A Proposal for the Performance, Classification, and Reporting of Lymph Node Fine-Needle Aspiration Cytopathology: The Sydney System.

BACKGROUND: The evaluation of lymph nodes (LN) by fine-needle aspiration cytology (FNAC) is routinely used in many institutions but it is not uniformly accepted mainly because of the lack of guidelines and a cytopathological diagnostic classification. A committee of cytopathologists has developed a system of performance, classification, and reporting for LN-FNAC. METHODS: The committee members prepared a document that has circulated among them five times; the final text has been approved by all the participants. It is based on a review of the international literature and on the expertise of the members. The system integrates clinical and imaging data with cytopathological features and ancillary techniques. The project has received the endorsement and patronage of the International Academy of Cytology and the European Federation of the Cytology Societies. RESULTS: Clinical, imaging, and serological data of lymphadenopathies, indications for LN-FNAC, technical procedures, and ancillary techniques are evaluated with specific recommendations. The reporting system includes two diagnostic levels. The first should provide basic diagnostic information and includes five categories: inadequate/insufficient, benign, atypical lymphoid cells of undetermined/uncertain significance, suspicious, and malignant. For each category, specific recommendations are provided. The second diagnostic level, when achievable, should produce the identification of specific benign or malignant entities and additional information by utilizing ancillary testing. CONCLUSION: The authors believe that the introduction of this system for performing and reporting LN-FNAC may improve the quality of the procedure, the report, and the communication between cytopathologists and the clinicians. This system may lead to a greater acceptance and utilization of LN-FNAC and to a better interdisciplinary understanding of the results of this procedure.

Role of fine needle aspiration biopsy cytology in the diagnosis of infections.

The role of fine needle aspiration biopsy (FNAB) cytology in diagnosing infections has expanded due to the increase in the number of immune compromised patients and the increasing role of FNAB in the developing world where infection is a major cause of illness. FNAB has become the first procedural test in cases where the clinical and imaging findings suggest an infectious lesion or where there is a differential diagnosis of infection or metastatic or primary tumor. This applies to FNAB of palpable or image directed or deep seated lesions accessed by EUS and EBUS. This article details a recommended approach and technique for FNAB of infectious lesions, and discusses the role of rapid on site evaluation and the application of ancillary testing including the rapidly expanding array of molecular tests based on FNAB material. The utility of recognizing suppurative and granulomatous infectious patterns in FNAB direct smears, and the specific cytomorphological features on routine Papanicolaou and Giemsa stains and on special stains of FNAB smears is described for a large number of bacterial, fungal, viral, parasitic, and protozoan infections. The role of cytopathologists is to now train cytopathologists in sufficient numbers to provide FNAB services, teach trainee cytopathologists and cytotechnologists, and to encourage our clinical colleagues to use FNAB in the diagnosis of infections and other lesions to the benefit of patients and the medical system. Diagn. Cytopathol. 2016;44:1024-1038. © 2016 Wiley Periodicals, Inc.

Stratified Mucin-Producing Intraepithelial Lesion of the Cervix: Subtle Features Not to Be Missed.

OBJECTIVES: Stratified mucin-producing intraepithelial lesion (SMILE) is an uncommon premalignant lesion of the uterine cervix. A detailed examination of preinvasive SMILE cases including a comparison of the cytologic features with usual-type adenocarcinoma in situ (AIS) and human papillomavirus (HPV) genotyping was performed. STUDY DESIGN: Excisions and preceding Papanicolaou (Pap) tests were retrieved from the files of 2 tertiary care centers. Histologic review estimated the lesional SMILE proportion. Pap tests were reviewed and assessed for architectural, cellular and background features. Cobas® HPV test was performed. RESULTS: 13 cases were identified. Mean/median patient age was 35/33 years (range 23-51 years). Concurrent high-grade squamous intraepithelial lesion was found in 10/13 (77%) and AIS in 8/13 (62%) cases. In 6 cases, SMILE was dominant (≥50%) and represented in 5/6 corresponding Pap tests. Cytology interpretations differed more often in the SMILE-dominant group (p < 0.05). SMILE and AIS had overlapping features. Feathering and prominent nucleoli were absent in SMILE. HPV DNA was detected in all 12 cases tested. HPV 18 was most common (7/12). Excisions with positive/suspicious margins were reported in 5/6 SMILE-dominant versus 3/7 nondominant cases. CONCLUSION: SMILE is best considered as an AIS variant for cytologic, etiologic and management purposes. Cytologic features overlap with AIS, but are more subtle and easily missed. HPV testing may play a role in facilitating SMILE detection.

