Differential NEUROD1, ASCL1, and POU2F3 Expression Defines Molecular Subsets of Bladder Small Cell/Neuroendocrine Carcinoma With Prognostic Implications

Abstract

Small cell carcinomas (SMC) of the lung are now molecularly classified based on the expression of transcriptional regulators (NEUROD1, ASCL1, POU2F3, and YAP1) and DLL3, which has emerged as an investigational therapeutic target. PLCG2 has been shown to identify a distinct subpopulation of lung SMC with stem cell-like and prometastasis features and poor prognosis. We analyzed the expression of these novel neuroendocrine markers and their association with traditional neuroendocrine markers and patient outcomes in a cohort of bladder neuroendocrine carcinoma (NEC) consisting of 103 SMC and 19 large cell NEC (LCNEC) assembled in tissue microarrays. Coexpression patterns were assessed and integrated with detailed clinical annotation including overall (OS) and recurrence-free survival (RFS) and response to neoadjuvant/adjuvant chemotherapy. We identified 5 distinct molecular subtypes in bladder SMC based on the expression of ASCL1, NEUROD1, and POU2F3: ASCL1+/NEUROD1- (n = 33; 34%), ASCL1- /NEUROD1+ (n = 21; 21%), ASCL1+/NEUROD1+ (n = 17; 17%), POU2F3+ (n = 22, 22%), and ASCL1- /NEUROD1- /POU2F3- (n = 5, 5%). POU2F3+ tumors were mutually exclusive with those expressing ASCL1 and NEUROD1 and exhibited lower expression of traditional neuroendocrine markers. PLCG2 expression was noted in 33 tumors (32%) and was highly correlated with POU2F3 expression (P < .001). DLL3 expression was high in both SMC (n = 72, 82%) and LCNEC (n = 11, 85%). YAP1 expression was enriched in nonneuroendocrine components and negatively correlated with all neuroendocrine markers. In patients without metastatic disease who underwent radical cystectomy, PLCG2+ or POU2F3+ tumors had shorter RFS and OS (P < .05), but their expression was not associated with metastasis status or response to neoadjuvant/adjuvant chemotherapy. In conclusion, the NEC of the bladder can be divided into distinct molecular subtypes based on the expression of ASCL1, NEUROD1, and POU2F3. POU2F3-expressing tumors represent an ASCL1/NEUROD1-negative subset of bladder NEC characterized by lower expression of traditional neuroendocrine markers. Marker expression patterns were similar in SMC and LCNEC. Expression of PLCG2 and POU2F3 was associated with shorter RFS and OS. DLL3 was expressed at high levels in both SMC and LCNEC of the bladder, nominating it as a potential therapeutic target.

Keywords: ASCL1; DLL3; NEUROD1; PLCG2; POU2F3; small cell carcinoma.

Figure 1.

A. Representative examples of ASCL1+, NEUROD1+ and POU2F3+ immunohistochemistry of neuroendocrine bladder cancers. Note the overall non-overlapping expression pattern of these markers. POU2F3+ tumors typically had low or no expression of traditional neuroendocrine markers such as synaptophysin. DLL3 was expressed in ASCL1+, and to a lesser extent in NEUROD1+ bladder tumors but was typically negative in POU2F3+ tumors. B. Distribution of markers expression sorted by order of ASCL1, NEUROD1 and POU2F3 binary expression. Each column represents one tumor sample and each raw corresponds to an immunohistochemical marker. For p53, positive indicates strong and diffuse expression (mutated pattern) and negative indicates complete loss of expression (null pattern). For Rb, negative indicates loss of expression and positive indicates retained expression. C. Correlation plot of novel and traditional markers. Points are scaled by the magnitude of correlation coefficients and colored by direction of the correlation. This plot shows Spearman Correlation coefficient values as calculated pairwise between continuous percent values each of marker.

Figure 2.

A. Whole slide staining of a tumor with ASCL1+ and POU2F3+ subpopulations, showing mutually exclusive expression patterns. Note synaptophysin expression in ASCL1+ tumor region and lack of expression in POU2F3+ areas. B. An example of small cell carcinoma with POU2F3 and PLCG2 co-expression. Note the low chromogranin expression. This tumor is additionally negative for ASCL1, NEUROD1 and DLL3. C. Oncoprint of frequently mutated genes, sorted by expression of novel endocrine markers ASCL1, NEUROD1 and POU2F3. Top panel is number of mutations per tumor among selected genes and right panel is gene alteration prevalence in the entire cohort. Bottom panel shows expression patterns of novel and traditional neuroendocrine markers. Results or p53, RB, PLCG2 and DLL3 expression are also shown. For p53, positive indicates strong and diffuse expression (mutated pattern) and negative indicates complete loss of expression (null pattern). For Rb, negative indicates loss of expression and positive indicates retained expression.

Figure 3.

Overall and Progression-free survival in POU2F3+ (A) and PLCG2+ (B) groups.