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Background: Metastatic breast cancers are frequently encountered in cytology and require immunocytochemistry (ICC). In this study, traditional and multiplex ICC (mICC) for GATA-binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP15), and mammaglobin (MMG) were performed with the aim of validating mICC in cell blocks, with further single-cell expression pattern analysis to identify the single markers and combinations of markers most sensitive in subtypes of breast cancer., Methods: GATA3, GCDFP15, and MMG were paired with OptiView 3,3'-diaminobenzidine and Ventana DISCOVERY Purple and Blue, respectively, with cyclical and serial staining. Bright-field imaging was performed with the Mantra 2 system and analyzed with the inForm Tissue Finder (Akoya Biosciences). Cell detection and phenotyping were further confirmed by two pathologists., Results: In the 36 cases studied, traditional ICC and mICC demonstrated good concordance (kappa coefficient, >0.5; p < .01) at three cutoffs (1%, 5%, and 50%), except for GATA3 at the 1% cutoff. Single-marker positivity outnumbered double-marker positivity and the exceedingly rare triple-marker positivity (<3%). GATA3 was the leading single marker-positive phenotype in all breast cancer subtypes, except for MMG in estrogen receptor-positive, progesterone receptor-positive, and human epidermal growth factor receptor 2-positive (ER+/PR+/HER2+) breast cancers. Limited to two markers, GATA3/MMG included the greatest number of tumor cells for luminal breast cancers (ER+/PR+/HER2+, 60.6%; ER+/PR+/HER2+, 31.4%), whereas HER2-overexpressed breast cancers (27.4%) and triple-negative breast cancers (26.4%) favored the combination of GATA3/GCDFP15., Conclusions: For a single marker, GATA3 displayed the highest sensitivity. The addition of MMG for hormone receptor-positive breast cancers and GCDFP15 for hormone receptor-negative breast cancers further increased sensitivity. The low proportion of multimarker-positive cells suggested that the coexpression observed with traditional ICC is attributable to intratumoral heterogeneity, not genuine coexpression., (© 2024 The Author(s). Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Background: The identification of BRAF mutations in thyroid cancer has prognostic and therapeutic implications. Although the gold standard for identifying BRAF mutations is molecular testing, the ability to perform BRAF p.V600E immunostaining on fine-needle aspiration (FNA) samples can facilitate the rapid triaging of patients to treatment options., Methods: A total of 50 thyroid carcinoma FNA samples, including papillary (29 samples), poorly differentiated (10 samples), anaplastic (9 samples), and Hurthle cell (2 samples) carcinomas, with a known BRAF p.V600E mutation status were selected for the current study. Immunostaining was performed on smears and cell block sections using an anti-BRAF p.V600E antibody (clone VE1). The results were compared with the known mutation status obtained by molecular testing and/or immunostaining of surgical pathology material from the same patient., Results: Of the total of 50 cases, 26 cases had smears available for the evaluation of BRAF p.V600E immunostaining; positive immunostaining was noted in 16 samples and negative immunostaining was noted in 4 samples, whereas 6 cases were equivocal. Of the 34 cases for which cell blocks were available for evaluation, BRAF p.V600E immunostaining was positive in 17 cases, negative in 16 cases, and equivocal in 1 case. The overall sensitivity and specificity of BRAF p.V600E immunostaining on the cell block preparation was 94.4% and 100%, respectively, whereas for the smears it was 80% and 63.6%, respectively., Conclusions: BRAF p.V600E immunostaining can be performed reliably on thyroid FNA cell block preparations. However, false-positive results on direct smears limit their utility and therefore need to be interpreted with caution. Cancer Cytopathol 2018;126:406-13. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

Background: Fine‐needle aspiration cytology (FNAC) is generally the initial sampling method for salivary gland neoplasms. The cytomorphologic features of acinic cell carcinoma (AciCC) of salivary gland can overlap with other neoplastic and nonneoplastic entities. AciCCs harbor a recurrent t(4;9) rearrangement with upregulation of nuclear receptor subfamily 4 group A member 3 (NR4A3). NR4A3 protein overexpression has been shown to be highly sensitive and specific for the diagnosis of AciCC in histologic specimens and cell block preparations. However, data on NR4A3 immunocytochemistry (ICC) on conventional smears or liquid‐based cytology are limited. Methods: The authors identified 18 FNAC of histologically proven AciCC cases between 2013 and 2019. FNAC samples of diagnostic mimickers were likewise retrieved and included in the study cohort for comparison. The NOR1/NR4A3 mouse monoclonal antibody was applied directly to FNAC slides using a standard ICC technique. Results: The cohort included ethanol‐fixed Papanicolaou‐stained cytologic smears and liquid‐based preparations from 18 AciCC, one secretory carcinoma, four mucoepidermoid carcinomas, four salivary duct carcinomas, five pleomorphic adenomas (PA), five Warthin tumors, five oncocytomas, one oncocytic hyperplasia, and five nonneoplastic salivary gland cases. Strong nuclear staining for NR4A3 was present in all AciCC, weak nuclear staining was present in one PA, and all other non‐AciCC were negative (sensitivity, 100%; specificity, 97%). Conclusions: NR4A3 ICC can be used directly on FNAC conventional smears and liquid‐based cytology to reliably distinguish AciCC from its mimickers. This marker may be useful in cases where a cell block preparation is unavailable or inadequate. NR4A3 immunocytochemistry can be used directly on fine‐needle aspiration cytology conventional smears and liquid‐based cytology to reliably distinguish acinic cell carcinoma from its mimickers. This may be useful in cases where a cell block preparation is unavailable or inadequate. [ABSTRACT FROM AUTHOR]

Background: The development of high‐throughput drug screening (HTS) using primary cultures provides a promising, clinically translatable approach to tailoring treatment strategies for patients with cancer. However, this has been challenging for solid tumors because of often limited amounts of tissue available. In most cases, in vitro expansion is required before HTS, which may lead to overgrowth and contamination by non‐neoplastic cells. Methods: In this study, hematoxylin and eosin staining and immunohistochemical staining were performed on 129 cytopathology cases from 95 patients. These cytopathology cases comprised cell block preparations derived from primary tumor specimens or patient‐derived xenografts as part of a pediatric precision oncology trial. Cytopathology cases were compared with the morphology and immunohistochemical staining profile of the original tumor. Cases were reported as tumor cells present, equivocal, or tumor cells absent. The HTS results from cytopathologically validated cultures were incorporated into a multidisciplinary tumor board report issued to the treating clinician to guide clinical decision making. Results: On cytopathologic examination, tumor cells were present in 77 of 129 cases (60%) and were absent in 38 of 129 cases (29%), whereas 14 of 129 cases (11%) were equivocal. Cultures that contained tumor cells resembled the tumors from which they were derived. Conclusions: Cytopathologic examination of tumor cell block preparations is feasible and provides detailed morphologic characterization. Cytopathologic examination is essential for ensuring that samples submitted for HTS contain representative tumor cells and that in vitro drug sensitivity data are clinically translatable. Routine cytopathology is able to establish the presence or absence of tumor cells in primary cultures derived from patients with pediatric cancer. Cytopathologic examination ensures the clinical relevance of in vitro drug sensitivity profiling in a pediatric precision oncology platform. [ABSTRACT FROM AUTHOR]