Multiplex sequencing for EZH2, CD79B, and MYD88 mutations using archival cytospin preparations from B-cell non-Hodgkin lymphoma aspirates previously tested for MYC rearrangement and IGH/BCL2 translocation.

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.

Enteropathy-associated intestinal T-cell lymphoma in cavitating mesenteric lymph node syndrome: fine-needle aspiration contributes to the diagnosis.

Cavitating mesenteric lymph node syndrome (CMLNS) is an infrequently reported manifestation of unrecognized/longstanding celiac disease and may be associated with enteropathy-associated intestinal T-cell lymphoma and hyposplenism. Unrecognized malignancy and life-threatening infections can pose a significant risk to patients in cases of delayed diagnosis. Fine-needle aspiration of the mesenteric lesions may contribute significantly to the correct diagnosis and can expedite patient management. We report on the cytologic characteristics of enteropathy-associated intestinal T-cell lymphoma first detected in a cyst fluid specimen obtained from a patient with cavitating mesenteric lesions. Image-guided fine-needle aspiration resulted in chylous fluid that contained a lymphoid cell population with neoplastic morphology and abnormal immunophenotype. Further work-up led to the diagnosis of enteropathy-associated intestinal T-cell lymphoma with bone marrow involvement. Cytologic assessment of the cyst fluid is an important part of the diagnostic cascade in patients with CMLNS to exclude clinically occult lymphoma.

Microcystic variant malignant mesothelioma presenting as a localized paraspinal mass.

A 58-year-old man presented with productive cough and fever. Computed tomography (CT) scan of the chest showed an upper right paraspinal mass. CT-guided fine-needle aspiration biopsy showed lobules of vacuolated cells against a background of myxoid material. The cells demonstrated moderate to severe nuclear atypia and occasional mitoses. Immunohistochemistry revealed tumor cells to be immunoreactive for calretinin, WT-1, D2-40, cytokeratin (CK) 7, AE1/AE3, high molecular weight keratin, vimentin and epithelial membrane antigen, and negative for thyroid transcription factor-1, Ber-EP4, carcinoembryonic antigen, S100 protein, CK20, and CDX2. The combined morphologic and immunohistochemical findings confirmed the diagnosis of microcystic variant of localized malignant mesothelioma. The subsequent lung resection showed a pleural-based mass in the right upper lobe and confirmed the diagnosis. Awareness of the existence of unusual morphologic variants and localized forms of mesothelioma are necessary to avoid misdiagnosis of fine needle biopsy samples. Recognition of characteristic cytomorphologic features along with optimal use of panel of immunohistochemistry studies is crucial for making a specific diagnosis.

Sample features associated with success rates in population-based EGFR mutation testing.

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.

Biophysical characterization of bladder cancer cells with different metastatic potential.

Specific membrane capacitance (SMC) and Young's modulus are two important parameters characterizing the biophysical properties of a cell. In this work, the SMC and Young's modulus of two cell lines, RT4 and T24, corresponding to well differentiated (low grade) and poorly differentiated (high grade) urothelial cell carcinoma (UCC), respectively, were quantified using microfluidic and AFM measurements. Quantitative differences in SMC and Young's modulus values of the high-grade and low-grade UCC cells are, for the first time, reported.

