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Introduction: Cytology samples are frequently relied upon for the diagnosis of advanced cancer such as lung cancer. As the recommendations for solid malignancies biomarker testing continue to expand, it becomes increasingly important to efficiently utilize limited specimens to minimize the need for additional sampling and its associated risks and costs., Materials and Methods: We performed molecular testing on fresh or CytoLyt-fixed supernatants derived from fine needle aspirates (FNAs) and compared its performance against the clinical specimen (including formalin-fixed paraffin-embedded cell blocks, residual PreservCyt and fresh samples). Supernatants were assessed for cellularity using Field-stained Cytospin (CS) preparations., Results: There was overall almost perfect agreement (41/45 cases, K = 0.822) and substantial to almost perfect agreement in molecular testing results of clinically actionable variants between fresh (20/23 cases, Κ = 0.742) and CytoLyt-fixed (21/22 cases, Κ = 0.908) and its clinical specimen counterpart. Interestingly, CS examination of the supernatants revealed viable tumor cells. Centrifugation for 1 minute at 300 rpm is optimal for overall or tumor cellularity recovery. Delayed molecular testing after 3, 4 and 7 days at 4 degrees Celsius showed identical molecular results., Conclusions: We validated the use of supernatants derived from FNA cytology samples as a substrate for molecular testing using next-generation sequencing and other molecular techniques., (Copyright © 2022 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Background: Testing for BRCA1/2 gene alterations in patients with high-grade serous carcinoma (HGSC) is a critical determinant of treatment eligibility for poly(adenosine diphosphate-ribose) polymerase inhibitors in addition to providing vital information for genetic counselling. Many patients present with effusions necessitating therapeutic drainage, and this makes cytologic specimens (CySs) the initial diagnostic material for HGSC, often before histologic sampling. Initiating somatic BRCA testing on a CyS allows the BRCA status to be determined sooner, and this affects clinical management., Methods: Retrospectively, 8 cases of formalin-fixed, paraffin-embedded (FFPE) CySs of peritoneal or pleural fluid from patients with HGSC and known BRCA1/2 alterations previously established by the testing of FFPE surgical specimens (SpSs) underwent next-generation sequencing (NGS). Prospectively, 11 cases of peritoneal or pleural fluid from patients with HGSC but an unknown BRCA1/2 status underwent NGS with fresh, alcohol-fixed, and FFPE CySs, and they were compared with subsequent NGS on 4 SpSs., Results: CySs yielded high-quantity and high-quality DNA for NGS analysis when sufficient tumor cellularity was present. Fresh, alcohol-fixed, and FFPE CySs were all suitable for NGS and provided identical NGS results. SpS and CyS BRCA testing was concordant in 10 of 12 cases. The 2 discordant cases showed low tumor cellularity and quality in the CyS and the SpS, respectively., Conclusion: Effusion CySs of HGSC are excellent sources for NGS testing for BRCA1/2 genetic alterations when sufficient tumor cellularity is present. Fresh, alcohol-fixed, and FFPE CySs are equivalent for NGS of BRCA1/2. NGS testing of HGSC CySs demonstrates good concordance with SpSs for the BRCA1/2 status., (© 2021 American Cancer Society.)

Objective: To assess consistency and long-term progress in thyroid biopsy performed by trained sonographers under supervision of a radiologist., Methods: Trained sonographers started performing thyroid biopsy at our institute in August 2011. The data for this study were extracted from a prospectively maintained database for ultrasound guided thyroid biopsy and included the number of thyroid fine needle aspiration biopsy procedures performed between August 2011 and 2016 and the final cytopathology report as per the Bethesda Classification. For the analysis, the study was divided into two time periods: initial postimplementation period (August 2011 to 2013) and late postimplementation period (2014-2016)., Results: In all, 5,538 thyroid biopsies were performed by trained sonographers in the period, 2,561 in the initial implementation period and 2,977 between 2014 and 2016. The unsatisfactory rates dropped from 21% to 10% in the two periods (P < .001), and the proportion of malignant nodules on cytopathology increased from 6% to 7% in the two periods (P = .010). Wait times for thyroid biopsies remained low during the period., Conclusion: Sonographers trained to perform ultrasound guided thyroid biopsies provide persistent improved patient care over a long-term period. This reinforces the role of physician extenders in targeted scopes of practice., (Copyright © 2020. Published by Elsevier Inc.)