Background: High‐risk human papillomavirus (HR‐HPV) status is critical for the diagnosis, prognosis, and treatment of patients with oropharyngeal squamous cell carcinoma (OPSCC). Patients often present with enlarged cervical nodes, and fine‐needle aspiration cytology (FNAC) is frequently the initial diagnostic procedure. Although p16 is the most widely used surrogate marker, problems with interpretation can limit its utility in FNAC. HR‐HPV RNA in situ hybridization (ISH) has emerged as a specific way to assess HPV status on cell block preparations of cervical nodes. The authors evaluated the utility of HR‐HPV ISH in conventional smears and liquid‐based cytology (LBC) preparations of metastatic head and neck squamous cell carcinoma (SCC). Methods: Thirty‐one aspirates of proven, HPV‐related SCC (confirmed by p16 and/or HR‐HPV ISH in corresponding surgical specimens) were selected. Ten aspirates of HPV‐negative SCC were also retrieved. HR‐HPV ISH was performed on 27 smears and 14 LBC preparations. All results were scored as positive, equivocal, or negative. Results: Eighty‐four percent of metastatic, HPV‐related SCCs were positive for HR‐HPV RNA ISH, with high number of signals (n = 19) and low number of signals (n = 7), whereas five HPV‐related SCCs were equivocal. All metastatic, HPV‐negative SCCs were negative for HR‐HPV ISH. Conclusions: HR‐HPV ISH can be reliably performed on smears or LBC preparations, particularly when cell blocks are unavailable or paucicellular. Results were easy to interpret when high numbers of signals were present but were challenging in aspirates with low or rare number of signals. The current study suggests that HR‐HPV ISH could be used as the initial testing modality for determining HPV status in FNAC specimens of metastatic SCC. High‐risk human papillomavirus RNA in situ hybridization can be reliably performed on conventional smears or liquid‐based cytology. It could also be used as the initial testing modality for determining human papillomavirus status in patients with metastatic head and neck squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Background: Acinic cell carcinoma (AcCC) is diagnostically challenging on fine‐needle aspiration because it can mimic several other neoplasms or even normal acinar tissue. Immunopositivity for DOG1, especially circumferential membranous staining, can support the diagnosis of AcCC but is not entirely specific, and it is prone to technical and interpretive challenges on small specimens. NR4A3 (nuclear receptor subfamily 4 group A member 3) translocation and nuclear NR4A3 overexpression were recently described in the majority of AcCCs. Here, the authors evaluate the performance of NR4A3 immunohistochemistry (IHC) and NR4A3 break‐apart fluorescence in situ hybridization (FISH) on cell block preparations and compare them with DOG1 IHC in distinguishing AcCC from other entities in the differential diagnosis. Methods: The authors identified 34 cytology cell blocks with lesional cells, including 11 specimens of AcCC (2 of which derived from 1 patient and showed high‐grade transformation) as well as 2 secretory carcinomas, 7 salivary duct carcinomas, 4 mucoepidermoid carcinomas, 3 oncocytomas, 3 renal cell carcinomas, and 6 specimens containing nonneoplastic salivary gland tissue. NR4A3 IHC, DOG1 IHC, and NR4A3 FISH were attempted for all cases. Results: NR4A3 IHC had 81.8% sensitivity and 100% specificity for AcCC, whereas NR4A3 FISH had 36.4% sensitivity and 100% specificity, although 4 cases (3 mucoepidermoid carcinomas and 1 salivary gland tissue sample) could not be analyzed because of low cellularity. Notably, no normal acinar tissue specimens showed NR4A3 positivity by IHC or FISH. In addition, DOG1 IHC had 72.7% sensitivity and 92% specificity. Conclusions: NR4A3 IHC is highly specific for the diagnosis of AcCC and is more sensitive than DOG1 IHC and NR4A3 FISH. In addition, NR4A3 IHC performance is not improved by the inclusion of DOG1 IHC. Finally, NR4A3 positivity resolves the perennial problem of distinguishing AcCC from normal acinar tissue. Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry (IHC) is sensitive and specific for the diagnosis of acinic cell carcinoma in cytology cell block preparations, with better performance than DOG1 IHC. NR4A3 fluorescence in situ hybridization is also specific for acinic cell carcinoma but is less useful with limited tissue and is less sensitive than NR4A3 IHC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal (GI) tract, and the majority contain KIT or PDGFRA activating mutations. Fine-needle aspiration biopsy (FNAB) is a valuable technique in the diagnosis of GIST and may allow for preoperative therapy with tyrosine kinase inhibitors (TKI). Because of the morphologic diversity of these tumors, routine diagnosis of GIST often relies on C-Kit immunohistochemical staining in addition to morphologic findings. However, up to 15% of GISTs are C-Kit negative. Antibodies with increased sensitivity and specificity for detection of C-Kit-negative GIST cases may be of value, especially because some of these cases may also benefit from TKI therapy. METHODS: Immunohistochemical staining for DOG-1, C-Kit (CD117) and protein kinase C theta (PKCθ) was performed on FNA cell-block preparations representing 30 GISTs, 17 leiomyosarcomas, 16 melanomas, 16 schwannomas, 11 adenoid cystic carcinomas, and 8 leiomyomas. RESULTS: DOG-1 was found to have 100% sensitivity and 100% specificity in diagnosis of GIST. C-Kit demonstrated 70% sensitivity and 76% specificity, and PKCθ showed 40% sensitivity and 86% specificity. When only spindle-cell neoplasms were considered (adenoid cystic carcinomas excluded), the specificity of C-Kit increased to 89%. Of interest, all C-Kit-negative cases showed DOG-1 positivity. CONCLUSIONS: DOG-1 was the most sensitive and specific of the 3 markers for the diagnosis of GIST in cell-block preparations and may be of particular use in the diagnosis of C-Kit-negative GIST. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society [ABSTRACT FROM AUTHOR]

Background: A peritheliomatous pattern (PP) in tumors is characterized by a sheath of viable tumor cells closely surrounding a central blood vessel. In the authors' cytology practice, such a PP has been recognized in several metastatic melanoma specimens. The aim of this study was to evaluate the frequency of a PP in cytology samples of melanoma in comparison with other tumors. Methods: Eighty archival fine‐needle aspiration (FNA) cases of metastatic melanoma were compared with 65 control cases (35 poorly differentiated/metastatic carcinomas, 15 lymphomas, and 15 recurrent/metastatic/high‐grade sarcomas). Cytologic findings were correlated with corresponding histologic specimens, which were available for 44 cases (55%) in the melanoma group and for 23 cases (35.38%) in the control group. All slides were examined for a PP and were semiquantitatively graded for comparison. Results: A PP was present in 51.3% of the cytologic preparations (n = 41) among the melanoma group cases, whereas in the control group, a PP was present in only 3.1% of the cases (n = 2). A PP was present in 65.9% of melanomas with available histologic sections (n = 29) and in 8.7% of tissue samples from the control group (n = 2). A PP was seen more often in cell blocks than direct smear preparations (51.3% vs 40.0%) from patients with melanoma. Conclusions: The presence of a characteristic PP can be helpful in diagnosing melanoma in FNA samples because it was present in almost half of the metastatic melanoma cases in this study and was rarely present in other tumor types. A PP is easier to recognize and more often presents in cell blocks than aspirate smears. Ancillary studies such as immunohistochemistry are helpful for excluding other entities that may exhibit a PP. In this study, the presence of a peritheliomatous pattern of tumor cells is shown to be helpful in diagnosing melanoma in fine‐needle aspiration smears and cell block preparations. This characteristic growth pattern is present in almost half of the metastatic melanoma cases in this study, and this highlights it as an important morphologic feature to recognize in fine‐needle aspirates of melanoma. [ABSTRACT FROM AUTHOR]

Genomic profiling of tumor from malignant effusions have conventionally been performed on tumor cells derived from cytologic preparations such as cell block preparations, direct smears, cytospin preparations and liquid based cytologic preparations. The study by Yang et al. exemplifies the rapid progress of molecular cytopathology as cytopathologists increasingly delve into novel sources for genomic testing in an attempt to provide the theranostic information necessary for patient care. [ABSTRACT FROM AUTHOR]

BACKGROUND: The success of cell block preparation is crucial for ancillarydiagnostic tests in cytology.However, achieving an optimal cell block can be challenging. The current study describes a self-clotting-based technique for fine-needle aspiration (FNA) cell block preparations and evaluates its usefulness in comparison with the conventional needle wash technique. METHODS: The clinical data, FNA procedure, and cellularity of cell blocks of the self-clotting group (37 cases) and the conventional needle wash group (33 cases) were compared. The cellularity was evaluated using a scoring system (0 indicated acellular, 1 indicated 1-50 cells, and 2 indicated >50 cells). RESULTS: Approximately 76% of cases in the self-clotting group received a score of 2 versus 36% in the conventional needle wash group. Approximately 14% received a score of 1 in the self-clotting group compared with 9% in the conventional needle wash group, whereas 11% in the self-clotting group received a score of 0 versus 55% in the conventional needle wash group. The differences between the 2 methods were statistically significant. CONCLUSIONS: The results of the current study demonstrate that the self-clotting method is superior to the conventional needle wash method for FNA samples. [ABSTRACT FROM AUTHOR]

