With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers.In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.

Context.— Somatic mutations in SMARCA4 (SWI/SNF–related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene and/or BRG1 (Brahma-related gene 1) loss identifies a subset of non–small cell lung carcinomas (NSCLCs) lacking alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), and ROS1 (ROS proto-oncogene 1) genes. Preliminary observations suggest responsiveness to immunotherapy and targeted therapies. Objective.— To study BRG1 loss in NSCLCs and elucidate the clinicopathologic profile of such SMARCA4-deficient NSCLCs. Design.— Non–small cell lung carcinomas diagnosed during 6 years were subject to immunohistochemistry for BRG1 and BRM (Brahma). Tumors with BRG1 loss were stained with antibodies against thyroid transcription factor 1 (TTF-1), p40, cytokeratins, hepatocyte paraffin 1 (Hep Par 1), Sal-like protein 4 (SALL4), CD34, SRY-box 2 (SOX2), chromogranin, synaptophysin, p53, integrase interactor 1, ALK, and ROS1. EGFR mutation testing was performed by polymerase chain reaction–based method. Results.— Among 100 NSCLCs tested, 4 cases (4%) showed BRG1 loss. The histology ranged from solid adenocarcinomas (n = 1) to large cell/poorly differentiated carcinomas (n = 3) with clear cell cytology in 2 cases. All showed loss/reduction of BRM with variable cytokeratin and SALL4 expression, and were negative for TTF-1, p40, Hep Par 1, ALK, ROS1, and EGFR mutations. CD34 and SOX2 were negative in all 4 cases. Isolated BRM loss was common (21%), distributed across all NSCLC subtypes including squamous cell carcinomas and a hepatoid adenocarcinoma. Conclusions.— BRG1 loss occurs in a subset of TTF-1/p40–negative poorly differentiated NSCLCs. Identification and follow-up will clarify the prognosis, diagnostic criteria, and potential for therapeutic personalization.

The cell block (CB) offers many advantages over other cytological preparations, particularly for immunocytochemical and molecular testing. However, inconsistent cellularity remains the most common reason for dissatisfaction among cytopathologists. In recent years, there has been a surge in the demand for CBs imposed by the increasing number of minimally invasive procedures performed to obtain material for diagnostic, prognostic and predictive purposes from advanced stage cancer patients. However, routine preparation of CBs significantly increases laboratory work load, operating cost and sample turn-around time. The objectives of our review were to: (a) identify scenarios where a CB is likely to improve diagnostic yield; (b) optimise CB preparatory methods; and (c) understand the factors influencing the success and validity of ancillary testing on various types of CBs. We performed an extensive literature search on CBs in cytology on internet search engines using the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. New CB methods, improvisations of previous CB methods, their utility for diagnosis, immunocytochemistry and molecular testing, and role in predictive biomarker testing are discussed in this review. CBs of good quality and cellularity outperform other cytology preparations in their reliability and versatility for ancillary testing. With many CB methods described in the literature, each with specific advantages and limitations, laboratories may choose to use one or more of the methods depending upon their infrastructure, expertise and workload.

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Introduction: Inactivating mutations of the SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene and/or loss of the BRG1 (brahma-related gene 1) protein defines SMARCA4-deficient thoracic sarcoma (SMARCA4-dTS), an aggressive neoplasm with a usually fatal outcome. Similar SMARCA4 mutations/BRG1 loss is also seen in a subset of non-small cell lung carcinomas (NSCLCs; SMARCA4-dNSCLCs) that lack alterations in currently targetable oncogenic drivers, that is, EGFR, ALK, and ROS1. There is limited knowledge on the cytomorphological features of these SMARCA4-deficient thoracic neoplasms. Methods: We retrospectively analysed the cytology of 2 cases each of SMARCA4-dNSCLC and SMARCA4-dTS to understand their cytomorphological overlap, if any, and identify features that would prompt testing for BRG1 loss. Results: All 4 patients were males presenting with advanced disease, with a mean age of 41.5 years (SMARCA4-dTS) and 58.5 years (SMARCA4-dNSCLC) at presentation. The cytology of the 2 SMARCA4-dTSs was strikingly similar, showing predominantly singly dispersed rhabdoid phenotype tumour cells with perinuclear cytoplasmic condensations in an inflammatory or necrotic background. The cytology raised suspicion for a wide range of differentials, including melanoma, high-grade lymphoma, germ cell tumour, undifferentiated carcinoma, and undifferentiated sarcoma. SMARCA4-dNSCLCs, on the other hand, were recognizable as poorly differentiated (adeno)carcinomas and were easily distinguished from SMARCA4-dTSs, with both cases showing cohesive clusters of frequently large tumour cells with abundant pale cytoplasm. Conclusion: A diagnosis of SMARCA4-dTS is possible on cytology with appropriate ancillary testing and a high index of suspicion. The cytology of SMARCA4-dNSCLCs does not overlap with SMARCA4-dTS; rather, it resembles that of any poorly differentiated (adeno)carcinoma in the limited numbers analysed in this study.

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COrona VIrus Disease 2019 (COVID-19). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-19 patients for other indications. IHC for CD61 and CD163 was performed for the assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. In a total of 55, 44 COVID-19 post-mortem lung samples were tested for ACE2, 36 for CD163, and 26 for CD61, compared to 15 non-covid 19 control lung sections. Quantification of immunostaining, random sampling, and correlation analysis were used to substantiate the morphologic findings. Our results show that ACE2 protein expression was significantly higher in COVID-19 post-mortem lung tissues than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels were positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

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Background In a significant percentage of patients with small cell lung carcinoma (SCLC), cytology samples represent the only source of tumor tissue. Ancillary immunocytochemistry (ICC) for neuroendocrine markers is an important adjunct for the diagnosis of SCLC. Insulinoma‐associated protein 1 (INSM1) is a novel neuroendocrine marker proposed as an economical single‐marker alternative to the traditional 3‐marker panel of chromogranin, synaptophysin, and CD56. To the authors’ knowledge, limited studies have evaluated INSM1 immunohistochemistry (IHC) for the diagnosis of SCLC and reported high sensitivities and specificities. The objective of the current study was to evaluate the sensitivity and specificity of INSM1 ICC on direct smears (DS) from patients with SCLC in comparison with IHC on small biopsies (SBs). Methods All available DS and SBs from patients with SCLC who were diagnosed over the previous year were retrieved. Immunostaining for INSM1 was performed on alcohol‐fixed DS and formalin‐fixed SBs wherever available. A total of 10 DS and SBs from patients with non–small cell lung carcinoma were included for comparison. Nuclear staining for INSM1 in ≥1% tumor cells was interpreted as positive. Results Among a total of 60 patients with SCLC who were included in the current study, a total of 37 underwent INSM1 IHC on SBs and 36 underwent INSM1 ICC on DS. ICC was noninterpretable in 3 DS due to necrosis. The sensitivity of INSM1 IHC was 97% (36 of 37 cases) whereas the sensitivity of INSM1 ICC was 91% (30 of 33 cases) for the diagnosis of SCLC. Among matched IHC and ICC results available for 11 patients, 91% of cases (10 of 11 patients) demonstrated concordant IHC‐ICC staining. All cases of non–small cell lung carcinoma were negative for INSM1 (100% specificity). Conclusions INSM1 appears to be a robust and reliable ICC marker for the confirmation of SCLC diagnosis on cytology smears.