Background: Programmed death ligand-1 (PD-L1) assessed by immunohistochemistry (IHC) is used as biomarker for pembrolizumab therapy in advanced stage lung cancer patients. However, data permitting direct performance comparison between cytology and surgical specimen types are limited since both specimens from a single tumor site are infrequently available. In addition, alcohol fixation used with cytology specimens requires technical validation of the PD-L1 IHC assay before clinical use. We here report our experience with implementation of the PD-L1 22C3 IHC pharmDxTM assay for cytologic samples at a large tertiary cancer center., Study Design: Archival formalin-fixed (FF), paraffin-embedded cell blocks (CBs) and subsequent lung tumor resections (LTRs) from the same anatomical site were used for a direct comparison of PD-L1 tumor proportion scores (TPSs). TPS values were independently determined by one surgical lung pathologist and two cytopathologists blinded to the specimen pairs. An interim analysis was performed to facilitate the pooling of expertise among observers. After PD-L1 22C3 IHC pharmDxTM implementation for FF cytology specimens, dual-processed samples were used for a prospective technical validation of CytoLyt® prefixation (CF). Digital image analysis was performed for a subset of dual-processed specimens., Results: Eighty-one CBs and LTRs were included for comparison of the specimen types. PD-L1 assessment in CBs had an accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 88.9/72.8, 66.7/73.5, 95.2/72.3, 80.0/65.8, and 90.9/79.1% for the ≥50/≥1% cutoff, respectively. The intraclass correlation coefficient was 0.84 (95% confidence interval [CI]: 0.76, 0.90), and it improved after interim analysis (before: 0.79 and after: 0.92). The overall concordance between CF and FF for the categories defined by the ≥50/≥1% cutoff values was 90.4% (95% CI: 79.0, 96.8). Similar assay performance was confirmed by digital analysis., Conclusions: PD-L1 22C3 IHC pharmDxTM shows good reliability if used with CB preparations. CF does not impact assay results significantly. Clinical validation with outcome data is needed, and digital methods of assessment should be further investigated., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Background: The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported., Methods: Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients., Results: Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months., Conclusions: ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing., (© 2019 American Cancer Society.)

Purpose: Fine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor., Patients and Methods: Eligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined., Results: Forty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield from CNB and FNB were 775 ng (interquartile range, 240 to 347 4ng) and 649 ng (interquartile range, 180 to1350 ng), respectively. Of 29 CNB/FNB pairs available for comparison, actionable mutation results were concordant in 28 (96%). Six of nine actionable mutations (67%) that were found by CNB, FNB, or both were detectable in ctDNA. Two additional actionable mutations were found exclusively in ctDNA., Conclusion: Optimally processed FNB and liquid biopsy can be used routinely for tumor mutation profiling to identify actionable mutations.

Objective: Fine needle aspiration (FNA) is widely used in the diagnosis of metastatic melanoma, both at initial presentation and in the setting of recurrent disease. The purpose of this study was to evaluate the performance of confirmatory immunohistochemistry (IHC) and molecular analysis of the BRAF mutation in cytological preparations of metastatic melanoma., Methods: A 2-year retrospective review of pathology reports was performed on cytological samples of metastatic melanoma at the University Health Network (Toronto, Canada) and the Santa Casa Medical School (Sao Paulo, Brazil). IHC was performed on cell block sections prepared from formalin-fixed, fresh samples or residuum of CytoLyt/PreservCyt post-fixed in formalin. BRAF V600E/K mutations were assessed by amplification refractory mutation system (ARMS) analysis., Results: A total of 104 samples (94 FNAs and 10 fluids) from 83 patients (20 women, 63 men) were included. IHC was attempted in 43 cases (41.3%) and successful in 41 (95.3%). The panel number of antibodies ranged from 1 to 15 (median 3). The most frequently used melanoma markers included HMB-45, melanoma cocktail and S100 protein, used in 25 (58.1%), 23 (53.5%) and 18 samples (41.9%). Thirty cases (69.8%) used three or fewer markers. The BRAF V600E/K mutation was tested in eight samples, being successful in seven (87.5%) and positive in three (37.5%)., Conclusions: Cytological samples are a reliable and sufficient source for IHC and subsequent molecular analysis, allowing a reduced diagnostic time and rapid, appropriate treatment options in patients with advanced melanoma., (© 2017 John Wiley & Sons Ltd.)

Background: Although many studies have assessed the diagnostic utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the context of a specific disease, few studies have assessed the overall diagnostic yield, sensitivity, and negative predictive value in patients with isolated mediastinal and hilar lymphadenopathy (IMHL)., Objective: We evaluated the performance of EBUS-TBNA for diagnosing IMHL in a population with a high prevalence of concurrent or preexisting non-pulmonary malignancy., Methods: A retrospective chart review of patients who underwent EBUS-TBNA from October 2008 to April 2014 was performed to identify patients with IMHL. Patients with known or suspected primary pulmonary malignancy were excluded. When available, EBUS-TBNA results were cross-referenced with further diagnostic investigation or clinical diagnosis based on follow-up., Results: EBUS-TBNA was used to sample 765 lymph nodes from 350 patients. One hundred and fourteen (33.3%) patients had a concurrent or preexisting non-pulmonary malignancy. The overall yield of EBUS-TBNA for specific diagnosis was 300/350 (86%). The diagnostic yield for sarcoidosis, lymphoproliferative disease, metastatic lymphadenopathy from extrathoracic malignancy, and necrotizing granuloma was 123/149 (83%), 27/33 (82%), 20/25 (80%), and 13/19 (68%), respectively. Amongst 50 patients with non-diagnostic EBUS-TBNA, 25 yielded an insufficient sample and another 25 yielded only benign lymphoid material which was not representative of the underlying pathology. Overall, EBUS-TBNA had a sensitivity of 89%, a diagnostic yield of 86%, and a negative predictive value of 79%., Conclusion: For patients with isolated hilar or mediastinal lymphadenopathy and a high background prevalence of concurrent and preexisting non-pulmonary malignancy, EBUS-TBNA is a reliable first-line diagnostic investigation., (© 2017 S. Karger AG, Basel.)