BACKGROUND The circulating tumor cell (CTC) field is rapidly advancing with the advent of continuously improving technologies for enriching these rare neoplastic cells from blood. CTC enumeration provides prognostic information, and CTC characterization has the potential to provide more useful information for the clinical decision-making process in this era of personalized medicine and targeted therapeutics. Proof-of-principle studies have shown that CTC samples can be characterized with a variety of techniques in the research laboratory environment. The goal of the current study was to validate routine cytologic techniques and immunohistochemical markers in CTC samples in a clinical cytology laboratory, using inducible phosphorylated signal transducer and activator of transcription 3 (pSTAT3) as a clinically important example and Ki-67 as a positive control. METHODS Whole blood from noncancer patients was spiked with breast cancer cell lines with constitutive or inducible pSTAT3 expression and underwent CTC processing in the CellSearch system. The resulting CTC samples were subjected to various cytologic/immunocytochemical techniques and were compared with non-CTC-processed cultured cell controls. RESULTS CTC-processed samples showed a morphology comparable to that of controls in cytospin, ThinPrep, and cell block preparations. Immunocytochemistry for Ki-67 and pSTAT3 provided biological information from CTC samples, showing uniform Ki-67 staining across all samples, pSTAT3 positivity in the constitutive and induced cells, and an absence of pSTAT3 expression in the noninduced cells, as expected. CONCLUSIONS CTC samples can be processed in the cytology laboratory with routine methods. CTC morphologic and immunophenotypic analysis can be easily integrated into the existing clinical workflow, moving the field closer to a true peripheral blood liquid biopsy for cancer patients. Cancer (Cancer Cytopathol) 2015;123:696-705. © 2015 American Cancer Society. [ABSTRACT FROM AUTHOR]

BACKGROUND: The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing. METHODS: This study reviewed variables associated with all in-house cytology cases (n5207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias. RESULTS: One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/lL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%. CONCLUSIONS: The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis. [ABSTRACT FROM AUTHOR]

BACKGROUND The use of immunocytochemistry as an important ancillary technique for the workup of fine-needle aspiration (FNA) specimens is an important component in the daily practice of cytopathology. Although cell block preparations are commonly used, immunocytochemistry cannot be completed if the cell blocks are hypocellular. As an alternative approach when conventional cell blocks fail, immunocytochemistry can be performed on cell-transferred direct smears. Multiple immunostains can be applied to a single direct smear using the cell transfer technique (CTT). METHODS A retrospective review of all FNA cases performed at the study institution over a 3-year period was conducted. All cases in which CTT was used for immunocytochemistry were identified. The contribution of CTT to the final diagnosis in these cases was assessed. RESULTS CTT was used in 152 of the 11,259 FNAs performed. A total of 431 immunocytochemistry stains, including 46 different antibodies, were performed using CTT. In 118 of 150 cases, the immunocytochemistry results contributed significantly to the final diagnosis. It provided no added value in the remaining 32 cases and failed in 2 cases. CONCLUSIONS CTT is a very useful tool for the workup and diagnosis of FNA specimens when conventional cell blocks lack adequate cellularity. Although CTT was used in only 1.4% of FNA cases in the current study, it contributed to the final diagnosis in 79% of the cases in which it was used. For these patients, CTT reduced the need for repeat FNAs, thereby reducing potential patient morbidity and health care costs. Cancer (Cancer Cytopathol) 2014;122:898-902. © 2014 American Cancer Society. [ABSTRACT FROM AUTHOR]

BACKGROUND Cystic pancreatic neuroendocrine tumors (cPanNETs) account for 13% to 17% of PanNETs. Although the value of endoscopic ultrasound (EUS) imaging and cyst fluid analysis (CFA) in their preoperative diagnosis has been well described, limited information is available about the diagnostic role of cytology samples obtained from fine-needle aspiration (FNA). METHODS Cytopathology records between 1992 and 2013 were searched for all reports of cysts interpreted as PanNET. Patient demographics, clinical and radiologic information, CFA, histopathology, and cytopathology findings were recorded. Performance characteristics of cytology and EUS for the accurate diagnosis of cPanNET were calculated. RESULTS In total, 35 FNAs from 33 patients with cPanNETs were identified, and 34 EUS were performed. Cytology made a specific diagnosis of a cPanNET in 71% of the biopsies compared with a specific diagnosis by EUS in 38% of cases. An interpretation of suspicious for cPanNET was given in 77% of cases by cytology and in 47% by EUS. Cytology identified 86% of the lesions as high-risk pancreatic cysts compared with 56% by EUS. Diagnostic morphology was present on both cytology and cell block preparations in 60% of aspirates, on cytology only in 20%, and on cell block only in 20%. CFA was performed on 51% cyst fluids. All cysts but 1 revealed low carcinoembryonic antigen levels (range, 0.2 to >500 ng/mL; mean, 29.5 ng/mL), and amylase levels were <500 U/L in all but 2 cases (range, 16-1493 U/L; mean, 205 U/L). CONCLUSIONS Cytology is the most accurate test for preoperative diagnosis of cPanNETs. EUS is insufficiently accurate for independent diagnosis, and carcinoembryonic antigen and amylase analyses are noncontributory. Cancer (Cancer Cytopathol) 2014;122:435-444. © 2014 American Cancer Society. [ABSTRACT FROM AUTHOR]

BACKGROUND The usefulness of GATA3 (GATA-binding protein 3 to DNA sequence [A/T]GATA[A/G]) as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. METHODS Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial, and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from the breast (30 specimens), female genital tract (13 specimens), gastrointestinal tract (7 specimens), lung adenocarcinoma (9 specimens), pancreas (1 specimen), kidney (1 specimen), and bladder (1 specimen). The breast carcinoma cases included 15 ductal carcinomas and 8 lobular carcinomas; the histology subtype was not available for 7 specimens. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and gross cystic disease fluid protein of 15 kilodaltons (GCDFP-15) to compare their sensitivity with GATA3. RESULTS Positive nuclear staining for GATA3 was found to be present in 90% of metastatic breast carcinoma specimens (27 of 30 specimens). All nonbreast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases demonstrated weak positivity of benign lymphoid cells. Staining results were unambiguous because all positive cases demonstrated intense nuclear staining in > 50% of tumor cells. Mammaglobin (57% staining; 17 of 30 cases) and GCDFP-15 (33% staining; 10 of 30 cases) were found to be less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only 1 additional case compared with those stained with GATA3. CONCLUSIONS GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration. Cancer (Cancer Cytopathol) 2014;122:307-312. © 2014 American Cancer Society. [ABSTRACT FROM AUTHOR]

