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This overview of the fifth edition of the WHO classification of thymic epithelial tumors (including thymomas, thymic carcinomas, and thymic neuroendocrine tumors [NETs]), mediastinal germ cell tumors, and mesenchymal neoplasms aims to (1) list established and new tumor entities and subtypes and (2) focus on diagnostic, molecular, and conceptual advances since publication of the fourth edition in 2015. Diagnostic advances are best exemplified by the immunohistochemical characterization of adenocarcinomas and the recognition of genetic translocations in metaplastic thymomas, rare B2 and B3 thymomas, and hyalinizing clear cell carcinomas. Advancements at the molecular and tumor biological levels of utmost oncological relevance are the findings that thymomas and most thymic carcinomas lack currently targetable mutations, have an extraordinarily low tumor mutational burden, but typically have a programmed death-ligand 1high phenotype. Finally, data underpinning a conceptual advance are illustrated for the future classification of thymic NETs that may fit into the classification scheme of extrathoracic NETs. Endowed with updated clinical information and state-of-the-art positron emission tomography and computed tomography images, the fifth edition of the WHO classification of thymic epithelial tumors, germ cell tumors, and mesenchymal neoplasms with its wealth of new diagnostic and molecular insights will be a valuable source for pathologists, radiologists, surgeons, and oncologists alike. Therapeutic perspectives and research challenges will be addressed as well.

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.

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.

High-grade neuroendocrine tumors (HGNET) have distinctive tumor biology/behaviour. Newer modalities of treatment (immunotherapy) for them have been included in recent NCCN guidelines. Detection of programmed death receptor-ligand 1 (PD-L1) expression by immunohistochemistry have made easy identification of patients eligible for immunotherapy. We aimed to ascertain expression of PD-L1 on small cell and large cell neuroendocrine carcinomas of lung and review existing literature. Eighty-five cases of HGNET lung (primary/metastatic), were retrieved and reviewed. Immunostaining for PD-L1 using clone SP263 was done. Any amount/intensity of membranous staining of > = 1% tumor cells was cut-off for positivity. Previously published studies using Google and/Pubmed search engines were reviewed. Of 85 cases, 70 were small-cell lung cancer (SCLC), 11 large-cell neuroendocrine carcinoma (LCNEC) and 4 combined SCLC. Median age was 46.5 years with male preponderance. No PD-L1 expression was seen in 91.6% cases. The 7 positive cases were 4 LCNEC, 2 SCLC and 1 combined SCLC. The percentage positivity varied from 1–100%; lower percentage positivity was seen in SCLC. PD-L1 expression on immune cells was seen in 31.3% cases. Sixteen studies evaluating 1992 NET were found; E1L3N PD-L1 clone was commonly used clone. PD-L1 positivity was associated with better prognosis in most studies. There are only a few studies available in literature related to PDL1 expression in high grade neuroendocrine carcinomas of lung. In general, PD-L1 positivity is highly variable and seen in lower percentage of these tumors. With the recent approval of immunotherapy, biomarkers other than PD-L1 should also be investigated in these tumors.

The recent development of immune checkpoint inhibitors (ICIs) has led to promising advances in the treatment of patients with NSCLC and SCLC with advanced or metastatic disease. Most ICIs target programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) axis with the aim of restoring antitumor immunity. Multiple clinical trials for ICIs have evaluated a predictive value of PD-L1 protein expression in tumor cells and tumor-infiltrating immune cells (ICs) by immunohistochemistry (IHC), for which different assays with specific IHC platforms were applied. Of those, some PD-L1 IHC assays have been validated for the prescription of the corresponding agent for first- or second-line treatment. However, not all laboratories are equipped with the dedicated platforms, and many laboratories have set up in-house or laboratory-developed tests that are more affordable than the generally expensive clinical trial–validated assays. Although PD-L1 IHC test is now deployed in most pathology laboratories, its appropriate implementation and interpretation are critical as a predictive biomarker and can be challenging owing to the multiple antibody clones and platforms or assays available and given the typically small size of samples provided. Because many articles have been published since the issue of the IASLC Atlas of PD-L1 Immunohistochemistry Testing in Lung Cancer, this review by the IASLC Pathology Committee provides updates on the indications of ICIs for lung cancer in 2019 and discusses important considerations on preanalytical, analytical, and postanalytical aspects of PD-L1 IHC testing, including specimen type, validation of assays, external quality assurance, and training.