The AFC in cytopathology program was created in 2012 to address the societal need in Canada for Cytopathology leaders with in-depth specific knowledge of Cytopathology diagnostics, laboratory management, quality assurance and risk management to ensure high quality and accurate patient outcomes. So far, the developed AFC program in Cytopathology has been successfully implemented in three academic centres, and it stands as a model for competency-based advanced training in Cytopathology., (Copyright © 2016 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To develop and implement a program where selected sonographers would be trained to perform thyroid biopsies independently under the supervision of a radiologist, with the goal of improving efficiency and quality., Materials and Methods: Institutional research ethics board approval was obtained for this retrospective study, with waiver of informed consent. After approval from the relevant regulatory bodies, four sonographers successfully completed a training program and began to perform all thyroid biopsies (with informed consent) in a room adjacent to the main radiologist-run biopsy room, where the radiologist was available for backup as needed. In the preimplementation period (January 2010 to April 2011), 1321 nodules were biopsied, 29 of which included on-site cytopathology assessment. In the postimplementation period (August 2011 to July 2012), 1347 nodules were biopsied, 103 of which underwent on-site cytopathology assessment. Wait times and adequacy rates were calculated for both periods., Results: Patient wait times decreased from a mean of 80-90 days before implementation of the thyroid biopsy specialist program to 20-30 days afterward. The percentage of adequate samples improved from 74.6% (985 of 1321 nodules) to 78.6% (1059 of 1347 nodules), with a P value of .015 (74.1% [957 of 1292 nodules] to 77.5% [964 of 1244 nodules] when excluding nodules with on-site cytopathology assessment, P = .0497). The percentage of malignant samples showed no significant change in the two time periods, 5.1% (68 of 1321 nodules) before implementation of the program versus 5.4% (73 of 1347 nodules) after implementation, P = .823 (5.1% [66 of 1292 nodules] vs 5.3% [66 of 1244 nodules] in the respective time periods when excluding nodules with on-site cytopathology assessment, P = .888). No major procedural complications occurred., Conclusion: Sonographers can be successfully trained to perform ultrasonography-guided thyroid biopsies safely under the supervision of a radiologist, which can improve wait times and adequacy rates.

Background: Gene rearrangements and specific translocations define some B-cell non-Hodgkin lymphoma (NHL) subtypes. Genome-wide mutational studies have revealed recurrent point mutations with prognostic implications. The goals of this study were to evaluate the feasibility of applying a multiplex mutation assay to archival cytospin preparations (CPs) and to investigate the rate of EZH2, CD79B, and MYD88 mutations in B-cell NHL samples previously tested for MYC rearrangement and/or IGH/BCL-2 translocation., Methods: DNA was extracted from archival CPs of B-cell NHL cases with previous fluorescence in situ hybridization (FISH) assays for MYC rearrangement and/or IGH/BCL-2 translocation. Multiplex sequencing was performed for the detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations. Sanger sequencing was applied to samples with positive results and failed assays., Results: Eighty-eight archival CPs were available from 40 patients. Alterations detected by FISH were: MYC rearrangement (10 cases), IGH/BCL-2 translocations (21 cases), dual translocations (6 cases), and other abnormalities for IGH/BCL-2 (23 cases) and for MYC (16 cases). DNA concentration ranged from 1.88 to 62.85 ng/µL (mean, 9.46 ng/µL). Successful results were obtained in 88.0% of the specimens submitted to multiplex sequencing. With Sanger sequencing, 2 additional mutated cases were found, and all cases with mutations were confirmed. Eight specimens showed mutations: 6 for EZH2, 1 for CD79B, and 1 for MYD88. Among them, 5 cases showed concurrent MYC and/or IGH/BCL-2 translocations and 2 revealed abnormal signals of IGH/BCL-2 and MYC., Conclusions: CPs archived for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Almost all B-cell NHL with point mutations showed concurrent chromosomal abnormalities., (© 2015 American Cancer Society.)