BACKGROUND: With the introduction of new treatment modalities and guidelines, it is important to subclassify primary pulmonary non-small cell carcinoma (NSCCA). Subsequent treatment and testing is dependent on accurate subclassification. Endobronchial ultrasound fine-needle aspiration (EBUS FNA) is used for primary evaluation and diagnosis, and can provide a cell block for ancillary testing. METHODS: EBUS FNA cases from primary pulmonary NSCCA with an immunohistochemical (IHC) panel performed on a cell block with concomitant surgical pathology biopsy were analyzed. Cell block preparations underwent an IHC panel including monoclonal antibodies for napsin A (nap-A), thyroid transcription factor (TTF-1), p63, and cytokeratin 5/6 (CK5/6). RESULTS: A total of 81 cases from 81 patients were identified. Of these, 69 cases (85%) were provided a specific diagnosis of adenocarcinoma (ADCA) or squamous cell carcinoma (SCCA) on the EBUS FNA. In 12 cases (15%), a diagnosis of NSCCA, not otherwise specified was provided. For specific subclassifications, there were 35 ADCA cases, 34 SCCA cases, and 12 NSCCA, not otherwise specified cases. For ADCA, nap-A showed granular cytoplasmic staining and TTF-1 nuclear staining. For SCCA, CK5/6 showed cytoplasmic staining and p63 nuclear staining. Surgical pathology concomitant material was present in 29 of 81 cases with 18 correlations and 11 noncorrelations. CONCLUSIONS: With new treatment guidelines for patients with primary pulmonary NSCCA, specific diagnosis is increasingly important. EBUS FNA with cell block provided a specific subclassification of NSCCA in 85% of cases when used in conjunction with a specific IHC panel including nap-A, TTF-1, CK5/6, and p63. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society. [ABSTRACT FROM AUTHOR]

Background: The objectives of this study were to investigate the utility of endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA)/endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) for the diagnosis of amyloidosis coupled with the feasibility of mass spectrometry (MS) for amyloid subtyping. Methods: All patients who had amyloid diagnosed by EBUS‐TBNA/EUS‐FNA at two tertiary care centers from 2011 to 2020 were retrieved along with the MS subtype, clinical findings, and outcomes. Results: Eight patients were included: seven underwent EBUS‐TBNA of mediastinal lymph nodes, and one underwent EUS‐FNA of a periportal lymph node. Ages ranged from 37 to 79 years (median, 69 years), with equal numbers of men and women. Presenting clinical history included one case each of follicular lymphoma, lymphoplasmacytic lymphoma, rheumatoid arthritis, possible sarcoid, cirrhosis, and chronic renal insufficiency, and one case each of suspected pulmonary and cardiac amyloidosis. All cases showed waxy, amorphous material on direct smears (n = 5) or ThinPrep slides (n = 3), which were confirmed as amyloid on Congo Red staining. Immunohistochemistry showed dominant lambda staining in two of three cases. MS was performed in all cases and identified five of the light‐chain (AL) type, one of the heavy‐chain/AL type, and two suggestive of AL amyloidosis. Bone marrow biopsy performed in seven patients demonstrated that three had monoclonal plasma cells and one had lymphoplasmacytic lymphoma. Two of four patients with systemic amyloidosis received chemotherapy and remained alive, whereas three with localized disease remained stable under observation. Conclusions: EBUS‐TBNA/EUS‐FNA is effective for amyloidosis diagnosis and provides adequate material for ancillary tests, including MS, which can identify the precursor amyloidogenic protein, leading to appropriate patient management. The utility of endobronchial ultrasound‐guided transbronchial needle aspiration and endoscopic ultrasound‐guided fine‐needle aspiration for the diagnosis of amyloidosis, including when clinically unsuspected, and the feasibility of subsequent mass spectrometry for subtyping of the amyloidogenic protein were investigated. Endobronchial ultrasound‐guided transbronchial needle aspiration/endoscopic ultrasound‐guided fine‐needle aspiration was able to provide an early and accurate assessment of unexpected mass‐like deposits of nodal amyloid with sufficient material to diagnose and classify the protein subtype. [ABSTRACT FROM AUTHOR]

Background: The management of cytological samples can significantly impact diagnostic interpretation. Cell blocks (CB) are a popular method due to their ability to provide additional morphological information and be used for immunocytochemistry and molecular tests. Recently, a new CB technique called the synthetic matrix CytoMatrix (CM) has been introduced, which can gather and hold cytological material within its three‐dimensional structure. Methods: In this study, 40 cytological samples from patients with melanoma metastases were analyzed to evaluate the diagnostic performance of CM compared to another CB method used in the laboratory. The researchers assessed the morphological adequacy of the two techniques, as well as their performance in immunocytochemical analysis and molecular. Results: This study showed that CM was quicker and equally effective compared to the other method, with the laboratory technician having less of an impact on the CM approach across all passages. Additionally, all CMs were adequate, whereas the other method was adequate in 90% of cases. Immunocytochemistry confirmed the diagnosis of melanoma metastases in all cases, and all 40 CMs and 36 of the other method were adequate for fluorescence in situ hybridization analysis. Conclusion: CM is a low‐time‐consuming technology that is unaffected by technicians during all setup phases, making it simpler to standardize the procedure. Moreover, a low loss of diagnostic cells ensures greater benefits for morphological analysis, immunocytochemistry, and molecular testing. Overall, the study highlights the potential of CM as a valuable technique for managing cytological samples. CytoMatrix is a low time‐consuming technique, not subject to the intervention of the technical operator in the various stages of setup, which allows a standardization of the cytological samples process. [ABSTRACT FROM AUTHOR]

Background: Claudin‐4 is a sensitive and specific marker for carcinoma in effusion cytology. The authors examined the diagnostic use of claudin‐4 versus MOC‐31 and Ber‐EP4 by comparing their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in differentiating carcinoma from mesothelioma and benign/mesothelial hyperplasia in effusion specimens. Methods: This retrospective study comprised a cohort of 229 cytology specimens, including 211 effusion fluid and 18 fine‐needle aspiration specimens. Cytologic categories included 134 carcinoma, 28 mesothelioma, 46 indefinite (suspicious and atypical), and 21 benign. Cell block sections were stained for claudin‐4 and compared with those previously stained for MOC‐31 and Ber‐EP4. Indefinite cases were further reclassified based on clinical and pathologic findings into benign (26 cases), mesothelioma (11 cases), and carcinoma (nine cases). Results: None of the mesotheliomas (0/39) or benign effusions (0/47) were positive for claudin‐4, whereas 134 of the 143 carcinoma specimens were positive. Compared to MOC‐31 and Ber‐EP4, claudin‐4 had the highest specificity and PPV (100% for each), followed by Ber‐EP4. Claudin‐4 showed high sensitivity (93.7%), albeit lower than MOC‐31. MOC‐31 had the lowest specificity and PPV but the highest sensitivity and NPV. Ber‐EP4 had the lowest sensitivity (91.6%). Conclusions: Claudin‐4 can be used as a single marker for carcinoma with high sensitivity and superior specificity compared with MOC‐31 and Ber‐EP4. Mesothelial lineage can be ruled out when claudin‐4 is positive. In equivocal cytology samples with few scattered cells of interest, a panel of claudin‐4 and Ber‐EP4 results in the highest combined sensitivity and specificity. The findings in this study demonstrate the high sensitivity and superior specificity of claudin‐4 as a single marker for carcinoma in cytology specimens and its ability to rule out mesothelial lineage with high certainty. The authors also identify the highly sensitive and specific combination of claudin‐4 and Ber‐EP4 in diagnosing equivocal specimens previously categorized as atypical or suspicious. [ABSTRACT FROM AUTHOR]

Background: Non–small cell lung carcinoma (NSCLC) patients with BRAF V600E–mutated tumors respond to targeted therapy. Testing for BRAF V600E is commonly performed with molecular methods; however, a mutation‐specific VE1 antibody clone can provide an alternative testing option using immunohistochemistry (IHC) for practices using single‐gene testing and in situations when the specimen is inadequate for molecular testing. This study evaluates the usefulness of VE1 IHC in screening for BRAF V600E mutations in NSCLC cytology specimens. Methods: The authors retrospectively identified cytology cases with a diagnosis of NSCLC that had BRAF V600E IHC performed on cell block sections with the monoclonal VE1 antibody clone. The BRAF V600E IHC results were compared with those of molecular testing performed with an amplicon‐based next‐generation sequencing assay. Results: There were 201 NSCLC cases evaluated. The VE1 IHC was positive in seven of seven BRAF V600E–mutated tumors (100%) and was negative in 158 of 158 nonmutated BRAF V600E tumors (100%). Thirty cases did not undergo molecular testing, primarily because of insufficient tissue or because molecular testing was performed on an alternative specimen. Six cases showed equivocal weak/focal staining: Two cases demonstrated BRAF V600E mutations, and four cases were negative by molecular testing. Conclusions: This study suggests that BRAF V600E IHC can be used reliably to screen NSCLC cytology specimens, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining. BRAF V600E immunohistochemistry can be used reliably to screen cytologic specimens of non–small cell lung carcinoma, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining. [ABSTRACT FROM AUTHOR]