The molecular pathogenesis of poorly differentiated sinonasal carcinoma received significant attention in recent years. As a consequence, several unclassified carcinomas in the morphologic spectrum of sinonasal undifferentiated carcinoma have been reclassified as distinctive genetically defined variants or entities. Among the latter are NUT-rearranged carcinoma and SMARCB1-deficient carcinomas. In this study, we further characterize a rare variant of sinonasal undifferentiated carcinoma-like tumors characterized by inactivation of the SWItch/Sucrose Nonfermentable chromatin remodeler SMARCA4 (BRG1) detectable by immunohistochemistry. Patients were 7 males and 3 females aged 20 to 67 years (median, 44). Tumors originated in the nasal cavity (6), nose and sinuses (2), or at unspecified site (2). Six tumors were initially misdiagnosed as small cell neuroendocrine carcinoma (SCNEC) or large cell neuroendocrine carcinoma (LCNEC). Histologically, the tumors were composed of small basaloid (3 cases) or large epithelioid (7) cells disposed into nests and solid sheets with extensive areas of necrosis. No glands or other differentiating features were noted. Abortive rosettes were seen in 1 case. Immunohistochemistry showed consistent expression of pankeratin and absence of CK5, p63, p16, and NUT in all tumors tested. Other tested markers were variably positive: CK7 (2/6), synaptophysin (9/10; mostly focal and weak), chromogranin-A (4/10; focal), and CD56 (3/5; focal). All tumors showed total loss of SMARCA4 and retained expression of SMARCB1/INI1. Co-loss of SMARCA2 was seen in 1 of 8 cases. Limited data were available on treatment and follow-up. Two patients received surgery (1 also radiotherapy) and 3 received chemotherapy. Metastases (cervical nodes, liver, bone, and lung/mediastinal) were detected in 3 patients; 2 were alive under palliative chemotherapy at 8 and 9 months while 1 died of progressive lung disease at 7 months. Three patients (1 with brain invasion) died soon after diagnosis (1 to 3 mo). In total, 4 of 6 patients (66%) with follow-up died of disease (median, 3 mo). This series characterizes SMARCA4-deficient sinonasal carcinoma as a genetically distinct aggressive entity in the spectrum of undifferentiated sinonasal carcinomas. These variants add to the spectrum of SWItch/Sucrose Nonfermentable-deficient sinonasal carcinomas, at the same time expanding the topographic distribution of SMARCA4-related malignancies.

Introduction: In effusion cytology, mesothelial cells can occasionally present with striking intracytoplasmic accumulation of rod- and crystal-like cytoplasmic lamellar inclusions (LIs). Their nature and function are poorly understood, and their diagnostic relevance is unknown. Objective: The aim of this study was to explore the nature of LIs in mesothelial cells and determine their prevalence and diagnostic utility in routine practice. Material and Method: We reviewed a consecutive series of cytological specimens of reactive (n = 102) and malignant effusions (n = 90), respectively. Malignant effusions included malignant mesotheliomas (n = 63) and carcinomas (n = 27). LIs of one effusion were analyzed by electron microscopy (EM). Results: LIs were found exclusively in benign mesothelial cells in 14% (14/102) of reactive and in 4% (1/27) of malignant effusions with carcinomatosis. They were absent in effusions of malignant mesothelioma. EM revealed mainly straight lamellar, less tubular, structures in cisternae of the hyperplasic rough endoplasmic reticulum (rER). Conclusion: Cytoplasmic LIs located within hyperplastic rER can be found in up to 14% of effusions restricted to benign mesothelial cells. They can be used as an indirect morphological clue favoring the diagnosis of benign effusion and helping the cytologist to differentiate between reactive and malignant mesothelial cells in daily practice.

The 2021 WHO Classification of Thoracic Tumours was published earlier this year, with classification of lung tumors being one of the chapters. The principles remain those of using morphology first, supported by immunohistochemistry, and then molecular techniques. In 2015, there was particular emphasis on using immunohistochemistry to make classification more accurate. In 2021, there is greater emphasis throughout the book on advances in molecular pathology across all tumor types. Major features within this edition are (1) broader emphasis on genetic testing than in the 2015 WHO Classification; (2) a section entirely dedicated to the classification of small diagnostic samples; (3) continued recommendation to document percentages of histologic patterns in invasive nonmucinous adenocarcinomas, with utilization of these features to apply a formal grading system, and using only invasive size for T-factor size determination in part lepidic nonmucinous lung adenocarcinomas as recommended by the eighth edition TNM classification; (4) recognition of spread through airspaces as a histologic feature with prognostic significance; (5) moving lymphoepithelial carcinoma to squamous cell carcinomas; (6) update on evolving concepts in lung neuroendocrine neoplasm classification; (7) recognition of bronchiolar adenoma/ciliated muconodular papillary tumor as a new entity within the adenoma subgroup; (8) recognition of thoracic SMARCA4-deficient undifferentiated tumor; and (9) inclusion of essential and desirable diagnostic criteria for each tumor. Crown Copyright (c) 2021 Published by Elsevier Inc. on behalf of International Association for the Study of Lung Cancer. All rights reserved.

