ALK Gene Mutation and ALK Protein Expression in Advanced Neuroblastoma and the Potential Value in Risk Stratification in Fine-Needle Aspiration Biopsy Samples.

OBJECTIVES: The protein ALK is targeted for therapy in neuroblastoma, and ALK mutation confers a poor prognosis. We evaluated ALK in a cohort of patients with advanced neuroblastoma diagnosed by fine-needle aspiration biopsy (FNAB). METHODS: Fifty-four cases of neuroblastoma were evaluated for ALK protein expression by immunocytochemistry and ALK gene mutation by next-generation sequencing. MYCN amplification by fluorescence in situ hybridization, International Neuroblastoma Risk Group (INRG) staging, and risk assignment was performed, and patients were managed accordingly. All parameters were correlated with overall survival (OS). RESULTS: ALK protein showed cytoplasmic expression in 65% cases and did not correlate with MYCN amplification (P = .35), INRG groups (P = .52), and OS (P = .2); however, ALK-positive, poorly differentiated neuroblastoma showed better prognosis (P = .02). ALK negativity was associated with poor outcome by Cox proportional hazard model (hazard ratio, 2.36). Two patients showed ALK gene F1174L mutation with 8% and 54% allele frequency and high ALK protein expression; they died of disease 1 and 17 months following diagnosis, respectively. A novel IDH1 exon 4 mutation was also detected. CONCLUSIONS: ALK expression is a promising prognostic and predictive marker in advanced neuroblastoma that can be evaluated in cell blocks from FNAB samples along with traditional prognostic parameters. ALK gene mutation confers a poor prognosis for patients with this disease.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis.

BACKGROUND: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation - plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. DESIGN: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ2 test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. RESULTS: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). CONCLUSION: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Lymphoreticular malignancies in serous effusions: Cytomorphologic, flow cytometric and immunocytochemical analysis.

INTRODUCTION: Involvement of body fluids by lymphoreticular malignancies (LRM) is rare and often associated with poor prognosis and decreased overall survival. The present study was conducted to analyze the characteristic cytomorphologic, flow cytometric and immunocytochemical features of LRMs in serous effusions. MATERIALS AND METHODS: This was a three-year retrospective study. A total of 218 effusion samples, reported as involved by lymphoreticular malignancies, on cytology, were reviewed. All the cases wherein the cytological diagnosis was confirmed by flow cytometric (FCM) and/or immunocytochemical (ICC) studies were retrieved and studied in detail. FCM and/or ICC were performed in a total of 51/218(23.4%) samples, including 30 pleural (58.8%), 18 peritoneal (35.3%), and 3 pericardial fluid (5.9%) samples. RESULTS: The cytomorphologic diagnoses included infiltration by non-Hodgkin lymphoma (NHL;n = 27), infiltration by LRM (n = 19), infiltration by chronic lymphocytic leukemia (CLL;n = 2), Hodgkin's lymphoma (HL;n = 1) and suggestive of infiltration by LRM (n = 2). FCM and/or ICC confirmed the diagnoses as infiltration by T-cell lymphoblastic lymphoma in 18; mature B-cell NHL in 10; Burkitt lymphoma in 7; diffuse large B-cell lymphoma in 4; follicular lymphoma, T- cell NHL and CLL in 2 samples each and hairy cell leukemia, plasmablastic lymphoma and HL in 1 sample each. 94.1% concordance was noted between the initial and final cytologic diagnosis. CONCLUSIONS: Involvement of body fluids and effusions by LRMs, though rare, carries an immense prognostic significance and hence the prompt detection is crucial. Detection of these malignancies by cytologic examination of effusions is challenging yet potentially useful and the least invasive method available to establish an early diagnosis.

Application of multicolour flow cytometry in the detection of metastatic carcinoma in serous effusions: Special emphasis in atypical cytology.