Background: The International System for Reporting Serous Fluid Cytopathology (ISRSFC) was published recently to provide standard reporting terminology for serous fluid. To date, several ISRSFC reclassification studies have reported a wide range of diagnostic category frequency and the associated risk of malignancy (ROM). Herein, the authors applied the ISRSFC to report pleural and peritoneal effusions retrospectively in a community hospital setting. Methods: With Internal Review Board approval, 446 peritoneal effusion specimens and 299 pleural fluid specimens from 576 patients in three community hospitals over a 12‐month period were reviewed and reclassified according to the ISRSFC. Results: After reclassification, in pleural effusions, 18 (5.0%) were nondiagnostic (ND), 273 (76.0%) were negative for malignancy (NFM), 18 (5.0%) were atypia of undetermined significance (AUS), 6 (1.7%) were suspicious for malignancy (SFM), and 44 (12.3%) were malignant (MAL). In peritoneal effusions, after reclassification, 11 (5.5%) were ND, 168 (77.1%) were NFM, 9 (4.1%) were AUS, 2 (0.9%) were SFM, and 27 (12.4%) were MAL. The calculated ROM was 0.0% for ND, 1.8% for NFM, 37.5% for AUS, 83.3% for SFM, and 100.0% for MAL in peritoneal effusions; and the ROM was 8.3% for ND, 1.2% for NFM, 44.4% for AUS, and 100.0% for both SFM and MAL in pleural effusions. Further analysis demonstrated notable heterogeneity among published ISRSFC reclassification studies, although the overall ROMs did not differ significantly from the ISRSFC‐determined ROMs (all p values were >.05 for mean ROM comparisons). Conclusions: The findings suggested the necessity for each laboratory to perform its own ROM analysis based on its statistics for ISRSFC‐tiered classification terminology. The International System for Reporting Serous Fluid Cytopathology was used to retrospectively report cytology in pleural and peritoneal effusion specimens in a community hospital setting. Although the system provided consistency of terminology as a tiered classification, the findings suggested the necessity for each laboratory to perform its own risk of malignancy analysis based on its own statistics. [ABSTRACT FROM AUTHOR]

Discordant p16 and human papillomavirus (HPV)‐specific testing results, although uncommon in oropharyngeal squamous cell carcinomas, present a quandary to pathologists and treating clinicians alike. This commentary outlines potential scenarios leading to p16/HPV discordance and highlights recent studies that shed some light on the prognostic significance thereof. [ABSTRACT FROM AUTHOR]

Background: Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC., Methods: In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases., Results: Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n = 13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n = 13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n = 4)., Conclusions: Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples., (© 2023 American Cancer Society.)

The new World Health Organization classification of endocrine tumors will include many updates on thyroid pathology. This summary highlights the changes that are most relevant for cytopathologists. [ABSTRACT FROM AUTHOR]

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN‐negative and 15 pN‐positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN‐negative and pN‐positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN‐positive versus pN‐negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin‐releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR‐154‐3p, miR‐299‐5p, miR‐376a‐3p, and miR‐302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting. ; Preoperative biomarkers predictive of aggressive behavior in thyroid cancer are needed. Our study indicate that a microRNA‐based approach is a feasible, methodologically robust, and accurate strategy to identify biomarkers to predict the presence of lymph node metastases in papillary thyroid carcinoma in the preoperative setting. [ABSTRACT FROM AUTHOR]

BACKGROUND: Fine‐needle aspiration (FNA) is a robust diagnostic technique often used for tissue diagnosis of metastatic carcinoma. For interpretation of FNA cytology, cell block immunohistochemistry (IHC) and clinicocytologic parameters are indispensable. In this review of a large cohort, the current report: 1) describes clinicocytologic parameters and immunoprofiles of aspirates of metastatic carcinoma, 2) compares the predictivity of immunostains and classical approaches for IHC interpretation, and 3) describes machine learning‐based algorithms for IHC interpretation. METHODS: Aspirates of metastatic carcinoma that had IHC performed were retrieved. Clinicocytologic parameters, IHC results, the corresponding primary site, and histologic diagnoses were recorded. By using machine learning, decision trees for predicting the primary site were generated, their performance was compared with 2 human‐designed algorithms, and the primary site was suggested in the historical diagnosis. RESULTS: In total, 1145 cases were identified. The 6 most populated groups were selected for machine learning and predictive analysis. With IHC input, the decision tree achieved a concordance rate of 94.5% and overall accuracy of 83.6%, which improved to 95.3% and 85.8%, respectively, when clinical data were incorporated and exceeded the human‐designed IHC algorithms (P <.001). The historical diagnosis was more accurate unless indeterminate diagnoses were regarded as discordant (P <.001). CDX2 and TTF‐1 immunostains had the highest weight in model accuracy, occupied the root of the decision trees, scored higher as features of importance, and outperformed the predictive power of cytokeratins 7 and 20. CONCLUSIONS: Cytokeratins 7 and 20 may be superseded in immunostaining panels, including organ‐specific immunostains such as CDX2 and TTF‐1. Machine learning generates algorithms that surpasses human‐designed algorithms but is inferior to expert assessment integrating clinical and cytologic assessment.; Machine learning generates algorithms that surpasses human‐designed algorithms but is inferior to expert assessment integrating clinical and cytologic assessment. Cytokeratin 7/20 may be superseded by organ‐specific immunostains, such as CDX2 and TTF‐1. [ABSTRACT FROM AUTHOR]

The recently published 2021 guidelines for the management of patients with anaplastic thyroid cancer from the American Thyroid Association provide updated recommendations and guidance for the treatment of this rare but lethal thyroid cancer and incorporate significant advances since the initial guidelines were published in 2012. In particular, the focus on prompt cytopathologic and immunohistochemical diagnosis with the identification of potentially actionable genomic/molecular targets affirms the central and key role of pathologists and cytopathologists in not only the diagnosis but also the management of these patients. [ABSTRACT FROM AUTHOR]

Blood‐based molecular diagnostics have emerged as diagnostic tools applicable to a broad spectrum of tumor types from early diagnosis to treatment monitoring and the detection of recurrent disease. Here, we discuss emerging data on HPV‐specific liquid biopsies and their utility as diagnostic tests for HPV‐associated oropharyngeal squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Background: Gynecologic sex cord‐stromal tumors (SCSTs) arise from sex cords of the embryonic gonad and may display malignant behavior. We describe the cytomorphologic features of SCSTs in females, including adult and juvenile granulosa cell tumors (AGCTs and JGCTs), Sertoli‐Leydig cell tumors (SLCTs), and steroid cell tumors (SCTs). Methods: We retrieved available cytology slides from females with a histologic diagnosis of sex cord‐stromal tumor between 2009 and 2020 from institutional archives and reviewed their cytoarchitectural features. Results: There were 25, 2, 2, and 1 cytology specimens from 19, 2, 2, and 1 patients (aged 7‐90 years, median 57 years) with AGCT, JGCT, SLCT, and SCT, respectively. Features common to all SCSTs included 3‐dimensional groups, rosettes, rare papillary fragments, abundant single cells and naked nuclei. Rosettes and a streaming appearance of cell groups were only seen in AGCTs, which also rarely featured eosinophilic hyaline globules and metachromatic stroma. AGCTs exhibited high nuclear:cytoplasmic (N:C) ratios, with mild nuclear pleomorphism, uniform nuclei with finely granular chromatin, nuclear grooves and small nucleoli; in contrast, other SCSTs lacked rosettes and nuclear grooves and had generally lower N:C ratios, greater nuclear pleomorphism, coarse chromatin and more abundant cytoplasm. Mitotic figures, necrosis, and inflammation were rarely identified. Conclusions: AGCTs show cytomorphologic features that are distinct from those of other SCSTs. Careful evaluation of the cytological features and ancillary studies (eg, immunochemistry for FOXL2, inhibin and calretinin, or sequencing for FOXL2 mutations) can aid in the accurate diagnosis of these tumors. Adult granulosa cell tumors show cytomorphologic features that are distinct from those of other types of sex cord‐stromal tumor, including microfollicular structures, eosinophilic hyaline globules, and longitudinal nuclear grooves in tumor cells. Careful evaluation of the cytologic features and ancillary studies (eg, immunochemistry for FOXL2, inhibin, and calretinin, or sequencing for FOXL2 mutations) can aid in accurate diagnosis of these tumors. [ABSTRACT FROM AUTHOR]