Objectives: To evaluate the effectiveness of CT texture analysis (CTTA) in (1) differentiating Thymoma (THY) from thymic hyperplasia (TH) (2) low from high WHO grade, and (3) low from high Masaoka Koga (MK)/International Thymic Malignancy Interest Group (ITMIG) stages. Methods: After institute ethical clearance, this cross-sectional study analyzed 26 patients (THY-18, TH-8) who underwent dual energy CT (DECT) and surgery between January 2016 and December 2018. CTTA was performed using TexRad (Feedback Medical Ltd., Cambridge, UK- www.fbkmed.com ) by a single observer. Free hand regions of interest (ROIs) were placed over axial sections where there was maximum enhancement and homogeneity. Filtration histogram was used to generate six first-order texture parameters [mean, standard deviation (SD), mean of positive pixels (MPP), entropy, skewness, and kurtosis] at six spatial scaling factors “SSF 0, 2, 3, 4, 5, and 6”. Mann–Whitney test was applied among various categories and p value < 0.05 was considered significant. Three-step feature selection was performed to determine the best parameters among each category. Results: The best performing parameters were (1) THY vs TH- Mean at “SSF 0” (AUC: 0.8889) and MPP at “SSF 0” (AUC: 0.8889), (2) Low vs high WHO grade - no parameter showed statistical significance with good AUC, and (3) Low vs high MK/ITMIG stage- SD at “SSF 6” (AUC: 0.8052 and 0.8333 respectively]). Conclusion: CTTA revealed several parameters with excellent diagnostic performance in differentiating thymoma from thymic hyperplasia and MK/ITMIG high vs low stages. CTTA could potentially serve as a non-invasive tool for this stratification. Advances in knowledge: This study has employed texture analysis, a novel radiomics method on DECT scans to determine the best performing parameter and their corresponding cut-off values to differentiate among the above-mentioned categories. These new parameters may help add another layer of confidence to non-invasively stratify and prognosticate patients accurately which was only previously possible with a biopsy.

Wilms tumor is the most common renal malignancy in children and difficult to differentiate from other paediatric abdominal tumors radiologically, necessitating an invasive procedure for diagnosis. Previous studies have shown the potential role of miRNA as biomarkers for diagnosis, histological subtyping and prognosis. In this study, we are exploring the role of miRNA in the histological subtyping of Wilms tumor in the Indian population. A total of 15 cases of Wilms tumor were evaluated for global miRNA expression analysis by microarray. Total RNA was extracted from fresh frozen tumor and miRNA expression analysis was performed using Agilent platform. Unsupervised clustering was done to analyse the data. Using unpaired student T test, top 10 significantly differentially expressed miRNA were selected which could differentiate among different histological subtypes by unsupervised hierarchical clustering and principal component analysis. The presence of necrosis, heterologous differentiation led to change in miRNA expression profile and led to a distinct cluster formation. A panel of 5 miRNAs (miR1, 133b, 299-3p, 499a-5p, 491-3p) could differentiate among different histological subtypes of Wilms tumor, thus avoiding an invasive procedure in children, however, further confirmation using real time PCR analysis will be needed.

Cytology specimens play an important role in the diagnosis and predictive testing of lung cancer. While morphological characterisation of small cell and non-small cell lung carcinomas (NSCLC) on cytology is possible, further subtyping of NSCLC into adenocarcinoma and squamous cell carcinoma morphology is also mandatory in the current era of personalised medicine. Notably, cytology specimens in different forms (fine needle aspiration, exfoliative, and cell block) with or without immunocytochemistry are reliable sources for accurate diagnosis of adenocarcinoma and squamous cell carcinoma as evidenced by numerous studies present in the literature. However, there are instances where subtyping of NSCLC based on morphology alone is challenging on cytology samples, especially non-cell block preparations. In this paper, we will discuss current concepts, advances, and challenges of subtyping NSCLC in cytology specimens.

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 COVID-19 (COrona VIrus Disease 2019). 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-9 patients for other indications. IHC for CD61 and CD163 were performed for assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. Quantification of immunostaining, random sampling, and correlation analysis was used to substantiate the morphologic findings. Our results show that among a total of 44 COVID-19 post-mortem lung tissues and 15 lung biopsies in non-COVID-19 patients included, ACE2 protein expression was significantly higher in COVID-19 patients 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 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.

OBJECTIVES: To study the histopathology of patients dying of COVID-19 using post-mortem minimally invasive sampling techniques. METHODS: This was a single-center observational study conducted at JPNATC, AIIMS. Thirty-seven patients who died of COVID-19 were enrolled. Post-mortem percutaneous biopsies were taken from lung, heart, liver, kidney and stained with hematoxylin and eosin. Immunohistochemistry was performed using CD61 and CD163. SARS-CoV-2 virus was detected using IHC with primary antibodies. RESULTS: The mean age was 48.7 years and 59.5% were males. Lung histopathology showed diffuse alveolar damage in 78% patients. Associated bronchopneumonia was seen in 37.5% and scattered microthrombi in 21% patients. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of renal biopsies. Seventy-one percent of liver biopsies showed Kupffer cell hyperplasia and 27.5% showed submassive hepatic necrosis. CONCLUSIONS: Predominant finding was diffuse alveolar damage with demonstration of SARS-CoV-2 protein in the acute phase. Microvascular thrombi were rarely identified in any organ. Substantial hepatocyte necrosis, Kupffer cell hypertrophy, microvesicular, and macrovesicular steatosis unrelated to microvascular thrombi suggested that liver might be a primary target of COVID-19.

