as compared to the non-cytopathologist group.


Electromagnetic navigation bronchoscopy-guided fine needle aspiration for the diagnosis of lung lesions

Shelley Redfern, MD 1 , Thomas Gildea, MD 2 , Deborah Chute, MD 1

1 Pathology and Laboratory Medicine Institute, 2 Respiratory Institute, Cleveland Clinic, Cleveland, Ohio

Introduction: Smaller lung nodules are being detected earlier due to the increasing use of improved imaging. Many peripheral lesions are beyond the reach of conventional bronchoscopes, and require CT-guided or open surgical biopsy, which carry increased risks to the patient. Electromagnetic navigation bronchoscopy (ENB) is a new technique, which uses an image-guided localization system to guide the bronchoscopic tools to predetermined points, within the bronchial tree. Preliminary studies have demonstrated the safety and feasibility of this technology. In this study, we investigate the sensitivity and specificity of ENB-guided fine needle aspiration (FNA) in the diagnosis of lung lesions.

Materials and Methods: All ENB-guided FNAs performed at one institution were included in the study. The superDimension / bronchus system (superDimension Inc, Plymouth, Michigan, USA) was used in all cases. The pathological reports of the ENB-guided FNAs, as well as all the other pathological material, obtained within an 18-month period, were reviewed. Patients with a positive ENB-guided FNA or malignancy within the same lobe were considered positive for malignancy. Patients with an atypical diagnosis but no definitive malignancy were considered negative for malignancy, for statistical purposes. Histological diagnosis was considered the gold standard when available.

Results: Ninety-one patients underwent 95 ENB-guided FNAs over a three-year period (32 RUL, 8 RML, 14 RLL, 28 LUL, 2 lingula, and 11 LLL). Thirty-four patients (37%) were positive for malignancy during the study period (14 adenocarcinoma, 9 squamous cell carcinoma, 7 non-small cell carcinoma NOS, 1 small cell carcinoma, 3 other). ENB had a sensitivity of 65% and a specificity of 86% for the detection of malignancy [Table 1]. Of the patients who were negative by ENB-guided FNA and had confirmed malignancy by another technique - four were positive on concurrent bronchial biopsy, four were positive on concurrent bronchial brushings, and two were positive on subsequent FNA. Of the patients negative for malignancy on all procedures - seven patients (12%) had findings consistent with an infectious process by at least one procedure (1 aspergillus, 6 granulomatous inflammation). ENB-guided FNA identified the infectious process in three patients (43%).

Conclusions: Electromagnetic navigation bronchoscopy is a new technique that is not widely used yet. In our institution, ENB-guided FNA has a sensitivity of 65% and specificity of 86% for the diagnosis of malignancy. The overall diagnostic yield from any procedure performed at the time of ENB has been 74%. ENB-guided FNA demonstrates good utility for the diagnosis of difficult-to-access lung lesions, especially when used in conjunction with other techniques.


Clinical experience in the application of a novel mRNA-based gene expression classifier in thyroid nodules with indeterminate fine needle aspiration cytology

Robert Monroe, MD 1 , Richard Lanman, MD 1 , Giulia Kennedy, PhD 1 , Jonathan Romanowsky, MBA 1 , Ken Brunt, BS 1 , Katie O'Reilly, MD 2 , Carola Zalles, MD 2 , S. Thomas Traweek, MD 2

1 Veracyte, Inc., South San Francisco, California; 2 Thyroid Cytopathology Partners, Austin, Texas

Introduction: The Afirma® Gene Expression Classifier is a multi-gene expression test for the analysis of fine needle aspirates (FNA) from thyroid nodules, with indeterminate cytopathology. Postoperatively, two out o three of these indeterminate nodules were diagnosed as benign. Preoperatively, the test identified indeterminate nodules that were benign, to avoid diagnostic thyroid surgery. However, performance of the test in clinical practice has not yet been reported.

Materials and Methods: Diagnoses were made by a panel of cytopathologists experienced in interpretation of slides utilizing liquid-based preparation as well as direct smears, and classified them in accordance with the bethesda system for reporting thyroid cytopathology. Indeterminate cytopathology included atypia or follicular cells of undetermined significance (AUS / FLUS), follicular and Hürthle cell neoplasm or suspicious for same (FN / SFN), and suspicious for malignancy. Reflex molecular testing was performed on FNAs with indeterminate cytopathology, utilizing the mRNA expression of 142 genes and a proprietary algorithm, as previously described.