INTRODUCTION: We evaluated the role of simultaneous use of multiple antibodies in flow cytometry (FCM) to detect metastatic carcinomas in effusion samples. METHODS: Cytological examination of 75 successive cases of effusion samples was performed. There were 48 peritoneal, 26 pleural and one pericardial fluid. Multi-coloured FCM examination was undertaken using a cocktail of CD45, CD14 and epithelial cell adhesion molecule (EpCAM), antibodies tagged with different fluorochromes. The percentage of EpCAM positivity was calculated in the CD45 and CD14 dual negative population by selective gating. The EpCAM value was correlated with the cytological findings, follow-up data and MOC-31 immunostaining. RESULTS: There were 20 benign, 35 malignant and 20 atypical cases diagnosed on cytomorphology. The primary sources of carcinomas were mainly from the ovary, followed by lung, gall bladder, intestine and other areas. Out of 20 cytologically benign cases, there were two malignant cases on the final follow-up, and EpCAM on FCM picked up all 18 benign cases and one malignant case. Out of 35 cytologically detected malignant cases, EpCAM picked up 32 malignant cases. The EpCAM detected 15/18 malignant and both benign cases out of 20 cytological atypical cases. EpCAM antibody by FCM showed 87% sensitivity, 100% specificity, 100% positive predictive value and 74% negative predictive value. CONCLUSION: This comprehensive study highlights the potential use of multi-coloured FCM along with cytological examination to diagnose metastatic carcinoma in effusion samples. Multi-coloured FCM is rapid and quantitative and is helpful in atypical cases.

Indian Academy of Cytologists Guidelines for Collection, Preparation, Interpretation, and Reporting of Serous Effusion Fluid Samples.

Cytological examination plays an important role in the initial work-up of the serous cavity effusion fluids to find out the possible etiology as benign or malignant. Among malignant effusions, cytology is helpful in determining the exact type, site, and stage of the tumor. However, for reporting effusion cytology specimens, there is no consistent and reproducible reporting system. AIMS: The aim of these guidelines is to provide a standardized format for effusion cytopathology right from sample receipt to its ultimate report sign-out for implementation in all cytopathology laboratories. The Indian Academy of Cytologists in consultation with experts across the country has prepared guidelines pertaining to collection, preparation, and diagnostic categories of effusion specimens to reduce reporting variability. The guidelines are made keeping in mind the different areas of practices in India, especially low- and medium-resource settings. The guidelines are broadly divided into essential, optimal, and optional categories for best usage and appropriate allocation of the precious specimens. In referral centers or well-established setups, essential ancillary techniques can be done for accurate and final diagnosis. By adhering to and implementing these uniform guidelines, it is hoped that clinical patient care and management in India will improve and be of uniformly good quality by enabling and facilitating good laboratory practices.

Molecular Testing for BRAFV600E and RAS Mutations from Cytoscrapes of Thyroid Fine Needle Aspirates: A Single-Center Pilot Study.

CONTEXT AND AIM: Molecular testing of thyroid FNA has been advocated in the indeterminate categories of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) 2018. The utility of cytoscrapes of thyroid FNA samples for BRAF V600E and RAS mutations was evaluated in this pilot study. METHODS AND MATERIALS: Thyroid FNA samples between 2015 and 2018 from TBSRTC categories 3-6 were included. DNA was extracted from one to two representative smears (cytoscrape). Real-time PCR for BRAF V600E and RAS (KRAS, NRAS, and HRAS) gene mutations was performed. Histopathology correlation was available in 44 cases. STATISTICAL METHODS: Chi-square test and calculation of sensitivity, specificity, and positive/negative predictive values were performed. RESULTS: A total of 73 thyroid FNA cases and 11 nodal metastases of papillary thyroid carcinoma (PTC) were evaluated. The DNA yield ranged from 1.9 to 666 ng/µl (mean 128 ng/µl) in 80 cases and was insufficient in four cases. Overall, mutations were seen in 45 (56.25%) cases with BRAF V600E, NRAS, HRAS, and KRAS in 21 (46.7%), 19 (42.2%), 4, and 1 cases, respectively. BRAF V600E mutation was seen in PTC (11/18, 61%), nodal PTC metastases (5/10, 50%), and occasionally in TBSRTC category 3 (1/18, 5.5%). NRAS mutations were seen across all categories and were maximum in the AUS/FLUS group (6/18, 33%). BRAF V600E /RAS testing had an overall sensitivity, specificity, and positive and negative predictive values of 61.7%, 80%, 91.3%, and 38%, respectively, for the detection of malignancy. In indeterminate thyroid nodules, the sensitivity, specificity, PPV, and NPV were 56.2%, 80%, 81.8%, and 53.3%, respectively. CONCLUSION: BRAF V600E/RAS mutation testing from cytoscrapes are useful as a rule-in test for indeterminate thyroid nodules and provide molecular confirmation in nodal metastases of PTC.

