Comprehensive Analysis of Neurod1, Ascl1, Pou2f3 and Yap1 Expression Signatures Reveal Unique Lcnec Subgroups With Potential Therapeutic Implications

Large-cell neuroendocrine carcinoma (LCNEC) can be genomically subtyped into small cell lung cancer (SCLC)- and non-SCLC (NSCLC)-like. NEUROD1, ASCL1, POU2F3, and YAP1 (NAPY) subtypes have been reported for SCLC. We immunohistochemically evaluated NAPY in LCNEC alongside relevant protein expression data. Tissue microarrays from 133 stage I-III resected LCNEC were reviewed and immunostained for NAPY, pRb, DLL3, cMYC and TTF1. H-score >10 was considered positive (+), and >50 dominant. Unsupervised clustering and spatial immune RNA profiling using GeoMX Digital Spatial Profiling (DSP) were performed. Clinical data were obtained from the Netherlands Cancer Registry. ASCL1 was dominant in 26%, NEUROD1 in 18% of LCNEC. pRb loss was observed in 75%, and DLL3+, cMYC+ and TTF1+ in 66%, 26%, and 70%, respectively. Unsupervised clustering identified five expression-based subgroups: NEUROD1high-ASCL1high (10%), ASCL1high (22%), POU2F3high (5%), YAP1high (11%), NAPYlow (51%). Both ASCL1high subgroups correlated with DLL3high and high neuroendocrine (NE) marker expression. YAP1high was enriched for pRb+. POU2F3high and YAP1high subgroups were NE marker low and DLL3low. DSP identified four upregulated genes involved in immune system and/or tumor development in the NEUROD1high-ASCL1high-POU2F3high-group. In this comprehensive evaluation of NAPY markers in LCNEC, we observed five expression-based subgroups: NEUROD1high-ASCL1high, ASCL1high, POU2F3high, YAP1high, and NAPYlow. The NE subgroups (NEUROD1high-ASCL1high and ASCL1high) were recognized with DLL3high expression. Compared to the proportion known in SCLC, more NAPYlow and YAP1high and fewer POU2F3high cases were identified. Application of NAPY in LCNEC provides a more modest discrimination of subgroups compared to SCLC. Further research on potential drug targets is warranted, i.e. differences in DLL3 and YAP1 expression could guide personalized treatment strategies.