Results: The first 1,025 consecutive FNA samples were submitted by 62 physicians practicing in academic (9%) and community settings (91%). The cytopathologists reviewed 928 FNAs and reported 70.5% as benign, 0.4% as malignant, 15.4% as indeterminate, and 13.7% as nondiagnostic. Ninety-seven FNAs were submitted directly to the gene expression classifier. The gene expression classifier results on the 226 indeterminate samples were: 53% benign and 38% suspicious (meaning not definitively benign), and 9% 'no result', [Figure 1]. All the 'no result' samples were AUS / FLUS. One hundred and twenty-four FNA samples with indeterminate subtype were classified in accordance with Bethesda by the cytopathology panel. Of these, 25% of the cytological suspicious for malignancy were molecular benign, as were 56% of the FN / SFN and 53% of the AUS / FLUS, [Table 1].

Conclusions: The benign, indeterminate, and nondiagnostic rates we report are consistent with the literature, however, our cytology malignant rate is lower. Fifty percent of the nondiagnostic samples were submitted by eight of 62 physicians (13%), suggesting that the nondiagnostic rates may be related to FNA technical skill or selection bias. There was little difference in the cytology subtype AUS / FLUS and FN / SFN by molecular analysis, which had molecular benign results of 53 and 56%, respectively. The cytology subtype suspicious for malignancy was benign molecularly 25% of the time, somewhat lower than the 38% benign found postoperatively in a recent meta-review. On the basis of the test specificity reported in two prospective validation studies, and the risk of malignancy in cytologically indeterminate nodules (34%), one would expect the molecular result to be benign in approximately 50% of the cases. This projection has now been accurately reflected in actual experience. A comparison of the rate of benign results by molecular analysis found slightly higher rates at the academic sites versus community-based sites, but the difference was not statistically significant. The molecular test has the potential to reduce the number of diagnostic thyroidectomies on nodules, with indeterminate FNA cytology, by more than 50%.


Identification of tumor proteins associated with early stage of lung adenocarcinoma by quantitative proteomics

Qing Li, MD, PhD, Yan Li, PhD, Frederic Askin, MD, Edward Gabrielson, MD, Hui Zhang, PhD

Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Introduction: Lung cancer is one of the leading causes of cancer-related deaths worldwide, and adenocarcinoma is now the most common histological variant. Although the targeted therapies are progressed rapidly in recent years, based on the discovery of molecular markers, the early detection of lung cancer and identification of prognostic biomarkers are still suboptimal. Over 60% of lung cancer patients present as locally advanced or metastatic disease at the time of diagnosis; and a progression-free survival rate is markedly different even between stage I and stage II cancers. Few previous studies have focused on the identification of tumor proteins in the early stage of lung cancer. In this study, we quatitatively analyzed and compared tumor proteins in both stage I and II lung adenocarcinomas using novel quantitative proteomics.

Materials and Methods: Paraffin-embedded lung adenocarcinoma tissues were collected and tumor cells were microdissected. Peptides from tumors, including eight cases of stage I and eight cases of stage II, were extracted and labeled with eight-channel iTRAQ reagents. The labeled peptides were identified and quantified by liquid chromatography-mass spectrometry (LC-MS) / MS using an LTQ Orbitrap Velos mass spectrometer. The candidate proteins were further evaluated by immunohistochemistry in lung cancer TMAs.

Results: A total of 1288 peptides (370 proteins) were identified and quantified. Among them, 173 proteins showed greater than a 1.5-fold difference between stage I and stage II tumors. Fifteen proteins showed significant changes in expression during tumor progression. The particularly interesting ones include fibrillin-2, annexin A5, myeloperoxidase, eukaryotic translation initiation factor 4A1 (eIF-4A1), and mucin-5B. Potential cellular functions of these proteins are summarized in [Table 1].

Conclusions: By using quantitative iTRAQ proteomic analysis, we are able to identify tumor proteins associated with early stage of lung adenocarcinoma. The significance of the differential expression of these staging-associated proteins is not well-studied in lung cancer, and they may be used as new potential protein biomarkers in both early detection of lung cancer and prediction of tumor progression. A further study is needed to validate these protein markers in lung cancers.


