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The International Academy of Cytology has joined with the International Agency for Research on Cancer to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians, and improve patient care. The WHO System describes 5 categories for reporting lung cytopathology: 'Insufficient/Inadequate/Nondiagnostic', 'Benign', 'Atypical', 'Suspicious for malignancy', and 'Malignant', each one with a clear descriptive term, a definition, a risk of malignancy, and a suggested management algorithm. The key diagnostic cytopathologic features of each of the lesions within each category have been established by consensus through an Expert Editorial Board, who are also the authors of this review and selected for each reporting system and chosen based on their expertise in the field and/or diversity of geographical representation. Many other co-authors from around the world also contributed. The assignment of writing and editing responsibilities used the same model as that used for the WHO Classification of Tumours (https://whobluebooks.iarc.fr/about/faq/). The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and guides in sampling and processing techniques to optimize the handling and preparation of specimens. The WHO System was created by the authors to be applicable globally and is based on cytomorphology with possibilities for additional diagnostic management of the patient. The authors are aware that local medical and pathology resources would differ, especially in low- and middle-income countries. The WHO Tumour Classification for Thoracic Tumors, Fifth Edition, is directly accessible through the online WHO System., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The use of RNA-based next-generation sequencing (NGS) assays to detect gene fusions for targeted therapy has rapidly become an essential component of comprehensive molecular profiling. For cytology specimens, the cell block (CB) is most commonly used for fusion testing; however, insufficient cellularity and/or suboptimal RNA quality are often limiting factors. In the current study, the authors evaluated the factors affecting RNA fusion testing in cytology and the added value of smears in cases with a suboptimal or inadequate CB., Methods: A 12-month retrospective review was performed to identify cytology cases that were evaluated by a targeted RNA-based NGS assay. Samples were sequenced by targeted amplicon-based NGS for 51 clinically relevant genes on a proprietary platform. Preanalytic factors and NGS quality parameters were correlated with the results of RNA fusion testing., Results: The overall success rate of RNA fusion testing was 92%. Of the 146 cases successfully sequenced, 14% had a clinically relevant fusion detected. NGS testing success positively correlated with RNA yield (P = .03) but was independent of the tumor fraction, the tumor size, or the number of slides used for extraction. CB preparations were adequate for testing in 45% cases, but the inclusion of direct smears increased the adequacy rate to 92%. There was no significant difference in testing success rates between smears and CB preparations., Conclusions: The success of RNA-based NGS fusion testing depends on the quality and quantity of RNA extracted. The use of direct smears significantly improves the adequacy of cytologic samples for RNA fusion testing for predictive biomarkers., (© 2020 American Cancer Society.)

Context.—: The approval of pembrolizumab for treatment of patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) advanced cancers has led to increased requests for MSI and/or MMR immunoperoxidase (IPOX) testing. Diagnoses for patients with advanced-stage cancer are frequently made from cytology specimens., Objective.—: To investigate the feasibility of using cell block (CB) preparations of effusions for MMR IPOX evaluation., Design.—: Surgical pathology cases of colorectal and endometrial carcinomas with known MMR/MSI status and matched effusions with available CBs were identified. Cell block sections were evaluated for adequacy and stained with MMR IPOX (MSH2, MSH6, MLH1, and PMS2). The CBs were reviewed, the number of tumor cells quantified, and MMR IPOX was interpreted as retained, lost, suboptimal, or noncontributory., Results.—: We identified 748 cases with MMR/MSI testing on surgical specimens having matched effusions. Of these, 131 cases (17.5%) had an available CB and 53 were deemed adequate for MMR IPOX staining. MMR IPOX results between effusion CBs and surgical pathology specimens were concordant in 45 of 53 (85%), inconclusive in 6 of 53 (11%), and discordant in 2 of 53 (4%) cases., Conclusions.—: There was high concordance of MMR IPOX testing between cytologic and surgical specimens, with no false-positive and 2 false-negative CB results. Limited tumor cells, staining in cells indefinite as tumor, tumor staining heterogeneity, and lack of internal control staining were problematic in some cases. Our findings indicate that cytologic effusion specimens may be suitable substrates for MMR IPOX biomarker testing; however, inconclusive cases need to be interpreted with caution., (© 2021 College of American Pathologists.)