Objective: This study investigated a published series evaluating the role of second-opinion diagnosis (SOD) or repeat fine-needle aspiration cytology (RFNA) for indeterminate thyroid aspirates., Study Design: Twenty-three studies were selected and the following parameters were analyzed: disagreement between SOD or RFNA and the original diagnosis (OD), reclassification of OD according to the Bethesda system for reporting thyroid cytopathology, the rate of definitive diagnosis and the diagnostic performance of SOD and RFNA., Results: 7,154 thyroid FNAs were retrieved from 9 studies that investigated the role of SOD, including 1,048 (14.6%) cases originally reported as indeterminate. The 14 studies that analyzed the role of thyroid RFNA comprised 67,581 FNAs and included 7,246 (10.7%) indeterminate cases. A definitive diagnosis was achieved by SOD in 450 cases (42.9%) and RFNA in 1,645 cases (57.2%, p=0.0001). Based on cases with histological follow-up, SOD demonstrated significantly higher rates of positive predictive value and accuracy than RFNA (55.8 vs. 37.7%, p=0.0001; 67.4 vs. 56.0%, p=0.0034, respectively)., Conclusions: Both SOD and RFNA demonstrated an improvement in the diagnosis of initially indeterminate thyroid FNAs. RFNA achieved a definitive diagnosis for the majority of indeterminate cases. Regarding histological follow-up, SOD was shown to be more accurate than RFNA., (© 2015 S. Karger AG, Basel.)

Background: Second-opinion diagnosis (SOD) on pathological material is an accepted practice before definitive therapy is considered for referred patients. The thyroid gland is an anatomical site prone to diagnostic disagreement between pathologists. We performed a review of the literature that addressed the role of interinstitutional SOD on thyroid fine-needle aspirations (FNAs)., Methods: Nine studies comprising second opinions on thyroid FNAs were selected. The parameters analyzed included: discordances between the initial diagnoses (IDs) and SODs; cytohistologic correlation; changes in the clinical management of the patients with thyroid nodules after SOD. The same parameters were applied to the "indeterminate" diagnostic category comprising cases initially reported as "atypia," "atypia of undetermined significance/follicular lesion of undetermined significance," "suspicious for a follicular neoplasm," "follicular neoplasm," "suspicious," and "suspicious for malignancy.", Results: A total of 7154 thyroid FNAs were retrieved, showing an overall discordance rate between ID and SOD of 28.6%. In general, SOD was better supported by clinical follow-up and histological diagnosis, showing higher diagnostic accuracy in comparison with ID. Almost one-third (30.4%) of the discordant cases resulted in changes in the clinical management of patients with thyroid nodules. Numerous thyroid FNAs initially categorized as "indeterminate" were definitively classified as benign or malignant by SOD, with an overall diagnostic resolution rate of 42.5%, sensitivity of 97.9%, and diagnostic accuracy of 73.7%., Conclusions: Second-opinion review of thyroid FNA improves diagnostic accuracy and potentially changes clinical management. SOD also demonstrates a significative rate of diagnostic resolution for thyroid FNAs originally diagnosed as "indeterminate.", (© 2014 American Cancer Society.)

Objective: To review cytomorphological criteria and clinicopathological findings in combination with ancillary tests for the specific diagnosis of pulmonary marginal zone lymphoma (MZL) in fine needle aspiration (FNA) specimens., Methods: Cases of pulmonary MZL diagnosed using cytological specimens from 2005 to 2012 were retrieved and reviewed by three cytopathologists. Results of immunophenotypic analysis, interphase fluorescence in situ hybridization (FISH) and molecular assays were collated, together with clinical information and imaging data. Concurrent surgical biopsies were also retrieved., Results: Fifteen lung FNA specimens were identified. The smears consisted predominantly of small centrocyte-like cells. Marked plasma cell differentiation was evident in 11 cases. All cases with slides available showed tissue fragments with lymphoid tangles (TFLTs). Multinucleated giant cells were present in nine cases, two of which showed granulomas. Immunophenotyping confirmed B-cell clonality in all cases. B-cell clonality was detected by polymerase chain reaction (PCR) in two samples. FISH identified MALT1 translocation in four of 10 cases tested and trisomy 3 in three of four cases. Concurrent surgical biopsies were diagnosed independently as MZL in seven cases., Conclusions: Cytology smears from lung FNA samples consisting of small lymphoid cells with a relative abundance of plasma cells or plasmacytoid cells and large TFLTs should prompt immunophenotyping and other ancillary studies, even if multinucleated giant cells and poorly formed granulomas are also identified. Specific diagnosis of pulmonary MZL in FNA samples can be rendered on the basis of morphological features coupled with the demonstration of B-cell clonality by immunophenotyping or PCR and cytogenetic abnormalities by FISH., (© 2013 John Wiley & Sons Ltd.)

Introduction: Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing., Methods: Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity., Results: From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%)., Conclusions: EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