Giant cell tumor of the larynx is an uncommon entity with only 44 cases reported in the literature. These tumors occur most commonly in the epiphysis of the long bones of female patients in third decade. Here in we report a case of 23 years old male patient who presented with an anterior neck swelling since past 4 months. Ultrasound and computed tomography of neck revealed a heterogenously enhancing lesion involving posteroinferior half of right thyroid cartilage with extension into the extra laryngeal strap muscle and intralaryngeal right true vocal cord and false vocal cord. The findings were suggestive of a neoplastic cartilagenous lesion. A fine needle aspiration of the right anterior neck mass was performed which showed many mononuclear cells along with multinucleated osteoclast type giant cells. No thyroid follicular cells or inflammatory cells were seen. A diagnosis of giant cell tumor of the thyroid cartilage was rendered on cytology. A biopsy was subsequently performed for the patient which confirmed the same. Hence, although giant cell tumor of the larynx is a rare entity, with very few cases reported in the literature, these tumors should be included in the differential diagnosis of giant cell lesions of the neck and aspiration cytology can offer an accurate and quick diagnosis in such cases.

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.

AIMS: In thymic carcinomas, focal clear cell change is a frequent finding. In addition to a prominent, diffuse clear cell morphology, some of these carcinomas show an exuberant hyalinised extracellular matrix, and therefore probably represent a separate entity. However, a characteristic genomic alteration remains elusive. We hypothesised that, analogous to hyalinising clear cell carcinomas of the salivary gland, hyalinising clear cell carcinomas of the thymus might also harbour EWSR1 translocations. METHODS AND RESULTS: We identified nine archived cases of thymic carcinoma with focal clear cell features and two cases that showed remarkable hyalinised stroma and prominent, diffuse clear cell morphology. These two cases expressed p40 and were negative for Pax8, CD5, and CD117. Programmed death-ligand 1 was highly positive in one case (70%), and negative in the other one. EWSR1 translocation was identified in both cases of hyalinising clear cell carcinoma, and was absent in all nine carcinomas that showed clear cell features without substantial hyalinisation. In one of the EWSR1-translocated cases, a fusion between exon 13 and exon 6 of EWSR1 and ATF1, respectively was identified by next-generation sequencing. CONCLUSIONS: These findings suggest that the EWSR1 translocation and possibly the EWSR1-ATF1 fusion might be unifying genomic alterations for thymic clear cell carcinomas with prominent hyalinised stroma, for which we propose the term 'hyalinising clear cell carcinoma of the thymus'. Because the immunophenotype is unspecific, testing for the EWSR1 translocation might be helpful in discriminating this entity from other thymic neoplasms or metastases, in particular those with clear cell change. ispartof: HISTOPATHOLOGY vol:75 issue:3 pages:431-436 ispartof: location:England status: published

Objective Langerhans cell histiocytosis (LCH) is a rare disease affecting predominantly children and young adults but can be found in any age group. Diagnosis of LCH is often difficult and can be delayed because of its rarity. The present study highlights the cytomorphological features in a large cohort of cases. An accurate cytological diagnosis may avoid unnecessary biopsy and guide appropriate management. Method Fourty seven (47) cases of LCH diagnosed on cytological material & fine-needle aspiration (FNA) over a period of 14 years (2003-2016) were retrieved from the archives. The cytological smears were evaluated and microscopic findings collected by semi-quantitative assessment done by two different pathologists RESULT: The age at the diagnosis of the patients ranged from 9 months to 28 years. The majority of cases were in the age group of 0-5 years. The most common site was head and neck region, which included cervical lymphadenopathy and scalp swelling. Two cases were diagnosed each from inguinal lymph node and bronchio-alveolar lavage (BAL). Cytological smears in the majority of the cases were moderate to highly cellular (58%) and showing abundant Langerhans cell in (72%) of cases. Areas of necrosis were seen in 38%, while 78% of cases showed giant cells. The majority of cases showed mild eosinophilia (61%), sparse lymphocytosis (83%) and mild neutrophilic infiltration (64%). There were 1-2 mitoses per 10 high power field in 12 cases (25.5%). No abnormal mitoses were identified. Conclusion The presence of cells with features of Langerhans cells associated with the expression of selected immunohistochemical markers allow the diagnosis of LCH on cytological samples, sparing more invasive procedure as a biopsy.

