The Clinicopathological And Molecular Characteristics Of Gastric Neuroendocrine Neoplasms

Objective 1.To explore the clinicopathological characteristics and prognostic factors of gastric neuroendocrine neoplasm(NEN).2.To detect the gene mutations and copy number variations(CNVs)of two components from gastric mixed adenoneuroendocrine carcinoma(MANEC),and explore the mechanisms of development and progression of gastric MANEC.Methods 1.Clinicopathological parameters and follow-up data collected from 117 patients with gastric NEN at Tianjin Medical University Cancer Institute and Hospital from March 2011 to December 2017 were reviewed,classified,and graded according to World Health Organization(WHO)2010 classification.Clinicopathological characteristics of different types and grades of gastric NEN were compared and survival analysis was performed.2.DNA was isolated from the neuroendocrine components,adenocarcinoma components and paired paracancerous samples,respectively,from collision-type gastric MANEC,and whole exome sequencing(WES)was performed.The gene mutations of paracancerous samples were detected by in-house,and the potential predispose genes were selected.The somatic mutations were detected in comparsion with paired paracancerous samples.The significantly mutated genes and the candidate driver genes were selected by Mu Sic and in-house.The signatures of gene mutations in the two components were in comparison between groups and in comparison within the same cases,respectively.3.Sanger sequencing was used to detect and verify the mutations of exons 5,6,7 and 8 of TP53 in the neuroendocrine and adenocarcinoma components of gastric MANEC,respectively.4.High-resolution copy number analyses were performed in the neuroendocrine and adenocarcinoma components of gastric MANEC using Onco Scan FFPE assay.All the altered chromosome arms and the genes located at these arms were selected,and then hierarchical clustering was performed.The signatures of CNVs in the two components were in comparison between groups and in comparison within the same cases,respectively.5.Quantitative real-time PCR(q RT-PCR)and fluorescence in situ hybridization(FISH)were utilized to validate the CNVs of CCNE1.Immunohistochemistry(IHC)staining was used to detect the expression of cyclin E.6.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of mutated genes and CNV-associated genes were performed,and the enriched pathways were selected to explore the mechanisms of development and progression of gastric MANEC.Results 1.Clinicopathological characteristics and prognostic factors of gastric NENs: an analysis of 117 cases 1.1 Among the 117 cases confirmed as gastric NEN,this entire cohort comprised 13 cases(11.1%)of neuroendocrine tumor(NET)G1,6 cases(5.1%)of NET G2,57 cases(48.7%)of neuroendocrine carcinoma(NEC),and 41 cases(35.1%)of MANEC.1.2 Gastric NET G1 and G2 typically consisted of multiple small tumors with shallow invasion and infrequent lymphatic and distant metastases at early stages at the time of diagnosis.The treatments of patients with gastric NET included endoscopic submucosal dissection and radical surgical resection.Precursor neuroendocrine lesions were detected in most cases.The patients with gastric NET G1 and G2 had a good prognosis.1.3 Gastric NEC and MANEC mostly consisted of single large tumors with deep infiltration,and common lymphatic and distant metastases at advanced stages when the diagnoses were confirmed.All the patients with gastric NEC and MANEC underwent surgical resection,and most received adjuvant therapy.Histopathological changes of gastric NEC were characterized by large cells and poorly differentiated tumors,while gastric MANEC had various forms of neuroendocrine and adenocarcinoma components.The prognosis of patients with gastric NEC and MANEC was poor for both;however,the predictors of progression-free survival(PFS)and overall survival(OS)were different between gastric NEC and MANEC.Distant metastasis and recurrence or metastasis during follow-up were the independent prognostic factors for poor PFS and OS of NEC,respectively.Lymphatic metastasis and distant metastasis were the independent prognostic factors for poor PFS of MANEC.Lymphatic metastasis was also the independent prognostic factor for poor OS of MANEC.2.The molecular characteristics of gastric MANEC 2.1 Due to the failure of database construction in one case,WES analysis was performed in 7 cases with gastric MANEC.CNV analysis was performed in 8 cases with gastric MANEC.2.2 Some gene mutations were detected in paracancerous tissues,including BPTF,ZFHX3,MAP3K1 and so on.The main type of somatic gene mutations was base substitution(C ? T/G ? A).The frequencies of somatic gene mutations in the neuroendocrine components and adenocarcinoma components were not both significantly different in comparison between groups and in comparison within the same cases(P>0.05).TP53 was listed in the significantly mutated genes and candidate driver genes in both neuroendocrine and adenocarcinal components,and was also the most common mutated gene in the two components of the same cases.The gene mutations of TP53 were confirmed by Sanger sequencing.2.3 Hierarchical clustering showed that the adenocarcinoma components from different cases were clustered,while the neuroendocrine components from different cases were not clustered.The average number of CNV in the neuroendocrine components was much greater than that in the adenocarcinoma components(P=0.007).The gains of CCNE1(19q12)were the most common CNV events simultaneously occurred in the neuroendocrine and adenocarcinoma components in the same cases.The gains of CCNE1 were then confirmed by q RT-PCR and FISH,and the over expressions of cyclin E encoded by CCNE1 were also further detected by IHC.2.4 KEGG enrichment analyses of mutated genes and CNV-associated genes were performed.PI3K-Akt signaling pathway,MAPK signaling pathway,nerve-associated pathways(including neuroactive ligand-receptor interaction,axon guidance,neurotrophin signaling pathway and glutamatergic synapse),cell cycle and regulation of actin cytoskeleton were the enriched pathways.There was no difference in the types of enriched pathways in the mutated genes between the neuroendocrine and adenocarcinoma components;however,the CNV-associated genes of neuroendocrine components were enriched in MAPK signaling pathway,nerve-associated pathways and cell cycle.Conclusions 1.Gastric NEN are a group of heterogeneous tumors with different clinicopathological features and prognosis.More multicenter studies with large sample sizes are still needed to improve the classification of gastric NEN and explore the prognostic factors.2.The molecular genetic results of the two components from the collision-type gastric MANEC suggest that the neuroendocrine and adenocarcinoma components of gastric MANEC are more likely to originate from the same endodermal pluripotent stem cells.The gene mutations,such as TP53,may play a role during the development of gastric MANEC.Meanwhile,CNVs,like the amplification of CCNE1,may also occur in the early stage of tumorigenesis.Furthermore,CNVs may play a greater role in the sequential differentiations of adenocarcinoma and neuroendocrine components.These conclusions need to be further verified in larger sample sizes,due to the small sample coverage in this study.