Secretory cell outgrowths, p53 signatures, and serous tubal intraepithelial carcinoma in the fallopian tubes of patients with sporadic pelvic serous carcinoma.

CONTEXT: High-grade serous carcinomas of ovarian, tubal, and peritoneal origin are together referred as pelvic serous carcinoma. The fallopian tubes, ovarian surface epithelium, and the tuboperitoneal junctional epithelium are all implicated in pelvic serous carcinogenesis. AIMS: The aim of this study is to identify putative precursor lesions of serous carcinoma including secretory cell outgrowths (SCOUTs), serous tubal intraepithelial carcinoma (STIC), and p53 signatures and assign its probable site of origin. SETTINGS AND DESIGN: Prospective case-control study of consecutive specimen comprising 32 serous carcinomas and 31 controls (10 normal adnexa, 10 benign and 6 atypically proliferative surface epithelial tumors, and 5 other carcinomas). SUBJECTS AND METHODS: Sectioning and extensive examination of the fimbrial end (SEE-FIM) protocol along with immunohistochemistry for Bcl-2, p53, and Ki-67 was employed for evaluating invasive carcinoma and precursor lesions in cases versus controls. RESULTS: SCOUT, p53 signatures, and STIC were most frequent in the serous carcinomas. p53 signatures and STIC were always seen in the fimbrial end. STICs were exclusively present in serous carcinomas, more common in ipsilateral tubes of cases with dominant ovarian mass. Multifocal p53 signatures with STIC were seen in 7 (21.9%) cases. STIC was present with or without an invasive carcinoma in 25% and in 6.25% of cases of pelvic serous carcinomas, respectively. The junctional epithelia did not show any lesion in any group. CONCLUSIONS: SEE-FIM protocol is recommended for evaluation of sporadicpelvic (ovarian/tubal/peritoneal) serous carcinoma. Based on the presence of STIC or invasive carcinoma, nearly 60% of all pelvic serous carcinomas are of fallopian tubal origin.

Precursor T-lymphoblastic lymphoma: Speedy diagnosis in FNA and effusion cytology by morphology, immunochemistry, and flow cytometry.

BACKGROUND: Precursor T-lymphoblastic lymphoma (T-LBL) is a rare lymphoma presenting clinically in children and adolescents with a rapidly enlarging mediastinal mass, dyspnea, and cervical lymphadenopathy requiring quick diagnosis. The objective of the current study was to report on the spectrum of cytomorphology and flow cytometric immunophenotyping (FCI). METHODS: The clinical profile, cytomorphological features, FCI, and cell block immunocytochemistry (CB-ICC) of all cases of T-LBL diagnosed from 2011 through 2013 were reviewed. RESULTS: Fifteen cases of precursor T-LBL (10 fine-needle aspiration samples and 5 pleural/pericardial fluid samples) were evaluated. Smears demonstrated dispersed lymphoblasts, with a high nuclear:cytoplasmic ratio and scanty basophilic cytoplasm. Nuclei demonstrated notches, clefts, and indentations. The chromatin was condensed in small and intermediate-sized blasts and dispersed in larger blasts. Nucleoli were present only in the larger blasts. Hand mirror-shaped cells and mitoses were variable. With regard to immunophenotyping, flow cytometry demonstrated positivity for CD2 (15 of 15 cases), surface CD3 (14 of 15 cases), cytoplasmic CD3 (15 of 15 cases), terminal deoxynucleotidyl transferase (TdT) (8 of 15 cases), CD5 (13 of 15 cases), CD10 (7 of 15 cases), and human leukocyte antigen-D related (HLA-DR) (1 of 15 cases). Dual CD4/CD8 positivity was observed in all cases forming a tight cluster, which is consistent with the cortical T-LBL subtype. CB-ICC demonstrated a uniform CD3-positive/TdT-positive/CD20-negative phenotype. In 7 cases in which TdT was negative by flow cytometry, CB-ICC was positive. CONCLUSIONS: Combining cytomorphology and FCI enables the accurate and rapid diagnosis of T-LBL on fine-needle aspiration and effusion cytology specimens, thereby obviating the need for a biopsy.

Flow cytometric immunophenotyping and cell block immunocytochemistry in the diagnosis of primary Non-Hodgkin's Lymphoma by fine-needle aspiration: Experience from a tertiary care center.