Objective Extranodal natural killer/T-cell lymphoma, nasal type (ENKTL) is an aggressive extranodal lymphoma of NK-cell or T-cell lineage. Its clinical features overlap with those of several sinonasal mass lesions. While the histopathological features are well described, diagnosis is often difficult, owing to presence of extensive coagulative necrosis, so that repeated biopsies may sometimes be necessary for correct diagnosis. Literature on cytological findings of ENKTL is limited. Methods Cytomorphological features of cases of histologically confirmed ENKTL having corresponding cytology samples were reviewed retrospectively, to identify distinctive features that could possibly suggest this entity. Results Aspirates from five patients were studied: four from cervical nodes, one from cheek swelling and one from pleural fluid. Two aspirates were reported as positive for malignancy, two as atypical lymphoid proliferation and one was non-diagnostic. Pleural fluid was reported as malignant, favouring a diagnosis of carcinoma. On cytology, aspirates showed medium to large cells with folded, indented nuclei and abundant pale cytoplasm, some with tongue-like cytoplasmic protrusions. A distinctive feature was presence of large loose clusters of tumour cells with arborising capillaries running through them. Interestingly, necrosis was consistently absent. Subsequent biopsies from palate (three cases) and nasal masses (two cases) confirmed the diagnosis of ENKTL. Conclusions Suspicion of ENKTL on cytology is crucial for timely diagnosis to avoid diagnostic delay, especially when only highly necrotic biopsy samples are available. Awareness of distinctive cytomorphological features is required to make fine needle aspiration an effective diagnostic tool for initial diagnosis and for evaluation of possible recurrences.

Background & objectives: Inhibitors of immune checkpoint regulators, programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), improve outcome in advanced non-small-cell lung carcinoma (NSCLC). Tumours expressing PD-L1 protein are more likely to benefit from this targeted therapy. Multiple concurrent clinical trials evaluating different anti-PD-1/PD-L1 therapies have validated five different immunohistochemistry (IHC) assays using varied antibody clones and staining conditions. This study was aimed at identification of a single harmonized PD-L1 assay for tumour tissue conservation and cost-effectiveness in patients with NSCLC. Methods: The performance of low-cost, manual, laboratory-developed technique (LDT) PD-L1 IHC assay using the easily available SP142 clone was compared with trial validated Ventana SP263 IHC performed on automated Ventana staining platform on tumour sections of NSCLCs. Results: Eighty cases of NSCLC were included. SP263 and SP142 stained both tumour cells and immune cells. The concordance rate of tumour cell staining was about 76 per cent, with SP263 detecting more tumour cells in 16 per cent of cases. The concordance rate of immune cell staining was only 61 per cent, with SP142 detecting more immune cells in 24 per cent of cases. The sensitivity, specificity, positive and negative predictive values of manual SP142 LDT assay against gold standard SP263 Ventana assay were 70, 94, 86 and 86 per cent, respectively, at positivity thresholds of ≥1 per cent tumour cell staining. Interpretation & conclusions: The study findings suggested that LDT using SP142 clone showed only moderate concordance with SP263 Ventana assay, and the two assays were not interchangeable. More such validation studies need to be done to generate information that can complement patient therapy in cases of NSCLC.

A significant proportion of sinonasal malignancies comprise poorly differentiated/undifferentiated carcinomas that defy accurate histologic classification and behave aggressively. Recent years have seen a refinement of this spectrum by inclusion of novel entities harboring specific genetic alterations, including SMARCB1 (INI1)-deficient sinonasal carcinoma (SDSC), characterized by inactivating alterations in SMARCB1 gene, as demonstrated by loss of INI1 immunoexpression. Cyclin D1 is a cell-cycle regulatory protein downstream of INI1. Loss of INI1 leads to derepression of cyclin D1 transcription, suggesting its role as a putative therapeutic target. However, cyclin D1 expression has not been assessed in SDSCs. We retrieved all sinonasal carcinomas, including sinonasal undifferentiated carcinoma, undifferentiated carcinoma, poorly differentiated squamous cell carcinoma, and adenocarcinoma. Histopathologic features were reviewed. INI1 immunohistochemistry was performed. Cyclin D1 was performed in cases showing INI1 loss. Loss of INI1 staining was seen in 13 cases (5.8%), including 11 males and 2 females (age range, 11-65 years). Original diagnoses included SDSC (3/13), sinonasal undifferentiated carcinoma (3/13), adenocarcinoma (3/13), poorly differentiated squamous cell carcinoma (2/13), and poorly differentiated carcinoma (2/13). Tumors were predominantly basaloid in 6 cases and plasmacytoid/rhabdoid in 5 cases. We identified 2 cases having oncocytoid cells arranged in a gland-like pattern. Significant cyclin D1 immunoexpression was absent. SDSC is a rare, emerging entity that resembles a poorly differentiated carcinoma. Histomorphologic spectrum of these tumors is evolving. In addition to basaloid and plasmacytoid/rhabdoid cells, oncocytoid/adenocarcinoma-like pattern can also be seen in SDSC and predicts INI1 loss. These histologic patterns can further be subjected to INI1 immunohistochemistry for correct diagnosis.