Background: Intraductal carcinoma of the salivary gland (IDC) is a rare cancer with potential actionable targets, including RET fusions. Histologic and molecular features of IDC were recently reported, but cytomorphologic data are limited. In the largest multi‐institutional fine‐needle aspiration (FNA) series, the authors describe the cytomorphologic features of 13 IDC cases with available clinical, radiologic, histopathologic, and molecular data. Methods: The cases included 13 FNAs for 9 low‐grade (LG) IDCs and 4 high‐grade (HG) IDCs with corresponding histopathology and available molecular, imaging, and clinical data. Smears and liquid‐based preparations available for 12 FNAs were semiquantitatively scored for key cytomorphologic findings and correlated with the corresponding resection. Results: LG IDC FNAs showed a cellular, biphasic population of large, atypical ductal cells with mildly pleomorphic nuclei in a clean background and a minor population of small, uniform myoepithelial cells. In contrast, all HG IDC FNAs showed predominantly ductal cells with marked nuclear pleomorphism, coarse chromatin, and necrosis. With the Milan system, most LG and HG IDC FNAs were classified as either salivary gland neoplasms of uncertain malignant potential (54%) or malignant (31%). Immunohistochemistry showed ductal epithelial reactivity with mammaglobin, androgen receptor, and S100, whereas myoepithelial cells were positive for p63 and/or calponin. Among cases with next‐generation sequencing, 4 LG IDCs showed NCOA4‐RET gene fusions, whereas an HG IDC showed HRAS and PIK3CA mutations. Conclusions: The cytomorphology of IDC overlaps with other benign and malignant salivary gland neoplasms. Immunohistochemistry limits the differential diagnosis, but definitive classification requires molecular analysis. A diagnosis of IDC has potential implications for patient management. The cytomorphology of low‐ and high‐grade intraductal carcinomas can overlap with other benign and malignant salivary gland neoplasms, and the diagnosis can affect patient management. Immunohistochemistry can limit the differential diagnosis, but molecular analysis is needed for definitive classification. [ABSTRACT FROM AUTHOR]

Background: The cytologic diagnosis of papillary lesions of the breast is challenging because of the diverse morphology, including epithelial hyperplasia, atypia, low‐grade malignancy, and neuroendocrine differentiation; also, traditional malignant features such as necrosis and myoepithelial cell loss can be lacking. Thus, the diagnostic criteria for papillary lesions may differ from those for other breast lesions. This study evaluated various cytologic parameters in a large cohort to identify useful diagnostic features. Methods: Cytologic preparations of papillary lesions with histologic follow‐up were reviewed for features related to cellularity, epithelial cohesiveness, cellular and stromal architecture, cytomorphology, and background. Corresponding histologic slides were also reviewed. Results: In all, 153 cases were included. Epithelial discohesion, solid and cribriform patterns, atypical nuclear features, and mitoses (P ≤.001 to P =.017) were associated with malignancy. Cell balls, monolayer sheets, and features of cystic change (P <.001 to P =.016) were associated with benign lesions. Complex (P =.031) and slender (P =.026) papillae and neuroendocrine features (P <.001) were associated with malignancy. Hemorrhage, background, and infiltrating neutrophils (P <.001 to P =.025) were associated with malignancy; fibrotic broad papillary stromal fragments (naked papillary fronds [NPFs]; P =.043) were associated with benignity. The presence of any single parameter, including the absence of myoepithelial cells within epithelial structure, the presence of cytoplasmic granules, an increased amount of cytoplasm, and a nuclear to cytoplasmic (N/C) ratio greater than 0.7, which were identified by principal component analysis, yielded a sensitivity of 95.1% and a specificity of 100.0% in predicting malignancy. Conclusions: Methodological assessment of multiple features is recommended. Myoepithelial cells, cytoplasmic granules, the amount of cytoplasm, and the N/C ratio are key features for diagnosis. Papillary breast aspirates are challenging and require methodological assessment of multiple features. Myoepithelial cells, cytoplasmic granules, the amount of cytoplasm, and the nuclear to cytoplasmic ratio are key features for diagnosis. [ABSTRACT FROM AUTHOR]

Malignant mesothelioma is a rare malignancy with a poor prognosis whose development is related to asbestos fiber exposure. An increasing role of genetic predisposition has been recognized recently. Pleural biopsy is the gold standard for diagnosis, in which the identification of pleural invasion by atypical mesothelial cell is a major criterion. Pleural effusion is usually the first sign of disease; therefore, a cytological specimen is often the initial or the only specimen available for diagnosis. Given that reactive mesothelial cells may show marked atypia, the diagnosis of mesothelioma on cytomorphology alone is challenging. Accordingly, cell block preparation is encouraged, as it permits immunohistochemical staining. Traditional markers of mesothelioma such as glucose transporter 1 (GLUT1) and insulin‐like growth factor 2 mRNA‐binding protein 3 (IMP3) are informative, but difficult to interpret when reactive proliferations aberrantly stain positive. BRCA1‐associated protein 1 (BAP1) nuclear staining loss is highly specific for mesothelioma, but sensitivity is low in sarcomatoid tumors. Cyclin‐dependent kinase inhibitor 2A (CDKN2A)/p16 homozygous deletion, assessed by fluorescence in situ hybridization, is more specific for mesothelioma with better sensitivity, even in the sarcomatoid variant. The surrogate marker methylthioadenosine phosphorylase (MTAP) has been found to demonstrate excellent diagnostic correlation with p16. The purpose of this review is to provide an essential appraisal of the literature regarding the diagnostic value of many of these emerging biomarkers for malignant mesothelioma in effusion cytology. Effusion cytology is often the only available specimen for diagnosis of malignant pleura mesothelioma (MPM), but the cytological features of MPM are not always straightforward, as reactive mesothelial cells can have an atypical appearance that overlaps with MPM. There are many biomarkers currently available to aid in the diagnosis of mesothelioma; however, because none of these markers is to be relied upon alone, they are best used in combination to increase their diagnostic yield. [ABSTRACT FROM AUTHOR]

Background: The distinction between mesothelioma with epithelioid features and metastatic carcinoma may be challenging, particularly on cytology. A novel 2‐hit Claudin‐4 and BRCA‐associated protein 1 (BAP1) panel was investigated. Methods: The objective of this study was to determine the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the panel on cytology from pleural effusions and matched biopsies, including 49 malignant mesotheliomas on cytology with 43 matched biopsies, 49 normal/reactive mesothelial proliferations, and 49 pleural metastatic carcinomas from different primaries with 21 matched pleural biopsies. The diagnostic role of the 4 categories obtained by crossing the immunostaining results was analyzed. Results: Claudin‐4 strongly stained all metastatic carcinomas and tested completely negative in normal mesothelium, benign reactive mesothelial hyperplasia, and malignant mesothelioma. All normal and benign mesothelial proliferations and all carcinomas except 1 were immunoreactive for BAP1, whereas BAP1 loss was observed in 88% of malignant mesotheliomas. The expression of Claudin‐4 alone excluded all benign and malignant mesothelial growth, consistently characterizing all metastatic carcinomas. Double negativity was evident in all malignant mesotheliomas, and double positivity was observed in all metastatic carcinomas. BAP1‐positive/Claudin‐4–negative status was observed only in malignant mesotheliomas and benign mesothelial proliferations. A single metastatic anal squamous cell carcinoma had BAP1‐negative/Claudin‐4–positive staining. Conclusions: Claudin‐4 expression was completely specific and sensitive for metastatic carcinoma, excluding mesothelial proliferations. BAP1 staining characterized 98% of metastatic carcinomas and 100% of benign mesothelial proliferations, whereas negativity was observed almost exclusively in mesotheliomas. This 2‐hit panel is probably the best compromise for differentiating malignant mesothelioma and metastatic carcinoma on either cytology or biopsy specimens. Claudin‐4 expression in effusion cytology is completely specific and sensitive for metastatic carcinoma, excluding mesothelial proliferations; otherwise, BRCA1‐associated protein 1 (BAP1) staining characterizes 98% of metastatic carcinomas and 100% of benign mesothelial proliferations, whereas negativity is almost exclusively disclosed in mesothelioma. This 2‐hit Claudin‐4/BAP1 panel is probably the best compromise in differentiating malignant mesothelioma and metastatic carcinoma on either cytology or biopsy specimens. [ABSTRACT FROM AUTHOR]

