SMAD4 Haploinsufficiency in Small Intestinal Neuroendocrine Tumors

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background </jats:bold>Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations in a single gene, <jats:italic>CDKN1B</jats:italic>, with heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.<jats:bold>Methods</jats:bold>Utilizing comparative genomic hybridization<jats:bold> </jats:bold>(CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. <jats:italic>SMAD4, </jats:italic>and <jats:italic>CDKN1B,</jats:italic> were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4<jats:sup>+/-</jats:sup> mouse model was used to detect entero-endocrine cell hyperplasia with IHC.<jats:bold>Results</jats:bold>Analyzing the CGH arrays we found loss of chromosome 18q and <jats:italic>SMAD4</jats:italic> in 71% of SINETs and that focal loss of chromosome 12 affecting the <jats:italic>CDKN1B </jats:italic>was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of <jats:italic>SMAD4</jats:italic>, but not <jats:italic>CDKN1B</jats:italic>, significantly correlated with reduced protein levels but hemizygous loss of <jats:italic>SMAD4</jats:italic> did not induce entero-endocrine cell hyperplasia in the Smad4<jats:sup>+/-</jats:sup> mouse model. <jats:bold>Conclusions</jats:bold> Hemizygous loss of chromosome 18q and the <jats:italic>SMAD4</jats:italic> gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression. Although <jats:italic>SMAD4 </jats:italic>haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.</jats:p>