Germline inactivating mutations in SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene encoding for BRG1 (Brahma related gene-1) are the molecular drivers in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) and in malignant rhabdoid tumors (MRT) that occur in the context of rhabdoid tumor predisposition syndrome-type 2. Somatic SMARCA4 mutations and/or loss of BRG1 have been identified in a variety of adult-onset epithelial and mesenchymal neoplasms. Among thoracic tumors, these include subsets of smoking-related non-small cell lung carcinoma (NSCLC) and a relatively rare, newly recognised tumor entity: thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Less than 100 cases of SMARCA4-UT have been reported to date. They present as large compressive and infiltrative mediastinal, lung and/or pleural masses in middle-aged male smokers. They are undifferentiated tumors composed of sheets of small/epithelioid and/or rhabdoid tumor cells variably expressing epithelial markers and consistently showing loss of BRG1 and the closely related protein, Brahma (BRM). Frequent expression of stem cell markers (SOX2, CD34, SALL4) is noted. Despite gene expression profiles similar to MRTs and SCCOHT, they show striking genomic overlap with SMARCA4-mutant NSCLC with frequent TP53, STK11, KEAP1, and KRAS mutations, high tumor mutation burden (TMB), and presence of smoking related molecular signatures in tumor cells. SMARCA4-UT show uniformly poor survival and are irresponsive to conventional therapies. Immunotherapy responses are variable but promising, although PDL1 expression appears to be of poor predictive value. Drugs exploiting genetic and epigenetic mechanisms of SMARCA4 antagonism hold promise for future targeted therapies.

Cytopathology has emerged as a promising platform in precision oncology especially after the revolutionary change in our understanding of the concept of lung cancer etiopathogenesis. With increasing use of minimally invasive techniques for sample acquisition, it becomes almost mandatory to utilize precious cytology samples maximally and judiciously by appropriate triaging of the specimen and timely action of the cytopathology team. Existing patient management protocols require accurate morphologic and molecular diagnosis of the lung cancer specimens which needs knowledge about evolving techniques related to specimen procurement, updates of genomic variants of lung cancer and recently developed molecular testing platforms and algorithms which are capable enough to use even miniscule amount of diagnostic material. This review provides a brief knowledge about advances in cytology of lung cancer which are helpful for developing correct clinical management strategies.

Immunohistochemistry is the technique by which antigens in tissues are detected by means of antigen-antibody reaction. The p40 antibody is directed against the ΔN domain of the ΔNp63 isoform of p63 and is a highly specific marker for the squamous cell carcinoma subtype of non-small cell lung carcinomas (NSCLC). As such, immunohistochemical detection of this antigen in NSCLC biopsies is extremely valuable to assess tumor histological subtype. Herein we describe a manual procedure for performing p40 immunohistochemistry on formalin-fixed paraffin-embedded tissue sections by the indirect polymer-based two-step technique using hydrogen peroxide and 3-3'diaminobenzidine detection system.

Introduction Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is required to determine the eligibility for pembrolizumab monotherapy in advanced NSCLC worldwide and for several other indications depending on the country. Four assays have been approved/ Communaute Europeene–In vitro Diagnostic (CV-IVD)–marked, but PD-L1 IHC seems diversely implemented across regions and laboratories with the application of laboratory-developed tests (LDTs). Method To assess the practice of PD-L1 IHC and identify issues and disparities, the International Association for the Study of Lung Cancer Pathology Committee conducted a global survey for pathologists from January to May 2019, comprising multiple questions on preanalytical, analytical, and postanalytical conditions. Result A total of 344 pathologists from 64 countries participated with 41% from Europe, 24% from North America, and 18% from Asia. Besides biopsies and resections, cellblocks were used by 75% of the participants and smears by 11%. The clone 22C3 was most often used (69%) followed by SP263 (51%). They were applied as an LDT by 40% and 30% of the users, respectively, and 76% of the participants developed at least one LDT. Half of the participants reported a turnaround time of less than or equal to 2 days, whereas 13% reported that of greater than or equal to 5 days. In addition, quality assurance (QA), formal training for scoring, and standardized reporting were not implemented by 18%, 16%, and 14% of the participants, respectively. Conclusions Heterogeneity in PD-L1 testing is marked across regions and laboratories in terms of antibody clones, IHC assays, samples, turnaround times, and QA measures. The lack of QA, formal training, and standardized reporting stated by a considerable minority identifies a need for additional QA measures and training opportunities.