BACKGROUND: Accurate diagnosis of Non-Hodgkin's Lymphoma (NHL) on fine-needle aspiration (FNA) specimen is challenging and requires ancillary testing. AIM: The feasibility of flow cytometric immunophenotyping (FCI) along with cell block immunocytochemistry (CB-ICC) as adjunct techniques in the diagnosis of NHL as per the current World Health Organization (WHO) classification was evaluated. MATERIALS AND METHODS: All cases of suspected lymphoma underwent FNA, and the sample was triaged for light microscopic evaluation, FCI, and CB-ICC, and each case was classified as per the current WHO classification. RESULTS: A total of 65 cases was analyzed which included 40 B-cell, 21 T-cell, and 4 unclassifiable lymphomas. Of 61 cases, FCI alone was contributory in 74% (45/61) cases whereas CB-ICC alone was contributory in 65.5% (40/61) cases in typing the lymphoma. In 11.4% (7/61) cases, the lymphoma could not be classified by either technique. Thus, in a total of 88.5% (54/61) cases a combination of FCI and CB-ICC from FNA enabled a diagnosis of lymphoma with its subtyping. CONCLUSION: Flow cytometric immunophenotyping and ICC on CBs are feasible on FNA material and are very useful in a suspected case of NHL especially when a biopsy may not be possible or feasible.

Primary Ewing's sarcoma/primitive neuroectodermal tumor of the kidney: Report of a case diagnosed by fine needle aspiration cytology and confirmed by immunocytochemistry and RT-PCR along with review of literature.

Primary Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) of the kidney is a distinct entity that can be mistaken for variety of round cell tumors. We report a rare case of ES/PNET of the kidney in a 35-year-old female patient diagnosed by fine needle aspiration cytology (FNAC) and confirmed by immunohistochemistry (IHC) and reverse-transcriptase polymerase chain reaction (RT-PCR). Ultrasound guided FNAC smears from the kidney mass showed a population of malignant small round cells with perivascular arrangement and focal rosette formation. IHC performed on the cell block, showed strong immunopositivity for CD99 (MIC2) and vimentin. Molecular analysis of the aspirate by RT-PCR confirmed the EWS-FLI type1 transcript. The application of RT-PCR on FNAC material for establishing a diagnosis of renal ES/PNET is being reported for the first time. FNAC also confirmed metastases in the right level I cervical lymph node. The utility of IHC and molecular techniques in diagnosis of such a rare case is stressed and relevant literature is discussed.

Reverse transcriptase-polymerase chain reaction as an ancillary molecular technique in the diagnosis of small blue round cell tumors by fine-needle aspiration cytology.

We evaluated the feasibility and usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) on fine-needle aspirates for categorization of small blue round cell tumors (SBRCTs). A total of 51 cases, including 25 Ewing sarcoma/peripheral primitive neuroectodermal tumors (PNETs), 11 rhabdomyosarcomas, 13 neuroblastomas, and 2 desmoplastic small round cell tumors (DSRCTs) were analyzed. The detection of the EWS-FLI1 (20/25) and EWS-ERG (4/25) fusion transcripts resolved 24 of 25 cases of Ewing sarcoma/PNET. The PAX3/7-FKHR fusion transcript was detected in 2 of 4 cases of alveolar rhabdomyosarcoma and the EWS-WT1 transcript in both cases of DSRCT. Tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (dopa) decarboxylase transcripts were demonstrated in 10 of 13 cases of neuroblastoma. In comparison, immunocytochemical analysis resolved 19 (76%) of 25 Ewing sarcomas, 9 (82%) of 11 rhabdomyosarcomas, 6 (46%) of 13 neuroblastomas, and 1 (50%) of 2 DSRCTs. Overall, RT-PCR resolved 38 (86%) of 44 vs 35 (69%) of 51 cases by immunocytochemical analysis. RT-PCR is easily applied to fine-needle aspirates of SBRCT and greatly facilitates accurate tumor typing.

Synovial sarcoma: diagnosis on fine-needle aspiration by morphology and molecular analysis.

