Comprehensive Analysis of Neurogenic Differentiation Factor 1 (NEUROD1), Achaete-Scute Homolog 1 (ASCL1), POU Class 2 Homeobox 3 (POU2F3), and Yes-Associated Protein 1 (YAP1) Expression Signatures Reveals Unique Large-Cell Neuroendocrine Carcinoma (LCNEC) Subgroups With Potential Therapeutic Implications

Large-cell neuroendocrine carcinoma (LCNEC) can be genomically subtyped into small-cell lung cancer (SCLC) and non-SCLC-like. Neurogenic differentiation 1 (NEUROD1), achaete-scute homolog 1 (ASCL1), POU class 2 homeobox 3 (POU2F3), and yes-associated protein 1 (YAP1) (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, protein retinoblastoma (pRb), delta-like ligand 3 (DLL3), cMYC, and thyroid transcription factor 1. An H-score of >10 was considered positive (+), and >50, dominant. Unsupervised clustering and spatial immune RNA profiling using GeoMx Digital Spatial Profiling (NanoString Technology) were performed. Clinical data were obtained from the Netherlands Cancer Registry. ASCL1 was dominant in 26% and NEUROD1 in 18% of LCNEC. pRb loss was observed in 75%, and DLL3+, cMYC+, and thyroid transcription factor 1+ in 66%, 26%, and 70%, respectively. Unsupervised clustering identified 5 expression-based subgroups: NEUROD1^high-ASCL1^high (10%), ASCL1^high (22%), POU2F3^high (5%), YAP1^high (11%), and NAPY^low (51%). Both ASCL1^high subgroups correlated with DLL3^high and high neuroendocrine (NE) marker expression. YAP1^high was enriched for pRb+. POU2F3^high and YAP1^high subgroups were NE marker low and DLL3^low. GeoMX Digital Spatial Profiling identified 4 upregulated genes involved in immune system and/or tumor development in the NEUROD1^high-ASCL1^high-POU2F3^high- group. In this comprehensive evaluation of NAPY markers in LCNEC, we observed 5 expression-based subgroups: NEUROD1^high-ASCL1^high, ASCL1^high, POU2F3^high, YAP1^high, and NAPY^low. The NE subgroups (NEUROD1^high-ASCL1^high and ASCL1^high) were recognized with DLL3^high expression. Compared with the proportion known in SCLC, more NAPY^low and YAP1^high and fewer POU2F3^high cases were identified. Application of NAPY in LCNEC provides a more modest discrimination of subgroups compared with SCLC. Further research on potential drug targets is warranted, ie, differences in DLL3 and YAP1 expression could guide personalized treatment strategies.