Assessment of nuclear nano-architecture characteristics for cervical cancer screening

Yang Liu, PhD 1 , Rajan Bista, PhD 2 , Shikhar Uttam, PhD 2 , Pin Wang, PhD 2 , Chengquan Zhao, MD 3

1 Departments of Medicine and Bioengineering, 2 Medicine, 3 Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Introduction: Cervical cancer is the second most common cancer in women worldwide. Papanicolaou (Pap) cytology is an established screening technique that has led to a significant reduction of incidence and mortality in cervical cancer. However, among the 55 million Pap cytology tests performed in the United States each year, only less than one percent of the patients have high-grade cervical intraepithelial lesions that require further extensive and invasive examination and treatment. Our recently developed novel optical technology - Spatial-domain Low-coherence Quantitative Phase Microscopy (SL-QPM) takes advantages of the ultra-sensitivity of the light interference effect and allows the detection of subtle structural alterations in the cell nuclei This technique is able to detect a minute change in nuclear structure, as small as 0.9 nm, on a scale ~1000 times smaller than what a conventional microscope can distinguish. This instrument can be directly used on the standard liquid-based Pap cytology slides without any modification. The goal of this pilot study is to evaluate the capability of SL-QPM-derived nuclear nano-architecture characteristics, to identify those patients who are likely to have high-grade squamous lesions on the follow-up colposcopy, in women with atypical Pap cytology diagnosis.

Materials and Methods: We performed SL-QPM on archived liquid-based Pap cytology specimens from 109 women (23 LSIL, 22 HSIL, 64 ASC-US) who underwent regular cervical cancer screening. Among those 64 ASC-US cases, 25 women had negative high-risk human papillomavirus (hrHPV) test (Hybrid Capture II) and 39 had positive hrHPV test (20 women had follow-up biopsies of CIN2 / 3). The slides were reviewed by a cytopathologist who marked the cells of interest for SL-QPM analysis. We analyzed the cell nuclei from approximately 15 to 20 cells per patient.

Results: A series of SL-QPM-derived nuclear nano-architecture were derived from both cytologically normal intermediate squamous cells and abnormal diagnostic cells, such as, average nuclear optical path length (a measure of nuclear density), intra-nuclear structural heterogeneity, and intra-nuclear entropy. For patients with HSIL and LSIL, these nuclear nano-architecture parameters were statistically different in normal, LSIL, and HSIL cells (P < 0.00001). For women with ASC-US cytology, they could distinguish cases with positive hrHPV from negative hrHPV with a very high level of statistical significance (P < 0.0001). More importantly, SL-QPM-derived nuclear nano-architecture parameters could distinguish those ASC-US cases with CIN2 / 3 on the follow-up biopsies with statistical significance (P < 0.05, 90% sensitivity and 74% specificity) from those ASC-US cases with positive hrHPV test without CIN2 / 3, in the follow-up biopsies.

Conclusions: The use of SL-QPM derived nuclear nano-architecture parameters represents a novel type of optical biomarker for cervical cancer screening. This optical biomarker could be incorporated into a diagnostic algorithm in which those patients with ASC-US Pap cytology would undergo SL-QPM analysis. Future studies are warranted to improve and evaluate this novel approach through the expansion of our study population and the identification of additional optical or other biomarkers to improve the prediction accuracy.


Uveal melanoma prognostication: Analysis of a method using fine needle aspiration and fluorescence in-situ hybridization

Abberly Lott Limbach, MD 1 , Arun Singh, MD 3 , Raymond Tubbs, DO 2 , Mary Turell, MD 3 , Dana Weber, CT(ASCP) 1 , Charles Biscotti, MD 1

1 Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio; 2 Molecular pathology, Cleveland Clinic Foundation, Cleveland, Ohio; 3 Ophthalmology, Cleveland Clinic Foundation, Cleveland, Ohio

Introduction: Uveal melanoma patients can be segregated into two prognostic groups. Approximately half will develop metastases, while the other half will be cured with treatment of the uveal lesion. Unfortunately, the prognosis is cast by the time of diagnosis. Most experts agree that occult metastases exist at the time of primary diagnosis. The presence of monosomy 3 identifies the poor prognosis patients. Fluorescence in-situ hybridization (FISH) can identify monosomy 3. Unfortunately, FISH methods have varied greatly. Cytological samples are well suited for FISH analysis because they lack the artifacts associated with tissue sections. We have prospectively analyzed a series of uveal melanoma patients, in part, to determine the effectiveness of a prognostication technique that includes fine needle aspiration (FNA) and FISH analysis.