Background: The International System for Reporting Serous Fluid Cytopathology was recently proposed as a tiered structure to provide consistent reporting terminology for serous effusions. Because of the variation in reporting practices for indeterminate serous effusions, namely, the atypia of undetermined significance (AUS) and suspicious for malignancy (SFM) groups, the authors retrospectively reviewed cases in these 2 categories at their institution and determined the associated risk of malignancy (ROM)., Methods: Pleural, peritoneal, and pericardial effusions that were reported as AUS or SFM over a 1-year period were reviewed, and their associated ROMs were calculated based on confirmation of malignancy by previous and/or subsequent fluid and/or tissue biopsy specimens from the same general location., Results: In total, 145 AUS and 98 SFM serous effusion cases were identified. The AUS category was used when the cells in question lacked the requisite quantitative (cell number) and/or qualitative (morphologic) features for a definitive diagnosis. Immunohistochemistry (IHC) or flow cytometry (FCM) was available in 15% of cases (n = 22) with inconclusive results. The ROM based on 69 cases with available follow-up results was 39%. In contrast, the SFM category demonstrated cells that were morphologically suspicious for malignancy but sparse, precluding IHC or FCM (n = 63; 64%) or yielding inconclusive results (n = 35; 36%). The ROM in the SFM category, based on follow-up results of 61 cases, was 64%., Conclusions: The ROM for SFM was significantly higher than that for AUS (P < .01), supporting separate diagnostic categories for these 2 groups., (© 2020 American Cancer Society.)

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material., Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235&#95;2249del15 p.E746&#95;A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798&#95;1799GT>AA p.V600K mutations at various allele frequencies (AFs)., Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798&#95;1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification., Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations., (© 2019 American Cancer Society.)

Objective: Interest in immune therapies has exploded since the 2014 approval of first-generation programmed cell death 1 blocking antibodies for use in advanced melanoma. Clinical trials have focused primarily on histological material as the gold standard for evaluating programmed death ligand 1 (PD-L1) by immunoperoxidase (IPOX) studies. Studies validating the use of cytological specimens in the assessment of PD-L1 by IPOX staining are needed to optimise tissue utilisation in complementary diagnostic testing., Methods: Twenty-three melanoma surgical biopsies (SBx) with an IPOX stain for PD-L1 clone 28-8, and a corresponding cytological specimen from the same patient, adequate for PD-L1 evaluation, were selected. Cell-transfer cell blocks (CBs) and conventional CBs were used to perform PD-L1 testing. Tumour proportion scores (TPS) were generated and the results were correlated with the corresponding SBx., Results: Overall agreement (OA) using a ≥1% TPS cut-off for SBx compared to CB was 88.9%, positive percent agreement (PPA) was 87.5%, and negative percent agreement (NPA) was 100%, OA using a ≥5% TPS cut-off was 55.6%, PPA was 42.9%, and NPA was 100%. SBx compared to cell-transfer CB using a ≥1% TPS cut-off had an OA of 65.2%, a PPA of 55.6%, and a NPA of 100%, while a ≥5% TPS cut-off generated an OA of 52.2%, a PPA of 35.7%, and a NPA of 77.8%., Conclusion: Our results demonstrate that cytological material, particularly conventional CB, is a viable alternative for evaluating PD-L1 in melanoma cases and suggest that a lower threshold (≥1%) may be beneficial when evaluating cytological material., (© 2018 John Wiley & Sons Ltd.)

