Clinicopathologic, Cellular, and Molecular Analyses of Pulmonary Neuroendocrine Carcinoma With High Expression of...

Pulmonary neuroendocrine carcinoma (NEC), including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), is highly aggressive and has a poor prognosis. The molecular subtyping of NECs has recently attracted attention, and we identified a new NEC subtype, the hepatocyte nuclear factor 4α (HNF4α) subtype. HNF4α, a transcription factor associated with gastrointestinal differentiation, and TTF-1 are mutually and exclusively expressed in lung adenocarcinomas; however, the characteristics of HNF4α-high NEC and TTF-1-high NEC have yet to be compared. We immunohistochemically examined the characteristics of HNF4α-high NEC in 83 surgically resected specimens (37 SCLCs and 46 LCNECs) and revealed that HNF4α-high and TTF-1-high NEC accounted for 15% (12/83) and 47% (39/83), respectively. In SCLCs, HNF4α-high cases (n = 3) and TTF-1-high cases (n = 20) were almost confined to the neuroendocrine phenotype with high ASCL1 expression, and the expressions of HNF4α, TTF-1, and POU2F3 were mutually exclusive. Similar results were obtained for LCNECs; however, some HNF4α-high cases were positive for TTF-1 or YAP1, possibly due to the heterogeneity of LCNEC. Therefore, we investigated the heterogeneity of LCNEC and performed a spatial transcriptome analysis of 1 HNF4α-high LCNEC case, which revealed a mutually exclusive mixture of different subgroups characterized by HNF4A and NKX2-1 (TTF-1) expressions. A whole-genome analysis of 10 LCNECs showed that NFE2L2/KEAP1 mutations were characteristic of HNF4α-positive LCNECs. A prognostic analysis revealed a significantly worse prognosis in HNF4α-high LCNECs than in HNF4α-low LCNECs. A cell line analysis showed that TTF-1-high-expressing (Lu139/H889/H510A) and HNF4α-high-expressing (VMRC-LCD/H810) lines were consistent with ASCL1-high-expressing lines. HNF4α knockdown/knock-in experiments in VMRC-LCD and SBC5 (HNF4α-negative) revealed that HNF4α promoted cell proliferation by inhibiting apoptosis. The HNF4α-subtype of pulmonary NEC is a unique subtype, characterized by a neuroendocrine phenotype with high ASCL1 expression and mutual exclusivity with the TTF-1/POU2F3 subtypes. NFE2L2/KEAP1 mutations and HNF4α itself are potential therapeutic targets for this subtype.