BACKGROUND: Synovial sarcoma (SS) is characterized by the t(X; 18) (p11.2; q11.2) translocation resulting in the SYT-SSX fusion transcript, detectable by reverse transcriptase polymerase chain reaction (RT-PCR). Fine-needle aspiration (FNA) cytology diagnosis of SS is challenging. We evaluated the applicability of RT-PCR on FNAs and to perform a detailed cytomorphological analysis in unequivocal cases of SS. METHODS: A prospective and retrospective analysis was performed over 4 years (2003-2007). Prospectively, FNAs positive for the SYT-SSX fusion transcript by RT-PCR (n = 6) and, retrospectively, cases proven on histopathology and immunohistochemistry (ICC; positivity for vimentin and epithelial membrane antigen [EMA]/cytokeratin) as SS (n = 10) were included in the study. A detailed cytomorphological analysis was carried out. RESULTS: There were 9 biphasic and 7 monophasic tumors. The aspirates from both biphasic and monophasic tumors were richly cellular in all cases with micro tissue fragments. Pericapillary arrangement of tumor cells was present in most cases. Attempted gland formation was seen in 7 of 9 biphasic tumors. The individual tumor cells were round, ovoid, or spindle shaped. Pleomorphism was mild; monophasic tumors displayed lesser pleomorphism as compared with the biphasic ones. Nuclear chromatin was bland in all cases except 1 and nucleolar prominence was seen in just 3 biphasic tumors. Mast cells were seen in 3 biphasic and 2 monophasic tumors. Scanty to moderate extracellular matrix material was seen in 5 cases. CONCLUSIONS: FNA cytology of SS shows a spectrum of cytomorphological features; the diagnosis is confirmed by RT-PCR on the aspirated material for the SYT-SSX fusion transcript.

Comparative evaluation of flow-cytometric immunophenotyping and immunocytochemistry in the categorization of malignant small round cell tumors in fine-needle aspiration cytologic specimens.

BACKGROUND: The precise diagnosis of malignant small round cell tumors (MSRCTs) in fine-needle aspiration (FNA) cytology is a challenge that requires ancillary investigations. In this study, the authors evaluated the applicability of flow-cytometric immunophenotyping (FCI) and compared it with immunocytochemistry (ICC) for the accurate categorization of MSRCTs. METHODS: In total, 37 consecutive MSRCTs that had been diagnosed with FNA cytology were analyzed by ICC and FCI using a panel of antibodies against desmin, vimentin, CD99/major histocompatibility class I-related antigen 2, neuron-specific enolase, and pancytokeratin. The final diagnoses included Ewing sarcoma (n = 17), rhabdomyosarcoma (n = 6; 4 embryonal and 2 alveolar subtypes), neuroblastoma (n = 10), desmoplastic small round cell tumor (n = 2), and retinoblastoma (n = 2). RESULTS: Accurate categorization was possible in 67.5% of cases by ICC and in 64.8% of cases by FCI. Concordant immunophenotyping results with either technique were obtained in 21 cases (59.4%). Low cellularity of the sample and negativity for all markers tested were some limitations to both techniques when applied on fine-needle aspirates. However, using a combination of both techniques, 86.4% (32 of 37 cases) MSRCTs were typed accurately. CONCLUSIONS: FCI is applicable on FNA material and complements ICC in accurate the typing of MSRCTs. This is particularly useful in advanced-stage disease, when neoadjuvant chemotherapy may be instituted promptly.

Cytoplasmic vacuolation, intracytoplasmic lumina, and DPAS staining in ductal carcinoma of the breast.

A retrospective study was carried out on 30 cases of histologically proven invasive ductal carcinoma of the breast with a prior fine-needle aspiration (FNA) cytology. On evaluating the May-Grünwald-Giemsa (MGG) FNA smears, cytoplasmic vacuolation was observed in 70% cases. Positivity with periodic acid Schiff-positive, diastase-resistant (DPAS) staining was observed in 90% of cases. The chi(2) value on a McNemar test was 4.16. Thus, DPAS staining was significantly superior to MGG staining for picking up cytoplasmic vacuoles (P < 0.05). In 56.67% cases, DPAS staining showed an improvement in score as compared to MGG smears. This was highly significant (P < 0.001) on Wilcoxon matched-pairs signed-ranks test. Applying the strict criteria of thick-walled cytoplasmic vacuoles with a central darkly stained dot, none of our cases revealed true intracytoplasmic lumina. Larger studies are required to establish a role for DPAS staining in separating borderline, in situ, and invasive breast lesions, and to see if such positvity can be incorporated into the grading systems for breast carcinoma.