Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are heterogeneous neoplasms characterized by poor outcome. Microsatellite instability (MSI) has recently been found in colorectal NECs showing a better prognosis than expected. However, the frequency of MSI in a large series of GEP-NEC/MANECs is still unknown. In this work, we investigated the incidence of MSI in GEP-NEC/MANECs and characterized their clinicopathologic and molecular features. MSI analysis and immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were performed in 89 GEP-NEC/MANECs (six esophageal, 77 gastrointestinal, three pancreatic, and three of the gallbladder). Methylation of 34 genes was studied by methylation-specific multiplex ligation probe amplification. Mutation analysis of BRAF and KRAS was assessed by PCR-pyrosequencing analysis. MSI was observed in 11 NEC/MANECs (12.4%): seven intestinal and four gastric. All but two MSI-cases showed MLH1 methylation and loss of MLH1 protein. The remaining two MSI-cancers showed lack of MSH2 or PMS2 immunohistochemical expression. MSI-NEC/MANECs showed higher methylation levels than microsatellite stable NEC/MANECs (40.6% vs 20.2% methylated genes respectively, P<0.001). BRAF mutation was detected in six out of 88 cases (7%) and KRAS mutation was identified in 15 cases (17%). BRAF mutation was associated with MSI (P<0.0008), while KRAS status did not correlate with any clinicopathologic or molecular feature. Vascular invasion (P=0.0003) and MSI (P=0.0084) were identified as the only independent prognostic factors in multivariate analysis. We conclude that MSI identifies a subset of gastric and intestinal NEC/MANECs with distinct biology and better prognosis. MSI-NEC/MANECs resemble MSI-gastrointestinal adenocarcinomas for frequency, molecular profile and pathogenetic mechanisms.

Colorectal poorly differentiated neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are well-recognized entities generally known to be associated with biological aggressiveness and poor patient survival. However, a few published papers have highlighted the existence of a subgroup of tumors with a better survival than expected; however, to date, there are no established parameters that usefully identify this category. In the present study we have investigated the morphologic features, the CpG methylator phenotype (CIMP), microsatellite instability (MSI), and the immunohistochemical profile, including the expression of transcription factors (TTF1, ASH1, CDX2, and PAX5), stem cell markers (CD117 and CD34), and cytokeratins 7 and 20, in a series of 39 carcinomas (27 NECs and 12 MANECs) to better characterize such neoplasms and to search for prognostic indicators. No different patient survival was observed between NECs and MANECs. Neoplasms showed a heterogenous spectrum of morphologic and immunohistochemical features; however, only large-cell subtype, significant peritumoral lymphoid reaction, CD117 immunoreactivity, vascular invasion, and MSI/CIMP+ status were significantly correlated with prognosis on univariable analysis. Furthermore, vascular invasion and CD117 immunoreactivity were independent prognostic markers on multivariable analysis. In addition to these prognostic features, neoplasms showed different expression of transcription factors, stem cell markers, and cytokeratins that should be considered for diagnostic purposes and, especially, for discriminating among possible differential diagnoses.

Article Mixed endocrine-exocrine tumors of the gut are a heterogeneous group of neoplasms with uncertain histogenesis showing different morphologic and clinical features. The aim of this work is to clarify the histogenesis of these tumors by studying the genetic profile of both the endocrine and exocrine components. We performed an allelotyping analysis of five mixed endocrine-exocrine tumors (two gastric and three colonic) and one rectal collision tumor, using 35 polymorphic microsatellite markers covering a total of six chromosomes, including 3, 5q, 6, 11, 17, and 18. The loss of heterozygosity (LOH) analysis showed concurrent losses of the same allele in both the endocrine and exocrine components in all of the five mixed tumors composed by a poorly differentiated endocrine carcinoma or a well differentiated endocrine carcinoma associated with adenocarcinoma or adenoma. Among these tumors an identical LOH pattern was frequently found on chromosomes 17p, 18q, and 5q. Additional allelic losses restricted to the poorly differentiated endocrine carcinoma were often observed. On the contrary, in the only collision tumor composed by a well differentiated endocrine carcinoma associated with adenocarcinoma, completely different allelotypes between the two components were detected. These findings confirm a close genetic relationship between the two distinct histologic components within mixed endocrine-exocrine tumors, supporting the hypothesis that a monoclonal mechanism of tumorigenesis is the most frequent genetic event in mixed exocrine-endocrine tumors. The clonal divergence observed in the only collision tumor, composed by a well differentiated endocrine carcinoma associated with an adenocarcinoma, confirms the existence of double tumors growing next to each other coincidentally but showing different histogenesis and different tumorigenetic pathways.