Subclassification of lymphoproliferative disorders in serous effusions: a 10-year experience.

BACKGROUND: Rare studies have reported the application of multiple ancillary tests to the diagnosis of lymphoproliferative disorder in serous effusions. In the current study, the authors evaluated the effectiveness of using an algorithm for the triage of serous effusions and the contribution of ancillary studies to achieve a specific subtype of lymphoproliferative disorder. METHODS: Serous effusion samples that had a final diagnosis of lymphoproliferative disorder or suspicious for lymphoma were selected from cases that were diagnosed between 2001 and 2010. Data were collected on patient and sample characteristics as well as results from immunophenotype and molecular studies. RESULTS: In total, 168 serous effusions were identified from 110 patients. The most common site of involvement was the pleural cavity (n = 133) followed by the peritoneal cavity (n = 30) and pericardial cavity (n = 5). The volume of serous effusions ranged from 2 mL to 1000 mL (mean, 238 mL). In 42 patients (38.2%), serous effusions were the primary source of diagnosis. In 129 patients who had a diagnosis of LPD, either generic (n = 82) or specific (n = 47) ancillary tests were performed as a single test in 58 samples (67.4%) or as a combination of multiple studies in 19 samples (23.2%). Immunophenotyping was successful in almost all samples that had a specific subtype with 16 B-cell and 4 T-cell lymphomas being diagnosed. More samples with a specific subtype of lymphoma underwent molecular tests compared with those who had a generic diagnosis (19.1% vs 13.4%). CONCLUSIONS: Successful, specific subtyping of lymphoproliferative disorders was achieved in approximately 33% of cases that were tested for ancillary studies following an approach for the triage and aliquoting of serous effusion samples.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples.

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.

Diagnosis and subclassification of lymphomas and non-neoplastic lesions involving mediastinal lymph nodes using endobronchial ultrasound-guided transbronchial needle aspiration.

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.

Diagnosis of B-cell non-hodgkin lymphomas with small-/intermediate-sized cells in cytopathology.

Fine needle sampling is a fast, safe, and potentially cost-effective method of obtaining tissue for cytomorphologic assessment aimed at both initial triage and, in some cases, complete diagnosis of patients that present clinically with lymphadenopathy. The cytologic diagnosis of B-cell non-Hodgkin lymphomas composed of small-/intermediate-sized cells, however, has been seen as an area of great difficulty even for experienced observers due to the morphologic overlap between lymphoma and reactive lymphadenopathies as well as between the lymphoma entities themselves. Although ancillary testing has improved diagnostic accuracy, the results from these tests must be interpreted within the morphological and clinical context to avoid misinterpretation. Importantly, the recognition of specific cytologic features is crucial in guiding the appropriate selection of ancillary tests which will either confirm or refute a tentative diagnosis. For these reasons, we here review the cytologic characteristics particular to five common B-cell non-Hodgkin lymphomas which typically cause the most diagnostic confusion based on cytological assessment alone: marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, and lymphoplasmacytic lymphoma. We summarize the most pertinent cytomorphologic features for each entity as well as for reactive lymphoid hyperplasia, contrast them with each other to facilitate their recognition, and highlight common diagnostic pitfalls.

Cyto-histologic agreement in pathologic subtyping of non small cell lung carcinoma: review of 602 fine needle aspirates with follow-up surgical specimens over a nine year period and analysis of factors underlying failure to subtype.

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.

The use of FTA cards for preserving unfixed cytological material for high-throughput molecular analysis.

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.

Negative images of crystalline immunoglobulin in crystal storing histiocytosis: A potential cytologic mimic of mycobacteria in smears.

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.

Maximizing the yield of lymph node cytology: Lessons learned from rapid onsite evaluation of image- and endoscopic-guided biopsies of hilar and mediastinal lymph nodes.

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.