Objective: Detection of Epstein-Barr virus (EBV) status might help in the diagnosis of EBV-related neoplasms. The rate of successful assays for the detection of EBV-infected cells in cytological preparations has not been fully explored. Our aims were to examine the rate of successful in situ hybridization (ISH) assays for EBV-encoded RNA (EBER) in cytological specimens and to explore reasons for failure., Methods: An electronic search selected cases with ISH-EBER assays performed on cytological preparations during a 10-year period. Data regarding patient age, gender and immune status, sample type and site, type of preparation, ISH-EBER results, immunophenotyping and immunohistochemistry results, final diagnosis and correspondent histopathological samples were retrieved., Results: Sixty specimens from 58 patients with diagnoses of lymphoproliferative disorder (n = 35), carcinoma (n = 24) and sarcoma (n = 1) were identified. ISH-EBER assays were performed on 50 cell block sections and on 10 cytospin preparations, with 22 positive and 32 negative results. Six tests (four cytospins and two cell block sections) failed owing to loss of material during the assay and background staining, with an overall failure rate of 10% and 4% if cytospins were excluded. Assays were performed on 13 cytology and surgical specimens from the same site, with only one discrepant result., Conclusions: Cell block sections had more successful ISH-EBER assays when compared with cytospins. Reasons for failure were loss of material on the slide and background staining. A high concordance rate with surgical specimens emphasizes the usefulness of cytological samples for determining EBV status in patients with exhausted or no histological material available., (© 2013 John Wiley & Sons Ltd.)

Introduction: The value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has been established for staging mediastinal lymph nodes in lung carcinoma patients with radiologically enlarged lymph nodes, but its utility for evaluation of primary lymph node disorders is not well defined. The objective of this study was to evaluate the usefulness of EBUS-TBNA with on-site assessment and triage of sample for multiple ancillary techniques, for the diagnosis and subclassification of lymphomas and non-neoplastic lesions involving mediastinal lymph nodes., Methods: One hundred and twenty consecutive patients who underwent EBUS-TBNA between January 2008 and August 2009 were reviewed. The final cytological diagnosis was based on air-dried Romanowsky and alcohol-fixed Papanicolaou stained direct smears, immunohistochemistry, immunophenotyping, and fluorescence in situ hybridization (FISH)., Results: A total of 38 cases were included in this study consisting of eight reactive lymphoid hyperplasia, 20 granulomatous lymphadenitis (17 non-necrotizing and 3 necrotizing granulomatous inflammations), 3 Hodgkin lymphomas and 7 non-Hodgkin lymphomas (1 small lymphocytic lymphoma (SLL), 1 SLL with scattered Reed-Sternberg cells, 1 marginal zone lymphoma, and 4 large B cell lymphomas). Cultures performed in 13 cases were negative for AFB and fungi. Immunophenotyping and immunohistochemistry for MIB1 in six cases, and FISH in five cases provided necessary information for subclassification., Conclusions: EBUS-TBNA is a minimally invasive procedure which provides sufficient sample for definitive primary diagnosis and classification of malignant lymphoma and granulomatous inflammation in patients with mediastinal lymphadenopathy. Rapid on-site specimen assessment is invaluable for appropriate assignment of sample to ancillary studies., (Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.)

Background: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay., Methods: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing., Results: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up., Conclusions: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377-386. © 2013 American Cancer Society., (© 2013 American Cancer Society.)

Two cases are described of crystal storing histiocytosis (CSH) associated with extranodal marginal zone lymphoma, presenting as lung and subcutaneous masses respectively. Fine-needle aspiration of subcutis and smears prepared from the resected lung masses showed negative images. Cytology slides of both cases were reviewed to identify cytomorphological features for the differential diagnosis between immunoglobulin crystals and mycobacteria. The crystals in CSH consist of straight and needle shaped rods with pointed or angular edges and are more variable in thickness than the uniformly thin mycobacteria. Mycobacteria show a haphazard distribution, whereas crystals are frequently present in parallel arrays. Small lymphoid or plasma cells are identified in the background of CSH, whereas a necrotic and inflammatory background is seen in mycobacteriosis. Additional samples for culture in the case of mycobacteriosis, or flow cytometry and molecular clonality testing in the case of CSH can provide critical data for a definitive diagnosis., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: The differential therapeutic efficacy and toxicity of targeted therapies has made subtyping of non-small lung cancer (NSCLC) mandatory. This study aimed to review the accuracy of NSCLC subtyping using lung fine needle aspirates (FNAs) in two periods (before and after the introduction of targeted therapy), checking the reasons for failure and the impact of the use of immunohistochemistry (IHC)., Methods: An electronic search retrieved all NSCLC FNAs with a corresponding surgical specimen from 2001 to 2009. NSCLC, NOS (not otherwise specified) cases from 2005 to 2009 (after targeted therapy) were reviewed to determine reasons for failure in subtyping and to further subtype based solely on cytomorphology. The number of cases in which IHC was performed and the antibodies used were also recorded., Results: Cytohistological agreement of 602 lung FNAs (341 adenocarcinomas, 93 squamous cell carcinomas and 168 NSCLC, NOS) was achieved in 93.80%. There was a significant decrease in the percentage of cases not subtyped in the period after the introduction of targeted therapy (35.07% versus 24.57%). Final percentage of cases not subtyped after morphological review was 17.03%. IHC was performed in 157 cases, with an increased use in recent years. The number of antibodies did not influence the overall success in subtyping and an average of 3 markers was used. Most frequent antibodies used were TTF-1, CK7, high molecular weight keratin and p63. More than half of cases not subtyped even after IHC corresponded to poorly or undifferentiated neoplasms in the surgical specimens. For the NSCLC, NOS which IHC was not performed, a cell block was produced in 106 cases (75.71%). Review of the cell block slides from 2005 to 2009 showed that the majority (70.7%) had rare, few or no tumor cells., Conclusions: Specific subtyping can be achieved in a high proportion of lung FNAs with high accuracy. The percentage of NSCLC, NOS has significantly decreased in recent years together with a trend for an increased use of IHC as well as increased number of cell blocks produced. An average of 3 IHC markers was used for subtyping and the number of markers did not influence the overall subtyping., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Novel high-throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols ("Classic" and "Elute") and assessed the feasibility of performing multiplex mutational screening using fine-needle aspiration (FNA) biopsy samples., Methods: Residual material from 42 FNA biopsies was collected in the cards (21 Classic and 21 Elute cards). DNA was extracted using the Classic protocol for Classic cards and both protocols for Elute cards. Polymerase chain reaction for p53 (1.5 kilobase) and CARD11 (500 base pair) was performed to assess DNA integrity., Results: Successful p53 amplification was achieved in 95.2% of the samples from the Classic cards and in 80.9% of the samples from the Elute cards using the Classic protocol and 28.5% using the Elute protocol (P = .001). All samples (both cards) could be amplified for CARD11. There was no significant difference in the DNA concentration or 260/280 purity ratio when the 2 types of cards were compared. Five samples were also successfully analyzed by multiplex MassARRAY spectrometry, with a mutation in KRAS found in 1 case., Conclusions: High molecular weight DNA was extracted from the cards in sufficient amounts and quality to perform high-throughput multiplex mutation assays. The results of the current study also suggest that FTA Classic cards preserve better DNA integrity for molecular applications compared with the FTA Elute cards., (Copyright © 2012 American Cancer Society.)