Materials and Methods: All patients had a clinical diagnosis of uveal melanoma based on the history and physical examination, including an ophthalmoscopic examination performed by one of the authors (ADS). The clinical assessment, most importantly the tumor size, determined whether the patients were treated with plaque radiotherapy or enucleation. FNA using a 25 G needle was performed at the time of plaque placement or enucleation by two of the authors (ADS and MET). In vivo samples were obtained by the transvitreal or transscleral approach, depending on the tumor location. Transvitreal samples were limited to a single pass. The aspirate samples were entirely rinsed into 2 - 3 ml of normal saline, visually examined, then submitted to cytology in CytoLyt (Hologic Corp, Marlborough, MA). ThinPrep® processing yielded four Papanicolaou stained slides per case. One of the authors (CVB) examined all the slides and depending on the cellularity, submitted up to three slides per case for FISH analysis. We counted CEP3 signals and used a 20% threshold for monosomy 3. Slides adequate for monosomy 3 evaluation had at least 200 cells counted.

Results: The 84 patients included 42 (50%) women and 42 men ranging in age from 26 to 91 years (mean 61 years). The tumors involved the choroid (59 cases, 70%), ciliochoroid (18 cases, 21%), iridociliary (4 cases, 4.8%), and iris (3 cases, 3.6%). Tumors ranged from 3.0 x 3.3 mm to 16 x 16 mm in basal dimensions and 1.2 mm to 15 mm in maximal height. FNA confirmed melanoma in 74 (88%) cases. Ten (12%) samples were insufficient for diagnosis. All diagnostic samples had melanoma cells well preserved for FISH analysis. Fifty-six (67%) patients had in vivo, transvitreal (55%) or transscleral (45%), aspirates. Thirty-seven (66%) of these had cellularity adequate for a 200 cell count FISH analysis. Twenty-eight (33%) patients had enucleation specimens aspirated and 26 (93%) of these had cellularity adequate for a 200 cell count FISH analysis. Overall, 43% of the tumors had monosomy 3.

Conclusions: Fine needle aspiration combined with FISH effectively identifies monosomy 3 in uveal melanomas. Overall, 88% of the cases in our series had diagnostic aspirate samples including cellularity adequate for a 200 cell count FISH analysis in 93% of the enucleation cases and 66% of the in vivo aspirate samples. The lower adequacy rate in the in vivo group is limiting, but not unexpected, as by definition, these tumors are smaller and thinner than those treated by enucleation.


Nanoscale nuclear architecture for urine cytology using spatial-domain low-coherence quantitative phase microscopy

Liron Pantanowitz, MD 1 , Rajan Bista, PhD 2 , Walid Khalbuss, MD, PhD 1 , Rajiv Dhir, MD 1 , Randall Brand, MD 2 , Sara Monaco, MD 1 , Yang Liu, PhD 3

1 Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 2 Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 3 Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania

Introduction: Definitive diagnosis of urothelial carcinoma in urine specimens based on cytomorphology alone may be challenging. Ancillary techniques such as fluorescence in-situ hybridization (FISH) have thus been employed to improve the sensitivity of cytology. We propose the use of a novel optical technique - spatial-domain low-coherence quantitative phase microscopy (SL-QPM) for analyzing nuclear architectural characteristics on the original unmodified cytology specimens with the goal of improving the sensitivity of urine cytology. This technique is able to detect a minute change in nuclear structure as small as 0.9 nm, a scale of about 1000 times smaller than what a conventional microscope detects. The aim of this study is to explore the ability of SL-QPM to improve the identification of malignant cells in urine cytology samples.

Materials and Methods: Urine samples were processed with ThinPrep® and submitted for FISH (Urovision). Urothelial cells from four benign (FISH-negative) and three positive (FISH-positive) cases for urothelial carcinoma were analyzed with SL-QPM, using Pap-stained ThinPrep® slides. The urothelial cells were classified into one of the three following scenarios: Benign cells in benign cases, morphologically benign-appearing cells in positive cases (i.e., uninvolved), and malignant cells in positive cases. SL-QPM-derived nanoscale nuclear architecture characteristics described by several optical parameters (i.e., optical path length, refractive index fluctuation ratio, intra-nuclear entropy, and uniformity) were analyzed from individual cell nuclei (4 - 25 cells / slide).

Results: All the SL-QPM-derived optical parameters were significantly different between morphologically benign and malignant urothelial cells (P = 5E-11). More importantly, the statistically significant differences for these optical parameters were also demonstrated among morphologically benign urothelial cells in both FISH-negative and FISH-positive cases (P = 0.03), as shown in [Figure 1].