Background: Undifferentiated carcinoma (UC) is a rare, aggressive subtype of endometrial carcinoma. Dedifferentiated carcinomas (DCs) are UCs associated with a component of well differentiated endometrioid carcinoma. The authors sought to describe the morphologic features of UCs and DCs in cytologic specimens. Methods: Cytologic specimens from 23 women (aged 46‐86 years; median age, 59 years) were reviewed, including cervicovaginal specimens (n = 7), peritoneal washings (n = 5), touch preparations of core biopsies from various sites (n = 5), fine‐needle biopsies of lymph nodes (n = 3), ascitic fluid (n = 1), pleural fluid (n = 1), and intrauterine fluid (n = 1). Results: There were 10 UCs (43%) and 13 DCs (57%). Tumor cells were arranged as single cells (9 UCs, 90%; 12 DCs, 92%) and 3‐dimensional groups (8 UCs, 80%; 11 DCs, 85%). Most cases showed high nuclear‐to‐cytoplasmic ratios. Nuclear molding was observed in 3 UCs (30%) and in 5 DCs (38%). Nuclear chromatin was often coarsely granular 6 UCs, 60%; 9 DCs, 69%). Nucleoli were inconspicuous in some cases (6 UCs, 60%; 8 DCs, 62%) but were appreciable in others. Necrosis was observed in 5 UCs (50%) and in 5 DCs (38%). Most cases exhibited clean backgrounds, and a few showed acute inflammation. Comparison of the cytologic features of UCs and DCs did not reveal any statistically significant differences. Conclusions: UCs and DCs have a spectrum of cytomorphologic appearances that are not pathognomonic, but the presence of some of these (relatively uniform population of predominantly singly dispersed cells with high nuclear‐to‐cytoplasmic ratios and variably conspicuous nucleoli) should prompt consideration of UC and DC in the differential diagnosis. [ABSTRACT FROM AUTHOR]

Background: Atypia of undetermined significance (AUS) is a category of the Milan System for Reporting Salivary Gland Cytopathology that refers to salivary gland fine‐needle aspiration (FNA) specimens that cannot be definitively diagnosed as neoplastic or nonneoplastic. Methods: The AUS FNA samples were selected from a large academic institution from 2008 through 2018. The AUS cases were divided into 6 subgroups. The risk of malignancy (ROM), risk of neoplasm (RON), and clinical outcomes for each subgroup were evaluated. Results: A total of 123 cases were found (76 males and 47 females with a mean age of 62 years [range, 6‐94 years]). The parotid gland was the most common FNA site (103 cases), followed by the submandibular gland (9 cases). The overall RON and ROM were 63% and 47%, respectively. Among the subgroups, salivary gland lymph nodes or lymphoid lesions was the most common diagnosis (42%), whereas mucinous cystic lesions with no or a scant epithelial component was the least common (2%). The specimens with preparation artifacts category had the highest RON and ROM (100% for both), whereas the reactive and reparative atypia indefinite for a neoplasm category had the lowest RON and ROM (7% for both). The salivary gland lymph nodes or lymphoid lesions indefinite for a lymphoproliferative disorder category had the second highest RON and ROM at 77% and 74%, respectively. Conclusions: The overall RON and ROM for the AUS category were 63% and 47%, respectively. The RON and ROM varied among the different AUS subgroups, being highest in the specimens with preparation artifacts category and lowest in the reactive and reparative atypia category, thereby demonstrating the importance of subgrouping in the AUS specimens. To the authors' knowledge, the current study is the first to evaluate the risk of neoplasm (RON) and risk of malignancy (ROM) in 6 subgroups of the atypia of undetermined significance (AUS) category. The authors report that the RON and ROM vary among the different subgroups, being highest in the specimens with preparation artifacts hampering the distinction between a nonneoplastic and neoplastic process and lowest in the reactive and reparative atypical indefinite for a neoplasm category. The results of this study suggest the importance of subtyping AUS cases, which can convey differential ROMs and facilitate clinical stratification. [ABSTRACT FROM AUTHOR]

Background: Pelvic washing specimens are relatively common and are submitted for patients undergoing gynecologic surgery to evaluate them for metastatic or occult disease. Psammoma bodies are a relatively uncommon finding in these specimens. To date, large‐scale studies of their cytologic‐histologic correlates and thus clinical significance have been limited. Methods: A 10‐year retrospective search for all pelvic washing specimens was performed, and all cases were reviewed for the presence of psammoma bodies. The findings for the corresponding surgical pathology specimens were then catalogued. Results: Psammoma bodies were present in 138 cases (3.6% of 3840 total pelvic washings). More than half of the cases (n = 73 [53%]) were associated with benign processes, including mesothelial hyperplasia (n = 44), endosalpingiosis (n = 11), endometriosis (n = 11), and ovarian cystadenoma/cystadenofibroma (n = 7). Nineteen cases (14%) were associated with serous borderline tumors. Malignancies were noted in a third of the cases (n = 46 [33%]) and were most frequently low‐grade serous adenocarcinomas (n = 15), high‐grade serous adenocarcinomas (n = 11), or endometrioid endometrial carcinomas (n = 8). Patients found to have benign processes (median age, 50 years) or borderline disease (median age, 56 years) were significantly younger than patients with malignancies (median age, 65 years; P <.0001). In addition, the correlation of cytologic interpretation categories with concurrent histopathologic findings showed that cytologic analysis was highly sensitive and specific in determining the nature of underlying processes. Conclusions: The majority of psammoma bodies noted in pelvic washings were associated with benign processes or borderline tumors, with approximately a third of the cases associated with malignancies. Psammoma bodies in pelvic washings from younger patients were significantly more likely to be associated with benign processes or borderline tumors. Psammomatous calcifications are a relatively uncommon occurrence in pelvic washing specimens and can be associated with both benign and malignant processes. Because of the limited literature on the incidence of this finding and the cytologic‐histologic correlates, a large, 10‐year retrospective study has been conducted to explore the significance of identifying psammoma bodies in pelvic washing fluids. [ABSTRACT FROM AUTHOR]

