Clinicopathological Characteristics And Multi-omics Study Of Gastroenteropancreatic Neuroendocrine Carcinoma

BackgroundsOriginating from peptidergic neurons and neuroendocrine cells,neuroendocrine neoplasms(NENs)are a class of rare tumors that have neuroendocrine functions and express neuroendocrine markers,with an incidence of 0.38/100000 in China,accounting for 1.24%of all malignant tumors.Gastroenteropancreatic neuroendocrine tumors(GEP-NENs),the most common among the digestive system,have a poor prognosis,with a five-year survival rate of only 16.3%for poorly differentiated neuroendocrine carcinomas.Treatment means and survival times of GEP-NENs with different sites and different pathological grades varied greatly.There may be differences in response rates to standard medical treatment regimens for primary and metastatic disease.A systematic multi-omics study of the clinicopathological molecular characteristics of their primary as well as metastases simultaneously is currently lacking.ObjectiveMulti-omics techniques were used to explore the molecular characteristics of GEP-NENS in different locations and pathological grades at multiple levels,such as gene,protein,and tumor microenvironment,to search for differences in genes and proteins between metastases and primary lesions,and to provide potential molecular targets for possible treatment options.MethodsA total of 177 GEP-NENs patients in Cancer hospital Chinese academy of medical sciences postoperative pathological diagnosis were retrospectively included to analyze the clinicopathological characteristics,and 14 GEP-NENs patients with both primary and liver metastases and peritumoral tissues were included,and the experimental requirements were fulfilled by quality control.Paraffin embedded samples from primary,metastatic and adjacent tissues of patients were collected for exome sequencing(WES),data independent acquisition(DIA)protein analysis and multiplex immunohistochemistry(mIHC)analysis to compare molecular mutations between primary and metastatic GEP-NENs at different locations.Differentially expressed proteins and immune infiltrates were identified,and survival analysis was performed.Immunohistochemistry(IHC)validation of the differential molecules was performed in 133 clinical specimens.Results1.Clinicopathological analysis of GEP-NENs revealed a positive correlation between higher Ki67 expression level and higher T stage(P=0.010,x2=6.70),and higher Ki67 expression was more likely to cause lymph node metastasis(P=0.020,χ2=5.39).In addition,HER-2-positive patients had higher pathological stage(P=0.010,x2=6.52).2.Differential gene analysis of primary,metastatic,and peritumoral tissues of GEP-NENs by WES revealed significant differences in mutation profiles.TP53,NCOR2 and ZNF genes were more frequently mutated in metastases compared to primary lesions,and tumor mutational burden(TMB)was significantly lower(P=0.047).The results of mismatch repair(MMR)related genetic testing showed a high rate of SNP and CNV variants in both primary and metastatic lesions(36.36%and 23.08%).Analysis of multi site tumor samples using peritumoral tissues as controls revealed that KRAS and APC genes in enteric neuroendocrine carcinoma(NEC)and ATRX genes in pancreatic neuroendocrine tumor(NET)were driver mutations and high frequency mutations shared between primary and metastatic lesions,and there was heterogeneity in the genetic variations among GEP-NENs of different site origin.3.An analytical approach employing proteomic DIA protein levels revealed marked heterogeneity in differential protein profiles in primary and metastatic GEP-NENs derived from different sites.Compared with the metastasis and primary lesion,the metabolic process and catalytic activity of enzymes were significantly changed in gastric NENs,intestinal NENs and pancreatic NENs.Analysis of HLA,a neoantigen related protein,revealed that HLA-A was significantly down regulated(P<0.05)in the metastases relative to the primary in the intestine,and HLA-DMB was also significantly down regulated(P<0.05)in the metastases relative to the primary in the pancreas NENs.4.Examination of the tumor microenvironment of GEP-NENs by the mIHC method revealed that the infiltration of B cells and M1 macrophages was significantly higher in the primary tumor than in the metastasis.Moreover,there are differences in the tumor microenvironment between primary and metastatic sites of GEP-NENs derived from different sites.In the tumor parenchyma,the infiltration of CD8+T cells,M2 type macrophages in intestinal NENs,and B cells in pancreatic NENs was significantly higher in the primary than in the metastatic lesion.5.IHC analysis of 133 clinical samples showed differences between primary and metastasis,with TP53 protein being significantly more positive in metastases(P=0.049)and MMR related protein loss in primaries compared to metastases(P=0.405).Conclusion1.Mutations of NCOR2 and ZNF genes in GEP-NENs,ATRX genes in pancreatic NENs,and KRAS and APC genes in intestinal NECSs may be closely related to the tumorigenesis and metastasis of their respective counterparts.Primary localization of GEP-NENs derived from different sites showed strong heterogeneity with respect to gene mutations,differential proteins,and immune infiltration characteristics of metastases.2.Decreased TMB,higher mutation rate of TP53 gene,lower expression of HLA-A and HLA-DMB proteins,and decreased B-cell infiltration in parenchyma in metastases of GEP-NENs compared with primary lesions suggest that the number of neoantigens is reduced in metastases,tumor antigen presentation capacity is diminished,DNA damage repair is dysfunctional,and the corresponding immune response is weakened.3.There are obvious differences in gene mutations,protein expression,and tumor microenvironment between primary and metastatic localization in the occurrence and development of GEP-NENs in different locations,and individual treatments should be performed according to multi-omics indexes to achieve better therapeutic outcomes.4.Through multi-omics study and clinical validation,GEP-NENs primary and metastasis differed in genomic and expression groups,suggesting that GEP-NENs may have unique clonal patterns in tumor metastasis.This conclusion provides a precision therapeutic basis for the clinical treatment of advanced metastatic GEP-NENs,along with research on targeted therapeutic targets and screening of applicable patients for immunotherapy.