The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

1st Department of Pathology and Experimental Cancer Research Semmelweis University Budapest Hungary

Department of Biomedical Engineering Lund University Lund Sweden

Department of Pathology Medical University of Vienna Vienna Austria

Department of Pathology University Hospital Ostrava and Faculty of Medicine University of Ostrava Ostrava Czech Republic

Department of Physics of Complex Systems Eotvos Lorand University Budapest Hungary

Department of Surgery University Hospital Ostrava and Faculty of Medicine University of Ostrava Ostrava Czech Republic

Department of Thoracic Surgery Comprehensive Cancer Center Medical University of Vienna Vienna Austria

Department of Thoracic Surgery Klinik Floridsdorf Vienna Austria

Department of Thoracic Surgery Semmelweis University and National Institute of Oncology Budapest Hungary

Diagnostic and Research Institute of Pathology Medical University of Graz Graz Austria

Division of Medical Oncology University of Colorado Anschutz Medical Campus Aurora CO USA

Division of Oncology Department of Internal Medicine and Oncology Semmelweis University Budapest Hungary

Institute of Clinical and Molecular Pathology Medical Faculty Palacky University Olomouc Olomouc Czech Republic

MTA SE NAP Brain Metastasis Research Group Hungarian Academy of Sciences Budapest Hungary

National Koranyi Institute of Pulmonology Budapest Hungary

Tisch Cancer Institute Center for Thoracic Oncology Mount Sinai Health System New York NY USA

Torokbalint County Institute of Pulmonology Torokbalint Hungary

University Clinic for Respiratory and Allergic Diseases Golnik Golnik Slovenia

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