Conclusions: We show that quantitative analysis of urothelial cell nuclei can be performed using original cytology slides without additional processing. Imaging of nanoscale nuclear architectural characteristics can readily identify malignant urothelial cells in Pap-stained urine specimens. The presence of nanoscale abnormalities in benign appearing cells from known cancer cases indicates a nuclear field defect that may not be evident by cytomorphology alone. This practical optical microscopy technique holds great promise as an ancillary technique to help improve the sensitivity of urine cytology of urothelial cancer and warrants further study.


Molecular diagnostics of metastatic melanoma utilizing cytological direct smears

Michael Roh, MD, PhD, Kim Hookim, MD, Joseph Willman, MD, Jeremiah Placido, MD, Helmut Weigelin, MLS(ASCP), Kristina Fields, BS, Judy Pang, MD, Bryan Betz, MD, Stewart Knoepp, MD

Pathology, University of Michigan Medical School, Ann Arbor, Michigan

Introduction: Activating mutations in the BRAF oncogene are present in at least 40% of the melanomas, with V600E and V600K representing the most commonly observed mutations. Clinical trials currently investigating the efficacy of targeted chemotherapy against mutant BRAF exemplify the heightened emphasis on personalized medical care. Hence, the use of fine needle aspiration (FNA) specimens of melanoma to assess BRAF mutations is increasing. Cell blocks are traditionally used for these studies. However, cell blocks exhibit variable cellularity, and in a significant proportion of cases, are of insufficient tumor cellularity. Direct smears represent an alternative platform for ancillary studies. Previously, we successfully demonstrated the application of immunocytochemistry as well as the epidermal growth factor receptor (EGFR) and KRAS mutational analyses to the cytological direct smears prepared from FNAs of non-small cell lung carcinomas. In this study, we investigate the application of immunocytochemistry and BRAF mutational analysis to direct smears prepared from FNAs of metastatic melanoma.

Materials and Methods: First, immunocytochemistry for S-100, HMB45, and Mart-1 was prospectively performed on air-dried, unstained, direct smears following brief formalin fixation (30 - 60 minutes), and also antigen retrieval in 16 consecutive FNAs of metastatic melanoma. Next, in 15 additional cases obtained from the archive, BRAF sequencing was performed using genomic DNA isolated from microdissected tumor cell-enriched areas on de-coverslipped, Diff Quik-stained smears. In parallel, BRAF sequencing was performed using DNA extracted from the corresponding cell blocks.

Results: Of the 16 melanoma FNAs in which immunocytochemistry was performed on unstained direct smears, S-100 positivity in the tumor cells was observed in all cases. HMB45 and Mart-1 positivity was seen in 80 and 88% of the cases, respectively. All three markers were positive in 75% of the cases [Figure 1]. Next, for the 15 archived melanoma FNAs, the cellular material microdissected from Diff-Quik® stained smears [Figure 2] yielded high quality genomic DNA in all cases. Mutations in BRAF were observed at the expected frequency; eight (53%) of the fifteen cases tested positive. Five and three melanomas harbored the V600E and V600K mutations, respectively [Figure 3]. Concordant sequencing results for cellular material obtained from direct smears and cell blocks were observed in 14 (93%) of the 15 cases; in one case, the mutation was not detected in the cell block material, whereas, the V600E mutation was detected in material obtained from the direct smear. There were no cases in which a BRAF mutation was detected in the cell block material, but not in the cellular material from the smears.

Conclusions: This study demonstrates that direct smears represent a robust and valuable source of cellular material for ancillary studies utilized in the cytological diagnosis of melanoma. Unstained, air-dried smears can be effectively utilized for confirmatory immunocytochemical studies. Furthermore, Diff-Quik® stained smears allow for direct visualization and microdissection of tumor-enriched areas for DNA isolation and subsequent BRAF mutation analysis. The application of these ancillary studies to cytological smears can be effectively applied, especially in scenarios when inadequate cell block cellularity is anticipated or encountered.


Emerin: A useful immunohistochemical marker for highlighting nuclear membrane irregularities in papillary thyroid carcinoma

Mary Kinsella, Cynthia Cohen, Momin Siddiqui

Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, Georgia

Introduction: The basic cytological diagnosis of papillary thyroid carcinoma (PTC) and its distinction from follicular neoplasia (FN) relies on a constellation of sometimes subtle morphological changes in the nucleus, including shape, chromatin distribution, and the presence of intra-nuclear inclusions and grooves. Immunohistochemical staining for Emerin, an integral inner nuclear membrane protein, has recently been shown to highlight fine nuclear membrane irregularities and structural changes in PTC that are not apparent with conventional H and E staining. The purpose of this study is to examine the diagnostic utility of emerin IHC in the cytological preparations of PTC and FN.