The evaluation of mediastinal and hilar lymph nodes for tissue diagnosis and staging of lung cancer is now commonly performed by minimally invasive, nonsurgical procedures such as computed tomography-guided fine-needle aspiration and endobronchial ultrasound-guided transbronchial needle aspiration. Ensuring that a sufficient quantity of cellular material has been acquired to enable multiple studies has become a priority issue in the era of personalized medicine, especially for patients with lung cancer, and this can be accomplished by rapid onsite evaluation (ROSE). This commentary focuses on the use of ROSE in guided procedures, especially for hilar and mediastinal lymph node aspirates, and describes an algorithm for handling these specimens., (Copyright © 2011 American Cancer Society.)

Background: B-cell lymphomas with concurrent IGH/BCL2 and MYC gene rearrangements, termed dual-translocation or double-hit lymphomas (DTLs), rarely are identified. They usually are characterized by highly aggressive behavior, a poor prognosis, and complex karyotypes. The objective of this study was to review and describe the cytomorphologic findings in different types of cytologic preparations and clinicopathologic characteristics of patients with DTLs., Methods: Patient samples with IGH/BCL2 and MYC rearrangements that were detected by fluorescence in situ hybridization during the period from October 2003 to September 2009 were selected for morphology review. Clinical data and results from additional studies were collected from patient reports., Results: Cytologic samples from 14 patients (5 men and 9 women) were reviewed. The most common cytomorphologic pattern was a mixed cell population consisting predominantly of large cells (88.2%), mainly centroblasts (94.1%), with dark blue cytoplasm (76.4%) accompanied by apoptotic bodies (64.7%), with marked cellular pleomorphism (94.1%). Nuclear segmentation was present in 64.7% of samples, conferring a "coffee bean" nucleus, and cytoplasmic vacuoles were observed in 46.6% of samples. Immunophenotyping revealed the expression of CD20, CD19, surface immunoglobulin, and CD10 in 13 samples. Other chromosomal aberrations were also identified. Seven patients died of their disease, and the time from progression to death ranged from 1 month to 16 months., Conclusions: Large cells with deeply basophilic cytoplasm, cytoplasmic vacuoles, and frequent segmented nuclei, particularly in fine needle aspirate smears and especially in patients with clinically aggressive and/or unusual clinical features, should trigger a fluorescence in situ hybridization analysis for IGH/BCL2 and MYC translocation to identify this entity., (Copyright © 2011 American Cancer Society.)