Background: NUT carcinoma (NC) is an aggressive neoplasm that often presents with alarge tumor burden and metastases; cytology is frequently one of the primary diagnostic modalities. Primary pulmonary NCs are very rare and cytology descriptions are limited. The current study was performed to analyze the cytomorphological features of primary pulmonary NCs in different cytology samples and preparations. Methods: A total of 15 cytology specimens from 10 patients with primary pulmonary NCs diagnosed primarily on histology were retrieved and reviewed. Results: Fifteen cytology samples, including aspirates from primary (5 samples) and metastatic (5 samples) sites, sputum (1 sample), and effusions (4 samples), that were prepared as direct smears, centrifuged smears, and cell blocks were reviewed. Aspirate smears from all cases were cellular and demonstrated fragments and cohesive clusters of primitive tumor cells with scant cytoplasm, ovoid nuclei with coarse granular chromatin, and consistently conspicuous single nucleoli in a frequently neutrophil‐rich necrotic background with dispersed bare tumor nuclei. In fluid cytology, tight, 3‐dimensional tumor clusters and singly lying tumor cells were observed. Squamous differentiation in the form of sheets and singly lying polygonal tumor cells with abundant dense cytoplasm was noted focally in rare cases. The diagnoses during original sign‐outs were poorly differentiated carcinoma, poorly differentiated squamous cell carcinoma, and malignant small round cell tumor. NUT‐1 (NUT family member 1 protein) immunocytochemistry performed on cell blocks demonstrated characteristic speckled nuclear staining in tumor cells. Conclusions: Pulmonary NC presents as a poorly differentiated carcinoma with focal to absent squamous differentiation on cytology. Cellular fragments of primitive tumor cells with conspicuous nucleoli should raise suspicion of NUT carcinoma and prompt ancillary testing. Pulmonary NUT carcinomas appear as fragments and cohesive clusters in aspiration specimens and as tight 3‐dimensional balls in fluid specimens. They are composed of primitive tumor cells with ovoid nuclei, coarse granular chromatin, consistently conspicuous single nucleoli, and scant cytoplasm in a frequently neutrophil‐rich inflammatory background with dispersed bare nuclei. A high index of suspicion with the judicious use of immunocytochemistry (p40, NUT) is necessary for an early and accurate diagnosis on cytology. [ABSTRACT FROM AUTHOR]

Background: NTRK‐rearranged thyroid carcinomas (NRTC), though rare, harbor a potential therapeutic target. The cytomorphologic features by fine needle aspiration (FNA) and the utility of preoperative molecular testing for NRTC remain largely uncharacterized. We provide a detailed cytomorphologic analysis of an institutional NRTC cohort with clinical, radiologic, histopathologic, and molecular correlations. Methods: Our NRTC FNA cohort included 21 specimens from 19 patients. The mean age and female‐to‐male ratio were 42 years and 2.2:1, respectively. Predominantly alcohol‐stained Papanicolaou smears and liquid‐based preparations were reviewed for 14 patients with available materials, and histologic review of subsequent resections was conducted for all 19 patients. Imaging and clinical data were accessed through electronic medical records. Results: Sonographically, NRTC were hypoechoic (87%), predominantly solid (53%) with limited central vascularity (27%), ill‐defined borders (67%), and microcalcifications (67%). Observed cytomorphologic features include mixed architectural patterns (79%), fibrosis (93%), oncocytic and vacuolated cytoplasm (36% and 43%, respectively), and abundant intranuclear pseudoinclusions (14%). Most NRTC FNAs were classified as suspicious for malignancy or malignant (89%). One case classified as atypia of uncertain significance underwent ThyroSeq sequencing where a NTRK1 fusion was identified. Conclusion: Although NRTC did not show a consistent cytomorphologic signature, mixed architectural patterns, prominent fibrosis and distinct cytoplasmic or nuclear features should raise suspicion for NRTC and, when accompanied by negative BRAFV600E by immunohistochemistry on cell block material, aid in selecting cases for molecular testing. This algorithmic approach may help identify potential NRTC, maximizing treatment options for patients, especially in patients for whom treatment planning is complicated. NTRK‐related thyroid carcinomas (NRTC) are amenable to targeted therapy. Identifying NRTC by preoperative fine‐needle aspiration may modify primary treatment options. [ABSTRACT FROM AUTHOR]

Predictive molecular testing has become an important part of the diagnosis of any patient with lung cancer. Using reliable methods to ensure timely and accurate results is inevitable for guiding treatment decisions. In the past few years, parallel sequencing has been established for mutation testing, and its use is currently broadened for the detection of other genetic alterations, such as gene fusion and copy number variations. In addition, conventional methods such as immunohistochemistry and in situ hybridization are still being used, either for formalin‐fixed, paraffin‐embedded tissue or for cytological specimens. For the development and broad implementation of such complex technologies, interdisciplinary and regional networks are needed. The Network Genomic Medicine (NGM) has served as a model of centralized testing and decentralized treatment of patients and incorporates all German comprehensive cancer centers. Internal quality control, laboratory accreditation, and participation in external quality assessment is mandatory for the delivery of reliable results. Here, we provide a summary of current technologies used to identify patients who have lung cancer with gene fusions, briefly describe the structures of NGM and the national NGM (nNGM), and provide recommendations for quality assurance. Standard methods for fusion testing such as immunohistochemistry and in situ hybridization are complemented by new parallel sequencing approaches in routine molecular pathology diagnostics for tissue and cytological specimens. The implementation of complex technologies requires continuous quality control and networking between institutions. [ABSTRACT FROM AUTHOR]

Background: Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated. Methods: Thyroid fine‐needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity. Results: Fifty‐six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P =.01), and storage time (median, 43 days; range, 7‐77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P =.01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0‐23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0‐42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell‐free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false‐negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics. Conclusions: Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors. Combined morphologic evaluation and molecular testing from material in a single vial is feasible and practical for fine‐needle aspiration biopsy specimens of the thyroid gland. The effect on quality and quantity of nucleic acid extracted from specimens collected in CytoLyt is evaluated for 2 key preanalytic variables: cellularity and storage time. [ABSTRACT FROM AUTHOR]

Background: High‐risk human papillomavirus–positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine‐needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy‐based techniques, the ability of liquid‐based assays was examined in the current study. Methods: The performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell‐free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization. Results: A total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty‐three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty‐two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell‐free supernatant fluid. Two cell‐free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas‐positive cell pellet was deemed to be a false‐positive result. The sensitivity of the cobas assay was 100% for pellet material and cell‐free supernatant fluid, with specificities of 94.7% and 100%, respectively. Conclusions: cobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell‐free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses. In the current study, the authors evaluated the performance of the Roche cobas HPV assay on cell pellet material obtained from needle passes of squamous cell carcinomas of the head and neck (HNSCCs) as well as on the corresponding cell‐free supernatant fluid specimens from these samples. The Roche cobas HPV test appears to be highly sensitive and specific for determining the high‐risk HPV status of patients with HNSCCs on both cellular cytologic specimens and their corresponding cell‐free supernatant fluid specimens. [ABSTRACT FROM AUTHOR]

Breast implants are surgically implanted by the hundreds of thousands every year worldwide for reconstructive or aesthetic purposes. Complications related to breast implants include early and late effusions that are often submitted for cytopathological analysis, particularly to exclude the possibility of breast implant–associated anaplastic large cell lymphoma (BIA‐ALCL), a rare disease that generally follows an indolent clinical course, although it is becoming clearer that a subset of patients with adverse features have a poorer prognosis. Since a late‐onset breast implant–associated effusion is the most common initial presentation of BIA‐ALCL, cytopathological analysis of these effusions is considered the cornerstone and gold standard for rapid, efficient, reliable diagnosis and is critical for appropriate management and treatment. The National Comprehensive Cancer Network recently published clinical guidelines for the diagnosis and management of BIA‐ALCL and stresses the essential role of cytopathological analysis, although it remains a matter of debate if all seromas should undergo immunocytochemistry or flow cytometry, particularly for assessment of expression of CD30 irrespective of morphological appearance on cytology. Herein, we review the current knowledge on BIA‐ALCL, review the key cytological findings of reactive and malignant effusions related to breast implants, and present a comprehensive cytopathological workup with the presence of atypical cells as the key and pivotal element triggering further ancillary studies. We believe this approach will ensure appropriate and cost‐effective management of effusion specimens from breast implants. We present a comprehensive cytopathological workup—including triage of the collected fluid, use of immunocytochemistry as indicated, and judicious use of molecular analysis and flow cytometry in selected cases—to arrive at a timely and cost‐effective diagnosis of breast implant‐associated anaplastic large cell lymphoma. The central element of this cytopathological workup is the identification of atypical cells on cytology, the key trigger for ancillary studies. [ABSTRACT FROM AUTHOR]