BackgroundThe Covid-19 pandemic began in China in December 2019. India is the second most affected country, as of November 2020 with more than 8.5million cases. Covid-19 infection primarily involves the lung with severity of illness varying from influenza-like illness to acute respiratory distress syndrome. Other organs have also found to be variably affected. Studies evaluating the histopathological changes of Covid-19 are critical in providing a better understanding of the disease pathophysiology and guiding treatment. Minimally invasive biopsy techniques (MITS/B) provide an easy and suitable alternative to complete autopsies. In this prospective single center study we present the histopathological examination of 37 patients who died with complications of Covid-19.MethodsThis was an observational study conducted in the Intensive Care Unit of JPN Trauma Centre AIIMS. A total of 37 patients who died of Covid-19 were enrolled in the study. Post-mortem percutaneous biopsies were taken by the help of surface landmarking/ultrasonography guidance from lung, heart, liver, and kidneys; after obtaining ethical consent. The biopsy samples were then stained with haematoxylin and eosin stain. Immunohistochemistry (IHC) was performed using CD61 and CD163 in all lung cores. SARS-CoV-2 virus was detected using IHC with primary antibodies in selected samples. Details regarding demographics, clinical parameters, hospital course, treatment details, and laboratory investigations were also collected for clinical correlation.ResultsA total of 37 patients underwent post-mortem minimally invasive tissue sampling. Mean age of the patients was 48.7years and 59.5% of them were males. Respiratory failure was the most common complication seen in 97.3%. Lung histopathology showed acute lung injury and diffuse alveolar damage in 78% patients. Associated bronchopneumonia was seen in 37.5% patients and scattered microthrombi were visualised in 21% patients. Immunostaining with CD61 and CD163 highlighted megakaryocytes, and increased macrophages in all samples. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of the renal biopsies but none of them showed evidence of microvascular thrombosis. 71% of the liver tissue cores showed evidence of Kupfer cell hyperplasia. 27.5% had evidence of submassive hepatic necrosis and 14% had features of acute on chronic liver failure. All the heart biopsies showed non-specific features such as hypertrophy with nucleomegaly with no evidence of myocardial necrosis in any of the samples.ConclusionsThe most common finding in this cohort is the diffuse alveolar damage with demonstration of SARS-CoV-2 protein in the acute phase of DAD. Microvascular thrombi were rarely identified in the lung, liver and kidney. Substantial hepatocyte necrosis, hepatocyte degeneration, Kupffer cell hypertrophy, micro, and macrovesicular steatosis unrelated to microvascular thrombi suggests that liver might be a primary target of Covid-19. This study highlights the importance of MITS/B in better understanding the pathological changes associated with Covid-19.

Introduction A grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma. Methods A multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I–III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics. Results The best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617). Conclusions A grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma.

IgG4-related disease (IgG4-RD) is a multi-organ immune-mediated disorder characterized by fibroinflammatory mass-forming lesions, mimicking malignancy or infection. While well-documented in salivary glands, orbit and thyroid in the head and neck, sinonasal IgG4-RD is rare. Cases of sinonasal IgG4-RD were retrieved, and clinicopathological features reviewed. Seven cases of sinonasal IgG4-RD were identified over a 2-year period, including three males and four females, with an age range of 13–48 years (median: 32 years). Patients presented with cheek swelling, pain and visual disturbances. Serum IgG4 levels were mildly elevated. Storiform fibrosis, obliterative phlebitis and plasma cell infiltration were seen in varying proportions. Destruction of bone and subepithelial mucoserous glands was present. ALK-1 negativity distinguished from inflammatory myofibroblastic tumor. Sinonasal IgG4-RD expands the growing spectrum of IgG4-RD. A high degree of suspicion is required to include IgG4-RD in differential diagnosis of sinonasal masses, and perform detailed histological and immunohistochemical workup for accurate diagnosis.

Metastatic deposit in the inguinal canal is an uncommon finding. Few tumors such as pancreatic cancer, rectal cancer, and ovarian cancer have been described previously with metastatic inguinal canal deposit. We present here a case of prostate cancer with an uncommon inguinal canal deposit that was detected on Ga-prostate-specific membrane antigen PET/CT.

Solitary fibrous tumor (SFT) is a fibroblastic tumor of the pleura, and subcutaneous and deep soft tissues of the extremities, abdomen, pelvis, and retroperitoneum. In the head and neck region, the sinonasal tract and soft tissues of the orbit are involved most frequently. However, the lacrimal sac is an extremely unusual location for this tumor, with occasional case reports in literature. Being superficially located and palpable, lacrimal sac masses are easily amenable to FNAC. Despite cytological features of SFT being well-documented, they are often underdiagnosed on FNAC. Immunocytochemistry for STAT6 is a sensitive and specific marker that can aid in accurate diagnosis, as is required for planning appropriate management. We describe the case of a 45-year-old lady who presented with a mass at the medial canthus of the right eye, and was diagnosed as SFT on FNAC, based on cytomorphological features and immunopositivity for STAT6. The diagnosis was subsequently confirmed on histological examination.

BACKGROUND Lung adenocarcinomas present as tight clusters and three-dimensional balls in effusion specimens. Unlike carcinomas of breast and stomach where singly lying malignant cells are seen in effusion samples, lung adenocarcinomas usually show cohesive morphology. This single-cell pattern may also be confused with reactive mesothelial cells. We studied the frequency of pulmonary adenocarcinoma with single-cell pattern cytomorphology in pleural effusion specimens. MATERIALS AND METHODS All cases reported as either suspicious or positive for malignancy on pleural effusion cytology (PFC) over the past 1 year were retrieved. The clinical details were obtained from requisition forms. Cases with predominant single-cell pattern, clinically suspicious of carcinoma lung were segregated. These were de-stained and immunocytochemistry (ICC) for TTF-1 was performed. RESULTS Of 103 cases reported as either suspicious or positive for malignancy on PFC, 29 had a predominant single-cell pattern. Of these, 13 (44.8%) were primary lung carcinoma. The rest were metastasis from ovary (5; 17.2%), breast (2; 6.9%), stomach (2; 6.9%), lymphoma (1; 3.5%), and Ewing's sarcoma (1; 3.5%). Five (17.2%) were those with unknown primary. All cases of lung carcinoma were positive for TTF-1 ICC. CONCLUSION Single-cell pattern of pulmonary adenocarcinoma is commoner than popularly believed. This pattern may be difficult to differentiate from carcinoma cells of other sites as well as from reactive mesothelial cells. A high degree of suspicion is therefore needed to perform relevant ICC to clinch the correct diagnosis.