Total laparoscopic hysterectomy (LH) is a minimally invasive technique, which results in comparable morbidity and better cosmesis compared with total abdominal hysterectomy. The literature is discrepant as to whether it is associated with a higher incidence of positive peritoneal cytology compared with total abdominal hysterectomy and recently, associated artifacts, including vascular pseudoinvasion (VPI), have been described. A retrospective histopathologic review of 266 hysterectomy specimens from 2 centers was performed. The observers, blinded to the surgical technique, assessed for the presence of artifactual changes including disruption of the endometrial lining, nuclear crush artifact, VPI, endomyometrial cleft artifact with or without epithelial displacement, inflammatory debris within vessels, serosal carryover, and intratubal contaminants. In addition, the rates of positive peritoneal washings over a 5-year period, and the use of immunohistochemistry (IHC) to aid in cell typing over a 3-year period, were compared between hysterectomies in which a uterine manipulator (UM) device had and had not (nonmanipulated hysterectomies) been used. The hysterectomies were performed for malignant (n=160) and benign (n=102) uterine disease or for ovarian or cervical disease (n=4), and included total abdominal (n=108), vaginal (n=17), laparoscopy-assisted vaginal (n=24), laparoscopy converted to laparotomy (n=10), nonrobotic laparoscopic (n=51), and robot-assisted laparoscopic (n=56) hysterectomies. One hundred and two (38%) of these hysterectomies involved the use of a UM. Artifactual changes of disruption of the endometrial lining, endomyometrial clefts, intratubal contaminants, nuclear crush artifact, intravascular inflammatory debris, and VPI were significantly more common with LH and with the use of a UM, independent of whether the endometrial pathology was benign or malignant. IHC to aid in endometrial cancer subtyping was more likely to be used in manipulated hysterectomies (P=0.0166). Furthermore, peritoneal washings were significantly more likely to be positive in hysterectomies in which a UM had been used (P=0.0061). Histologic artifacts are significantly more common in LH and specifically in hysterectomies in which a UM is used. Such artifacts impair the pathologists' interpretation of cell type requiring an increased use of IHC, and displaced epithelial fragments present within vessels or artifactual clefts may result in the misinterpretation of prognostic and staging parameters. Furthermore, there is a significantly higher rate of positive peritoneal cytology in cases that are subjected to uterine manipulation, suggesting dissemination of malignant cells into the abdominal cavity. The clinical significance of this finding needs to be determined.

Background: Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B-cell non-Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations., Methods: FISH results from assays performed on cytospin preparations from NHL FNAs over a 6-year period (2003-2009) were selected. Immunophenotyping, clinical data, and cytomorphologic data were reviewed according to the current World Health Organization (WHO) classification system. Hybridized probes, the purpose for the assay (subtyping or prognosis), and the cytogenetic abnormalities observed were retrieved from cytology reports. Data was categorized according to specific rearrangements and other chromosomal abnormalities., Results: Successful results were obtained in 284 (95.3%) of 298 cases from 282 patients. Abnormalities were found in 216 (76%) cases and 68 (24%) did not show alteration. Among cases submitted for subtyping, 198 showed FISH-positive results, and specific gene rearrangements were found in 122 (61.6%) cases as follows: follicular 82, mantle cell 21, marginal zone 3, "dual hit" 13, and Burkitt lymphoma 3. In 21 cases, abnormalities were useful for prognosis. Nonspecific alterations alone or in combination with translocations were found in 98 cases., Conclusions: FISH performed on cytospin preparations was useful for confirmation of specific subclasses of NHL and may also provide valuable prognostic information. Cytogenetic abnormalities other than specific translocations were frequently found and could provide supportive evidence for a definitive diagnosis of lymphoma in FNA., (© 2010 American Cancer Society.)

Background: MIB-1 proliferation index (PI) has proven helpful for diagnosis and prognosis in non-Hodgkin lymphomas (NHLs). However, validated cutoff values for use in fine-needle aspiration (FNA) samples are not available. We investigated MIB-1 immunocytochemistry as an ancillary technique for stratifying NHL and attempted to establish PI cutpoints in cytologic samples., Methods: B-cell NHL FNA cases with available cytospins (CS) MIB-1 immunocytochemistry results were included. Demographic, molecular, immunophenotyping and MIB-1 PI data were collected from cytologic reports. Cases were subtyped according to the current World Health Organization classification and separated into indolent, aggressive, and highly aggressive groups. Statistical analysis was performed with pairwise Wilcoxon rank sum test and linear discriminant analysis to suggest appropriate PI cutpoints., Results: Ninety-one NHL cases were subdivided in 56 (61.5%) indolent, 30 (33%) aggressive, and 5 (5.5%) highly aggressive lymphomas. The 3 groups had significantly different MIB-1 PIs from each other. Cutpoints were established for separating indolent (<38%), aggressive (> or =38% to < or =80.1%) and highly aggressive (>80.1%). The groups were adequately predicted in 76 cases (83.5%) using the cutpoints and 15 cases showed discrepant PIs., Conclusions: MIB-1 immunohistochemistry on CS can help to stratify B-cell NHL and showed a significant increase in PI with tumor aggressiveness. Six misclassified cases had PIs close to the cutpoints. Discrepant MIB-1 PIs were related to dilution of positive cells by non-neoplastic lymphocytes and to the overlapping continuum of features between diffuse large B-cell lymphoma and Burkitt lymphoma. Validation of our approach in an unrelated, prospective dataset is required., (Copyright 2010 American Cancer Society.)

