Expression of YAP1 delineates distinct subtypes of pulmonary large cell neuroendocrine carcinoma with divergent therapeutic implications

Abstract

Background: Molecular subtyping [Type I (non-small cell lung carcinoma-like, NSCLC-like) and Type II (small cell lung carcinoma-like, SCLC-like)] and YAP1 expression analysis show promise for precision oncology in pulmonary large cell neuroendocrine carcinoma (LCNEC). However, their clinical utility and prognostic value remain unvalidated, and the aim of this study is to evaluate the disparity of clinicopathologic features, survival outcomes and treatment-relevant biomarkers between these two classifications.

Methods: We retrospectively analyzed 42 surgically resected pulmonary LCNECs. Using next-generation sequencing (NGS) and immunohistochemistry on archival specimens, we classified tumors by molecular subtype and YAP1 protein expression. Clinicopathological features, survival outcomes, and therapeutic biomarkers were compared between these classifications.

Results: Molecular subtyping stratified 61.5% (24/39) as Type I and 38.5% (15/39) as Type II LCNECs. YAP1 immunoreactivity was positive in 71.4% (30/42) of specimens, defining YAP1-negative (n=12) and YAP1-positive (n=30) cohorts. No significant association existed between YAP1 status and molecular subtypes (P=0.15). ASCL1 overexpression (P=0.02) and diffuse prominent nucleoli (P=0.04) were enriched in YAP1-negative tumors, whereas NEUROD1-positive tumors favored YAP1 expression (P=0.04). Type II tumors exhibited elevated POU2F3 expression (vs. Type I, P=0.04) and higher tumor mutation burden (TMB) (P=0.002). YAP1 negativity correlated with upregulated ASCL1 (P=0.003) and DLL3 (P=0.003), whereas positivity associated with major histocompatibility complex class II (MHC II) overexpression (P=0.04). Quantitative analysis revealed that YAP1 expression level correlated positively with NEUROD1 (rho=0.349, P=0.02) but inversely with ASCL1/MHC I (rho=-0.326, P=0.04; rho=-0.338, P=0.03). Neither molecular subtype nor YAP1 status significantly impacted survival.

Conclusions: YAP1 expression was independent of molecular subtypes. YAP1-positive tumors may exhibit enhanced responsiveness to immunotherapy/NEUROD1-targeted therapy, while YAP1-negative tumors could benefit from ASCL1/DLL3-targeted approaches. Prospective validation is warranted.

Keywords: DLL3; Pulmonary large cell neuroendocrine carcinoma (pulmonary LCNEC); biomarkers; immunotherapy; molecular subtype.

Figure 1

Demographic and clinicopathological characteristics of LCNEC molecular subtypes. Genetic profiling identified TP53 mutations in 79.5% (31/39) and RB pathway genomic alterations in 46.2% (18/39). Immunohistochemical analysis revealed p53 positive expression in 84.6% (33/39) and Rb protein loss (mutant phenotype) in 46.2% (18/39). Based on these molecular signatures, cases were classified into molecular subtypes: Type I (61.5%, 24/39) and Type II (38.5%, 15/39). Unsupervised hierarchical clustering of transcription markers (ASCL1, NEUROD1, POU2F3, and YAP1) demonstrated concurrent expression of SCLC-defining lineage markers across both molecular subtypes of LCNEC. LCNEC, large cell neuroendocrine carcinoma; NSCLC, non-small cell lung carcinoma; Rb, retinoblastoma; SCLC, small cell lung carcinoma; TMB, tumor mutational burden; TNM, tumor-node-metastasis.

Figure 2

Intergroup comparative analysis of therapeutic markers. (A) Type II LCNEC demonstrated elevated POU2F3 expression (vs. Type I, P=0.04) and higher TMB (P=0.002). YAP1-negative tumors correlated with upregulated ASCL1 (P=0.003) and DLL3 (P=0.003), whereas YAP1 positivity associated with MHC II overexpression (P=0.04). (B,C) Histopathological micrographs demonstrating a case of Type I LCNEC, and representative images of a case with LCNEC tumors of Type II. The first column panels depict, from top to bottom: HE staining, p53 expression, and Rb expression. The second column panels show ASCL1 expression, NEUROD1 expression, and POU2F3 expression. The third column panels display YAP1 expression, DLL3 expression, and MHC-II expression (×400). LCNEC, large cell neuroendocrine carcinoma; MHC II, major histocompatibility complex class II; TMB, tumor mutation burden.

Figure 3

Association between YAP1 expression and therapeutic biomarkers. Correlations between YAP1 immunoreactivity (H-score) and therapeutic biomarkers were investigated. Quantitative analysis revealed YAP1 levels demonstrated a statistically significant positive correlation with NEUROD1 expression (rho=0.349, P=0.02). Conversely, inverse correlations were observed with ASCL1 (rho=−0.326, P=0.04) and MHC I expression (rho=−0.338, P=0.03). No significant associations were detected between YAP1 and other evaluated biomarkers. LMR, lymphocyte-to-monocyte ratio; MHC II, major histocompatibility complex class II; NLR, neutrophil-to-lymphocyte ratio; PD-L1, programmed death-ligand 1; PLR, platelet-to-lymphocyte ratio; PNR, platelet-to-neutrophil ratio; TMB, tumor mutation burden.

Figure 4

Kaplan-Meier risk-stratified survival curves for final multivariate model parameters. Kaplan-Meier analysis was used to compare the risk stratification of subgroups according to these prognostic factors, which were included in multivariate analysis. The results showed that early-stage disease (OS: P=0.004; DFS: P=0.01), absence of vascular invasion (OS: P=0.02; DFS: P=0.03), and STAS-negative status (OS: P<0.001; DFS: P=0.01) demonstrated significant associations with favorable outcomes. Paradoxically, tumor necrosis exceeding 30% correlated with improved OS (P=0.02). Adjuvant chemotherapy exhibited adverse survival outcomes, with treated patients demonstrating poorer prognosis (P=0.04). ASCL1 protein expression showed positive correlation with DFS advantage (P=0.02), while DLL3 expression and stromal CD8⁺ lymphocyte infiltration lacked statistical significance in survival outcomes. DFS, disease-free survival; OS, overall survival; STAS, spread through air spaces; TNM, tumor-node-metastasis.

Figure 5

Kaplan-Meier analysis in relation to molecular subtypes (Type I vs. II) and YAP1 expression status. Kaplan-Meier analysis of molecular subtypes showed overlapping survival curves for both OS and DFS (OS: P=0.44; DFS: P=0.63). Similarly, YAP1-negative and YAP1-positive cohorts demonstrated comparable survival outcomes (OS: P=0.46; DFS: P=0.85). DFS, disease-free survival; OS, overall survival.