Objective Molecular testing in non-small cell lung carcinoma (NSCLC) patients is validated mainly on formalin fixed tissue. In resource limited laboratories where only conventional cytology may be practiced without facilities for biopsies, cell blocks or microdissection, cytology smears are vastly underutilised for molecular testing. We aimed to evaluate the efficacy of EGFR mutation testing on cytology smears and discuss in detail the pre-analytic factors for successful testing. Methods Direct smears from diagnosed cases of NSCLC patients over a period of 6 years were retrieved. Decoverslipped smears were marked for tumor areas and slides were scraped using sterile blade. DNA was extracted using commercial kits and hot spot EGFR mutations were detected using real time-PCR based method. Matched small biopsies were also subjected to mutation testing. Results Seventy-one cytology smears from NSCLC patients were subjected to DNA extraction including 52 alcohol fixed and 19 air dried smears. Four smears with very low DNA concentration ( Conclusion Cytology smears are a reliable source of high-quality DNA for EGFR mutation testing and should be exploited for routine molecular testing. Particularly in low resource laboratories with limited histopathology processing, the practice of molecular testing on lung cytology samples will expedite predictive testing and allow early institution of specific treatment.

The assessment of capsular invasion is an essential but challenging step in the diagnosis of encapsulated follicular thyroid neoplasms. Therefore, interobserver agreement in the assessment of capsular invasion in these tumors was investigated among 11 thyroid pathologists by using virtual slides of 20 cases in which the original diagnosis considered the differential diagnosis of definite capsular invasion versus questionable capsular invasion. The assessment of capsular invasion was divided into three categories: (1) non-invasive, (2) questionable invasive, and (3) clear-cut invasive. The interobserver agreements for clear-cut invasive and non-invasive categories were fair (Kappa value = 0.578 and 0.404, respectively), whereas agreement for the questionable invasion was poor (Kappa value = 0.186). Disagreements in the assessment of invasion resulted in variable final pathological diagnoses. For example, the agreement for a diagnosis of malignancy was only fair (Kappa value = 0.545). Moreover, pathologists did not have a uniform approach for rendering a final diagnosis in cases with questionable capsular invasion, though nine of 11 pathologists did use the follicular tumor of uncertain malignant potential diagnosis as proposed by the World Health Organization classification of endocrine organs published in 2017. In conclusion, this study revealed considerable interobserver variation in the evaluation of capsular invasion, especially in follicular neoplasms with questionable invasion.

Composite tumor of larynx, a recently included entity in the current WHO classification, is often a difficult pathological diagnosis, especially in small biopsies. We report a case of laryngeal composite tumor, initially misdiagnosed as squamous cell carcinoma, which later turned out to be composite in nature, with associated neuroendocrine (small cell carcinoma) component. This report emphasizes the need for obtaining deeper biopsies and their thorough pathological examination to improve the diagnostic accuracy. Keywords: combined small cell carcinoma; composite tumor; larynx; small cell carcinoma; squamous cell carcinoma.

Aims Adenoid cystic carcinoma (AdCC) is frequent in the sinonasal region. The recently described human papilloma virus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) histopathologically resembles solid AdCC, but has a better outcome. Thus, clinical and pathogenetic differences between HMSC and sinonasal AdCC necessitate their distinction. We conducted this study to assess p16 immunoexpression in previously diagnosed AdCC cases, and to identify HMSC cases in p16 immunopositive cases. Methods and results Cases diagnosed as sinonasal and nasopharyngeal AdCC were retrieved. Histomorphological features were reviewed. Immunohistochemistry (IHC) for p16 was performed. HPV testing was performed in p16-positive cases by mRNA in-situ hybridisation (mRNA ISH) and polymerase chain reaction (PCR) assay. MYB rearrangement was assessed by fluorescence in-situ hybridisation. One hundred and two AdCC cases were retrieved. Six cases (5.9%) showed diffuse p16 positivity. HPV mRNA ISH and PCR were negative in p16-positive cases. Two cases showed MYB rearrangement. p16-positive cases were composed of basaloid cells demonstrating a cribriform pattern, at least focally. The predominant pattern was cribriform in three and solid in three cases. One case showed two distinct components: keratinising squamous cell carcinoma and cribriform AdCC. Other morphological patterns seen were tubular, reticular, epithelial-myoepithelial carcinoma-like, and glomeruloid, forming a minor component of the tumour area. Conclusions p16 staining alone, even when diffuse and strong, cannot be used as a surrogate for HPV testing to distinguish sinonasal AdCC from HMSC. p16 IHC should be accompanied by more specific methods, such as mRNA ISH, so as not to erroneously diagnose HMSC over sinonasal AdCC, bearing in mind the highly aggressive nature of the latter.