Materials and Methods: Alcohol- or formalin-fixed, paraffin-embedded cell blocks from FNAs of PTC or FN were stained with anti-Emerin polyclonal antibody and were subsequently evaluated for nuclear grooves, circumferential nuclear membrane irregularities (garlands), inclusions, deep stellate membrane invaginations, crescents, and nuclear shape variation. Additional clinical and pathological data, including patient gender and age, tumor size, and final histological diagnosis, were also obtained.

Results: A total of 34 FNA cases were evaluated, 24 were cytologically diagnosed as PTC and 10 as FN (three follicular variants of PTC on surgical follow-up). The final total number of PTC cases examined was 27, with seven FN. Emerin IHC of PTC revealed a predominantly oval nuclear shape in a majority of the cases (88%), with FN demonstrating round nuclei and FV of PTC showing a roughly equal distribution of round and oval shapes. In addition to the oval nuclear shape (P = 0.004), the presence of Emerin-positive nuclear grooves (P < 0.001), circumferential Emerin nuclear garlands (P = 0.006), and chromatin clearing on H and E staining (P = 0.02), in combination, were significant predictors of PTC by regression analysis. The average age of the patients was 49 years, and the average tumor size was 2.5 cm.

Conclusions: Emerin IHC of thyroid FNA specimens serves as a useful adjunct to conventional H and E staining in the diagnosis of PTC and its distinction from FN, by delineating the diagnostic nuclear membrane irregularities (garlands), nuclear grooves, and a characteristic oval nuclear shape. For diagnostically challenging cases with limited cellularity, emerin staining can help in providing a definitive diagnosis of PTC.


Methylation of O6-methylguanine-DNA methyltransferase as a chemosensitive biomarker in glioblastoma in fine needle aspiration biopsy samples

Bin Yang, MD, PhD, Rosemarie Read, PhD, Raymond Tubbs, DO

Pathology, Cleveland Clinic, Cleveland, Ohio

Introduction: O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that specifically removes promutagenic alkyl groups from the O6 position of guanine in DNA. Repair of O6-alkylguanine adducts by tumor cells has been implicated in drug resistance, as it reduces the cytotoxicity of alkylating chemotherapeutic agents. Recent studies indicate that patients with MGMT methylation have a better response to temozolomide and their median survival nearly doubles compared to those with lack of MGMT methylation. In order to provide this molecular biomarker clinically in triage patients for chemotherapy, we have developed and validated the MGMT methylation assay with pyrosequencing on the Fine Needle Aspiration Biopsy (FNAB) samples of glioblastoma.

Materials and Methods: Promoter methylation of MGMT was quantitatively analyzed by Pyro Q96. MGMT methylation was tested and validated with 33 cases of glioblastoma and 10 cases of non-neoplastic brain tissue. The analytical sensitivity and minimal requirement of the DNA amount was evaluated with two cell lines, a cancer cell line harboring of MGMT methylation and a normal cell line with lack of MGMT methylation. The analytical sensitivity between pyrosequencing and methylation-specific polymerase chain reaction (PCR) is also compared.

Results: Methylation of MGMT was identified in 33% (11 / 33) of the cases of glioblastoma and none in the epilepsy brain tissue. The average methylation level of CpG islands in the MGMT promoter was > 30% in glioblastoma and < 3% in epilepsy brain. By a series dilution of a methylated cancer cell line with an unmethylated normal cell line, the analytical sensitivity for pyrosequencing detection of MGMT methylation was at 5%. The minimal amount of genomic DNA required to successfully detect MGMT methylation by pyrosequencing was at 100 ng (approximately 3,000 cells). In comparison with MSP, pyrosequencing was comparably sensitive with less false-positive cases and also provided a quantitative methylation value for each CpG island.

Conclusions: We have validated the MGMT methylation assay clinically in providing a powerful biomarker for triaging patients with glioblastoma for chemotherapy. This assay can be performed both on FNAB tissue blocks and on cytological smears. We demonstrate that pyrosequencing detection of MGMT methylation has an analytical sensitivity suitable for clinical utility.[Figure 1]


  [Figure 1]


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