Pleural effusion supernatant: a reliable resource for cell-free DNA in molecular testing of lung cancer.

INTRODUCTION: DNA extracted from malignant pleural effusion (PE) sediments is the traditional source of tumor DNA for predictive biomarker molecular testing (MT). Few recent studies have proposed the utility of cell-free DNA (cfDNA) extracted from effusion cytology centrifuged supernatants (CCS) in MT. The aim of this study was to assess the feasibility and utility of molecular testing on cfDNA extracted from PE CCS in lung cancer patients. MATERIALS AND METHODS: The study was of prospective design. All PE CCS were collected and stored. Subsequently, in patients confirmed as primary lung adenocarcinoma (LUAD) and where patient matched effusion sediment/tissue biopsy/plasma was being tested for EGFR mutations, cfDNA extraction and EGFR MT by real-time polymerase chain reaction (qPCR) were performed. Custom panel targeted next-generation sequencing (NGS) (Ion Torrent; Thermo Fisher, Carlsbad, CA) was also performed wherever feasible. RESULTS: Out of 299 PE CCS collected, 20 CCS samples were included in the study. Concordant EGFR mutations were detected in pleural effusion CCS of 10 of 11 (91%) EGFR mutant cases as per qPCR performed on the matched sediment DNA (n = 8), lung biopsy (n = 2), and plasma (n = 1) samples. In 1 positive sample, CCS detected additional EGFR T790M mutation. Among 10 CCS samples also tested by NGS, additional EGFR mutations missed by qPCR were picked up in 2 (2 of 10). Success of mutation detection in CCS cfDNA did not correlate with cfDNA quantity or tumor fraction in sediment. CONCLUSIONS: cfDNA from effusion CCS is a reliable and independent source of tumor DNA highly amenable for MT and complement results from other tumor DNA sources for comprehensive mutation profiling in LUAD patients.

Mapping the immune landscape in small cell lung cancer by analysing expression of immuno-modulators in tissue biopsies and paired blood samples.

Small cell lung carcinomas (SCLC) are aggressive tumors with high propensity to metastasize. Recent NCCN guidelines have incorporated immunotherapy in extensive stage SCLC. Limited benefit in few patients compounded by side effects of unwonted immune-checkpoint-inhibitor (ICPI) usage necessitates identification of potential biomarkers predicting response to ICPIs. Attempting this, we analysed expression of various immunoregulatory molecules in tissue biopsies and paired blood samples of SCLC patients. In 40 cases, immunohistochemistry for expression of immune inhibitory receptors CTLA-4, PD-L1 and IDO1 was performed. Matched blood samples were quantified for IFN-γ, IL-2, TNF-α and sCTLA-4 levels using immunoassay and additionally for IDO1 activity (Kynurenine/Tryptophan ratio) using LC-MS. Immunopositivity for PD-L1, IDO1 and CTLA-4 was identified in 9.3%, 6.2% and 71.8% cases, respectively. Concentration of serum IFN-γ (p-value < 0.001), TNF-α (p-value = 0.025) and s-CTLA4 (p-value = 0.08) were higher in SCLC patients while IL-2 was lower (p-value = 0.003) as compared to healthy controls. IDO1 activity was significantly elevated in SCLC cohort (p-value = 0.007). We proffer that SCLC patients show immune suppressive milieu in their peripheral circulation. Analysis of CTLA4 immunohistochemical expression along with s-CTLA4 levels appears prospective as biomarkers for predicting responsiveness to ICPIs. Additionally, evaluation of IDO1 appears cogent both as prognostic marker and potential therapeutic target as well.

IDH1/2 Mutations in Sinonasal Undifferentiated Carcinomas : Previously Undescribed IDH2 R172K and R140x Variants.

Sinonasal undifferentiated carcinoma (SNUC) is a rare, poorly defined sinonasal epithelial neoplasm from which several genetically defined entities are emerging. IDH1/2 mutations were recently identified in a subset of SNUC. However, the ideal method for the detection of these mutations remains to be established. Cases diagnosed as SNUC between 2010 and 2020 were retrieved. Immunohistochemistry was performed using IDH1/2 mutant-specific antibody MsMab-1. Quantitative real-time polymerase chain reaction (qPCR) was performed on genomic DNA extracted from formalin-fixed paraffin-embedded tissue using 2 kits to detect IDH1/2 mutations. Sanger sequencing was performed in a subset of cases. Thirty-eight cases of SNUC were identified, 18 of which showed IDH1/2 mutations by qPCR (47.4%). IDH2 R172K and R140x were most frequent, each seen in 6 cases (33.3%). Sanger sequencing identified IDH1/2 mutations in 4 out of 21 cases (19%) and did not detect mutations identified by qPCR in 7 cases. On immunohistochemistry, strong IDH positivity was present in 2 cases (5.3%), 1 of which had IDH2 mutation, while no mutation was detected in the other. Our results demonstrating IDH2 R172K and IDH2 R140x variants are a novel finding in SNUC. Immunohistochemistry and Sanger sequencing have low sensitivity for detection of IDH1/2 mutations, and qPCR-based assays may be utilized, particularly in resource-limited settings where access to sophisticated sequencing techniques are difficult.

SMARCA4/BRG1-Deficient Sinonasal Carcinoma.

CONTEXT.­: Molecular analysis of poorly differentiated/undifferentiated sinonasal neoplasms has resulted in identification of a growing number of genetically defined tumors. SMARCA4-deficient sinonasal carcinoma is one such recently described entity that emerged from within sinonasal undifferentiated carcinoma (SNUC), neuroendocrine carcinoma (NEC), and teratocarcinosarcoma (TCS). OBJECTIVE.­: To identify SMARCA4-deficient sinonasal carcinomas from a large institutional cohort of poorly differentiated/undifferentiated carcinomas and evaluate their clinicopathologic features. DESIGN.­: SMARCA4/BRG1 immunohistochemistry was performed on all tumors diagnosed as SNUC, poorly differentiated carcinoma, NEC, and TCS during a 12-year period. SMARCA2/BRM and INSM1 immunostaining was performed in SMARCA4-deficient cases. RESULTS.­: Twelve SMARCA4-deficient sinonasal carcinomas were identified among 299 cases. Morphologically, 5 cases were large cell NEC, 2 cases were small cell NEC, and 5 were TCS. SMARCA4 loss was diffuse and complete in 10 cases, while 2 cases showed focal retention. Most cases showed diffuse cytokeratin staining accompanied by weak, usually focal staining for chromogranin and synaptophysin. INSM-1 showed negativity in most cases. All cases showed retained SMARCA2 expression. IDH1/2 mutation was absent in all cases analyzed. Four of 7 patients died of disease, and aggressive multimodality treatment provided better outcome. CONCLUSIONS.­: SMARCA4-deficient sinonasal carcinomas are morphologically akin to sinonasal poorly differentiated NECs and TCS, display cytokeratin positivity and only focal staining for neuroendocrine markers, and have aggressive biological behavior. Inclusion of SMARCA4 in the immunohistochemical panel for diagnostic workup of all sinonasal NEC and TCS phenotypes will facilitate their early recognition. Comprehensive germline and somatic mutational analyses of these tumors are necessary for further insights into their molecular pathogenesis.