Comprehensive Molecular and Clinicopathologic Analysis of 200 Pulmonary Invasive Mucinous Adenocarcinomas Identifies Distinct Characteristics of Molecular Subtypes

PURPOSE: Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically. EXPERIMENTAL DESIGN: A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS). RESULTS: Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R-NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P \u3c 0.0001). CONCLUSIONS: This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset

Diagnosis of thoracic SMARCA4-deficient undifferentiated tumor in cytology

INTRODUCTION: Although alterations in SMARCA4-deficient occur in non-small cell lung carcinoma (SD-NSCLC), thoracic SMARCA4-deficient undifferentiated tumor (TSDUT) is recognized as a distinct entity in the 2021 World Health Organization Classification of Thoracic Tumors because of unique morphologic, immunophenotypic and molecular features, and worse survival compared with SD-NSCLC. Cytologic diagnosis of TSDUT is clinically important because of its aggressive behavior and because it is often diagnosed by fine-needle aspiration because TSDUTs are usually unresectable at presentation. Here, we identify cytologic features that can be used for recognition of TSDUT and distinction from SD-NSCLC. MATERIALS AND METHODS: Cytomorphologic features were investigated in cytology specimens from patients with TSDUT (n = 11) and compared with a control group of patients with SD-NSCLC (n = 20). RESULTS: The presence of classic rhabdoid morphology, at least focally, was entirely specific for TSDUT (n = 6, 55%) compared with SD-NSCLC (n = 0) in this study. TSDUT more frequently showed tumor necrosis (n = 11, 100% vs. n = 8, 40%; p = .001), dominant single-cell pattern on aspirate smears or touch preparation slides (n = 8 [of 9], 80% vs. n = 3, 15%; p = .010), nuclear molding (n = 5, 45% vs. n = 1, 5%; p = .013), and indistinct cell borders (n = 11, 100% vs. n = 5, 25%; P \u3c .001) compared with SD-NSCLC, respectively. CONCLUSIONS: Cytomorphologic features occurring more frequently in TSDUT include tumor necrosis, dominant single-cell pattern, nuclear molding indistinct cell borders, and focal rhabdoid cells. Presence of these features in a cytology specimen of an undifferentiated tumor, particularly in a patient with a thoracic mass, should raise suspicion for TSDUT and prompt appropriate ancillary workup

Three-Dimensional Histologic, Immunohistochemical and Multiplex Immunofluorescence Analysis of Dynamic Vessel Co-Option of Spread Through Air Spaces (STAS) in Lung Adenocarcinoma

BACKGROUND: Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma, associated with tumor recurrence and poor survival. The spatial orientation of STAS cells/clusters to the lung alveolar parenchyma is not known. The aim of this study was to utilize high resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemistry (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma. METHODS: Four lung adenocarcinomas: 3 micropapillary (MIP) predominant and 1 solid (SN) predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from the formalin fixed paraffin-embedded (FFPE) blocks. 350 serial sections were obtained and stained with hematoxylin and eosin (H&E) (100 slides), IHC (200 slides), and multiplex IF (50 slides) with the following antibodies: CD31, collagen type 4, TTF-1 and E-Cadherin. Whole slide images (WSIs) were reconstructed into 3D images for evaluation. RESULTS: Serial 3D image analysis by H&E as well as IHC and IF showed the MIP clusters and SN nests of STAS focally attached to alveolar walls away from the main tumor. CONCLUSION: Our 3-D reconstructions demonstrated STAS tumor cells can attach to alveolar walls rather than appearing free floating as seen on 2D sections. This suggests that tumor cells detach from the main tumor, migrate through air spaces and reattach to alveolar walls through vessel co-option allowing them to survive and grow. This may explain the higher recurrence rate and worse survival for STAS positive tumors undergoing limited resection compared to lobectomy

The Genomic Landscape of SMARCA4 Alterations and Associations with Outcomes in Patients with Lung Cancer

PURPOSE: SMARCA4 mutations are among the most common recurrent alterations in NSCLC, but the relationship to other genomic abnormalities and clinical impact has not been established. EXPERIMENTAL DESIGN: To characterize SMARCA4 alterations in NSCLC, we analyzed the genomic, protein expression, and clinical outcome data of patients with SMARCA4 alterations treated at Memorial Sloan Kettering. RESULTS: In 4813 tumors from patients with NSCLC, we identified 8% (n= 407) patients with SMARCA4-mutant lung cancer. We describe two categories of SMARCA4 mutations: Class 1 mutations (truncating mutations, fusions and homozygous deletion) and Class 2 mutations (missense mutations). Protein expression loss was associated with Class 1 mutation (81% vs 0%, (P \u3c 0.001)). Both classes of mutation co-occured more frequently with KRAS, STK11, and KEAP1 mutations compared to SMARCA4 wildtype tumors (P \u3c 0.001). In patients with metastatic NSCLC, SMARCA4 alterations were associated with shorter overall survival, with Class 1 alterations associated with shortest survival times (P \u3c 0.001). Conversely, we found that treatment with immune checkpoint inhibitors was associated with improved outcomes in patients with SMARCA4-mutant tumors (P = 0.01), with Class 1 mutations having the best response to ICIs (p = 0.027). CONCLUSIONS: SMARCA4 alterations can be divided into two clinically relevant genomic classes associated with differential protein expression as well as distinct prognostic and treatment implications. Both classes co-occur with KEAP1, STK11, and KRAS mutations, but individually represent independent predictors of poor prognosis. Despite association with poor outcomes, SMARCA4-mutant lung cancers may be more sensitive to immunotherapy

The Genomic Landscape of SMARCA4 Alterations and Associations with Outcomes in Patients with Lung Cancer

Purpose: SMARCA4 mutations are among the most common recurrent alterations in non-small cell lung cancer (NSCLC), but the relationship to other genomic abnormalities and clinical impact has not been established. Experimental Design: To characterize SMARCA4 alterations in NSCLC, we analyzed the genomic, protein expression, and clinical outcome data of patients with SMARCA4 alterations treated at Memorial Sloan Kettering. Results: In 4,813 tumors from patients with NSCLC, we identified 8% (n ¼ 407) of patients with SMARCA4-mutant lung cancer. We describe two categories of SMARCA4 mutations: class 1 mutations (truncating mutations, fusions, and homozygous deletion) and class 2 mutations (missense mutations). Protein expression loss was associated with class 1 mutation (81% vs. 0%, P < 0.001). Both classes of mutation co-occurred more frequently with KRAS, STK11, and KEAP1 mutations compared with SMARCA4 wild-type tumors (P < 0.001). In patients with metastatic NSCLC, SMARCA4 alterations were associated with shorter overall survival, with class 1 alterations associated with shortest survival times (P < 0.001). Conversely, we found that treatment with immune checkpoint inhibitors (ICI) was associated with improved outcomes in patients with SMARCA4-mutant tumors (P ¼ 0.01), with class 1 mutations having the best response to ICIs (P ¼ 0.027). Conclusions: SMARCA4 alterations can be divided into two clinically relevant genomic classes associated with differential protein expression as well as distinct prognostic and treatment implications. Both classes co-occur with KEAP1, STK11, and KRAS mutations, but individually represent independent predictors of poor prognosis. Despite association with poor outcomes, SMARCA4-mutant lung cancers may be more sensitive to immunotherapy