Whole-section digital analysis of immune profiles in surgically resected small cell lung carcinoma and their associations with molecular subtypes

Background: The molecular subtype-specific features of the tumor immune microenvironment (TIME) in small cell lung carcinoma (SCLC) remain poorly understood. We aimed to analyze the immune profiles in surgically resected SCLC and their associations with molecular subtypes. Methods: Tumor samples from 83 treatment-naive SCLC patients who underwent surgical resection were analyzed. The protein expression of subtype-defining markers (ASCL1, NEUROD1, POU2F3, and YAP1) and nine immune-related markers were assessed using whole-section immunohistochemistry. Digital image analysis was employed for precise quantification of immune cell infiltrates and distributions. The findings were subsequently correlated with clinicopathological parameters and patient prognoses. Results: Unsupervised hierarchical clustering categorized the tumors into four molecular subtypes: achaete-scute homologue 1-dominant (ASCL1; SCLC-A, 71.1%, n=59), neuronal differentiation factor 1-dominant (NEUROD1; SCLC-N, 12.1%, n=10), POU class 2 homeobox 3-dominant (POU2F3; SCLC-P, 10.8%, n=9), and quadruple-negative (SCLC-QN, 6.0%, n=5). Expression of major histocompatibility complex class I (MHC I) and class II (MHC II; P=0.02, P=0.02), tumor programmed death-ligand 1 (PD-L1; P=0.006), and an inflamed phenotype characterized by CD8+/CD3+ T cells (P=0.001, P=0.003) were more prominent in SCLC-P tumors compared to other subtypes. Additionally, SCLC-P tumors demonstrated the highest levels of MHC II (P=0.04) and PD-L1 expression on both tumor and stromal cells (P=0.003, P=0.01). The tumor proportion score of PD-L1 positively correlated with tumor expression levels of POU2F3 (rho=0.297, P=0.006) and MHC I (rho=0.239, P=0.03), as well as the combined positive score of PD-L1 (rho=0.222, P=0.04; rho=0.433, P<0.001). Intra-tumoral tertiary lymphoid structures (intra-TLS) and peri-tumoral TLS (peri-TLS) were observed in 60.2% (n=50) and 96.4% (n=80) of patients, respectively. High intra-TLS density was more frequently associated with SCLC-P tumors (P=0.02). Notably, low peri-TLS density and stromal PD-L1 expression were linked to improved overall survival (OS) and progression-free survival (PFS), respectively. Conclusions: This study highlights the heterogeneity of the TIME across molecular subtypes of SCLC. The SCLC-P subtype and MHC I expression may serve as predictive biomarkers for immunotherapy response, while peri-TLS density and stromal PD-L1 expression might serve as prognostic indicators in resected SCLC.