The immunophenotype of lymphoid cytologic samples obtained by laser scanning cytometry (LSC) and flow cytometry (FC) was compared in 72 cases composed of a series of 23 cases with simultaneous LSC and FC immunophenotyping and a second series of 49 cases in which nonsimultaneous immunophenotyping was performed. In both series, no discordance in the population immunophenotype was found that would result in changes in diagnostic classification, although minor discordance in some antigens was found, predominantly affecting FMC7, CD11c, and CD23. The immunophenotype obtained by LSC shows a high degree of concordance with that obtained by FC and generates results that are diagnostically equivalent. Potential explanations for the discordant markers include differences related to the techniques, differences in the fluorochrome-labeled antibodies, technical factors, differences in antigen expression related to anatomic sites, temporal variations, and interpretive variances.

Hairy cell leukemia, a chronic low-grade lymphoproliferative disorder first recognized in mid 1920's typically involves the lymphoreticular system, especially the spleen and the bone marrow. Clinically, it tends to present with pancytopenia and splenomegaly. Liver involvement, if present, is usually in a sinusoidal and a periportal pattern. We report a case of hairy cell leukemia associated with a liver mass 20 years after the initial presentation. To our knowledge, this is the first case of hairy cell leukemia presenting as a solid liver mass diagnosed by a percutaneous ultrasound guided fine needle aspiration biopsy to be reported in the English literature., ((c) 2008 Wiley-Liss, Inc.)

Pulmonary infiltrates in immunocompromised patients frequently represent infections. This study was undertaken to evaluate cytologic examination of bronchoalveolar lavage samples and the value of different preparations and silver staining. Over 6 mo, 336 samples were collected from 155 immunosuppressed patients in whom both cytologic and microbiologic studies were performed. In 27 samples, the cytology, microbiology or both demonstrated the presence of infectious agents. In four cases, cytology identified neoplastic process. Cytology had a sensitivity of 34.6% for detection of infection, which increased to 42.9% when Cytomegalovirus was excluded. Cytology detected 6 of 15 cases of Aspergillus, including three cases not detected by microbiology and 3 of 4 cases of Pneumocystis, but did not identify any of the Cytomegalovirus cases. The type of preparations did not affect detection of the organism when present in cytologic samples and silver staining did not appear to have added value in examination of these samples., ((c) 2007 Wiley-Liss, Inc.)

Cutaneous metastases from thyroid carcinoma are rare. This report describes four cases of thyroid carcinoma metastatic to the skin. Two cases were medullary carcinoma and two were papillary thyroid carcinoma. In two cases, skin metastases were the presenting feature of the underlying thyroid carcinoma. Examination of the skin lesions by conventional light microscopy suggested the possibility of metastatic carcinoma and immunohistochemical tests confirmed the diagnosis. Subsequent investigations identified primary thyroid lesions. In two cases, the skin metastasis was the first evidence of the recurrence of known thyroid carcinoma. These cases identify a novel presentation of thyroid carcinoma.

Objective: Prophylactic oophorectomy (PO) is an accepted treatment strategy for women who are at high risk for the development of ovarian carcinoma, particularly women who are BRCA mutation-positive. This study sought to assess the utility of peritoneal lavage cytology at the time of PO in detecting occult malignancy in this group of patients., Methods: Thirty-five high-risk women, who were not suspected of having any malignancy or ovarian mass, underwent peritoneal lavage at the time of PO. Thirty-one of the thirty-five women had undergone BRCA mutation analysis (BRCA1+, 18; BRCA2+, 10; BRCA-, 3). Intensive histopathologic examination was used in all 35 cases to identify occult carcinoma. Lavage specimens were reviewed for the presence of malignant cells and endosalpingiosis. The cytologic review was conducted without knowledge of either the histopathologic or BRCA results., Results: In 32 of the 35 lavage specimens no malignancy was detected. In the remaining three cases malignant cells were detected; in two of these cases histopathologic examination confirmed an ovarian/tubal occult carcinoma. Two of these women were BRCA1 mutation positive. Endosalpingiosis was detected in the peritoneal lavage specimens of 7 of the 32 cases showing no evidence of malignancy. All of these 7 women were BRCA mutation positive or unknown., Conclusion: Peritoneal lavage cytology can detect occult carcinoma at the time of PO and should be performed at PO. The significance of occult carcinoma detected by either histopathologic or cytopathologic examination is uncertain. Whether the prevalence of endosalpingiosis detectable by lavage cytology is increased in BRCA mutation-positive patients requires further study.

Hyalinizing trabecular tumors of the thyroid are interesting but uncommon neoplasms. They have been classified as benign hyalinizing trabecular adenomas or malignant hyalinizing trabecular carcinomas. They share both epidemiologic and morphologic features with papillary carcinoma, and there has been much speculation about the relationship between these two entities. Because RET/PTC gene rearrangements are specific to papillary thyroid carcinoma, the authors examined the presence of RET/PTC-1, -2, and -3 in eight hyalinizing trabecular tumors using reverse transcription-polymerase chain reaction with Southern hybridization and immunohistochemistry. They detected the presence of a RET/PTC gene rearrangement in six of the eight hyalinizing trabecular tumors. This confirms the long-standing suspicion that hyalinizing trabecular tumors do indeed represent a morphologic variant of papillary carcinoma.