There has been a paradigm shift in the practice of cytopathology with the advent of highly sensitive molecular tests using small amounts of tissue that can provide diagnostic, prognostic, and predictive information for clinical management. The cytopathologist plays a key role in providing a timely and accurate diagnosis as well as ensuring appropriate processing and handling of the specimen and judicious triaging of the tissue for molecular testing that guide therapeutic decisions. As the era of "precision medicine" continues to evolve and expand, cytopathology remains a dynamic field with advances in the practice of molecular cytopathology providing new paradigms in clinical care., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1) receptor and its ligand, programmed death ligand 1 (PD-L1), have emerged as a therapeutic approach for patients with non-small cell lung carcinoma (NSCLC). PD-L1 expression, assessed by immunohistochemistry (IHC), is used to select patients for PD-1/PD-L1 inhibitor therapy. Most studies have been performed with histology specimens, with limited data available on the performance in cytology specimens. This study evaluated PD-L1 in cytology specimens and compared the results with those from paired core-needle biopsy for concordance., Methods: Forty-one NSCLC fine-needle aspiration cases that had paired core-needle biopsy specimens with PD-L1 IHC were selected. A Papanicolaou-stained direct smear and a cell block section from each case were stained with a Dako PD-L1 pharmDx antibody (clone 22C3). Only slides with 100 or more tumor cells (37 smears and 38 cell blocks) were evaluated. Tumor proportion scores (TPS) were assessed on the basis of the partial/complete membranous staining of tumor cells and were correlated with those of paired core-needle biopsy., Results: All 9 smears that were negative for PD-L1 staining showed 100% concordance with the paired core-needle biopsy, whereas 28 smears with PD-L1 expression showed a similar TPS, except for 1 smear that was discordant. In contrast, 10 negative paired core-needle biopsy cases corresponded to 9 concordant negative cell blocks, whereas 1 cell block had a TPS of 1% to 5%. The remaining 28 cell blocks demonstrated PD-L1 expression, with 22 cases showing a TPS similar to that of the paired core-needle biopsy, whereas 6 cell blocks were discordant, likely because of intratumoral heterogeneity., Conclusions: The results show that NSCLC cytology samples evaluated for PD-L1 have high concordance with paired core-needle biopsy samples and can be used for assessing PD-L1 expression. Cancer Cytopathol 2018;126:342-52. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

Background: Cytology smears and cytospin preparations are increasingly being used for molecular testing. With these limited samples, optimizing tissue extraction to maximize the DNA yield is, therefore, critical. This study examined 2 common methods of tissue extraction and compared DNA yields from different types of glass slides., Methods: The H226 lung cancer cell line and 5 clinical samples of cellular effusions were used to prepare Diff-Quik-stained cytospins on 4 types of glass slides: fully frosted (FF), nonfrosted (NF), positively charged (PC), and silane-coated (SC). Tissue extraction was performed by either scalpel-blade scraping or cell lifting with the Pinpoint Slide DNA Isolation System (Zymo Research). DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) and was quantified with the Quant-iT PicoGreen Kit (Life Technologies)., Results: The DNA yield in cell-line cytospins was significantly lower from FF slides versus NF, PC, and SC slides with both scraping and cell-lifting methods. In addition, scraping yielded significantly more DNA than cell lifting (P = .005). DNA yields from 5 clinical effusion cases with FF and NF slides showed results similar to the results for cell-line samples, with scraping consistently yielding more DNA than cell lifting and with NF slides outperforming FF slides., Conclusions: Optimizing the DNA yield extracted from cytology specimens maximizes the chances of successful molecular testing and is critical in cases of low or marginal cellularity. This study demonstrates the following: 1) scraping yields more DNA than cell lifting, and 2) NF slides yield more DNA than FF slides., (© 2015 American Cancer Society.)

Objective: In the past decade molecular diagnostics has changed the clinical management of lung adenocarcinoma patients. Molecular diagnostics, however, is largely dependent on the quantity and quality of the tumor DNA that is retrieved from the tissue or cytology samples. Frequently, patients are diagnosed on cytology specimens where the tumor cells are scattered within the cell block, making selecting for tumor enrichment difficult. In the past we have used laser capture microdissection (LCM) to select for pure populations of tumor cells to increase the sensitivity of molecular assays. This study explores several methods for semiautomated computer-guided LCM., Study Design: Hematoxylin and eosin- or TTF-1-immunostained slides from a pleural effusion cell block with metastatic lung adenocarcinoma were used for LCM with either AutoScan or a recently described pattern-matching algorithm, spatially invariant vector quantization (SIVQ), to define morphologic predicates (vectors) to select cells of interest., Results: We retrieved pure populations of tumor cells using both algorithm-guided LCM approaches with slight variations in cellular retrievals. Both methods were semiautomated, requiring minimum technical supervision., Conclusion: In this study we demonstrate the first semiautomated, computer-guided LCM of a cytology specimen using SIVQ and AutoScan, a first step towards the long-term goal of integrating LCM into the clinical cytology-molecular workflow., (Copyright © 2012 S. Karger AG, Basel.)