Studying The Clinicopathological Characteristics And Molecular Mechanisms Of Early-Onset Gastric Cancers

Background and AimGastric cancer is one of the most common cancers in China with poor 5-year survival rate.Early-Onset gastric cancer is defined as younger than 45 years GC.It is often diagnosed with advanced stage.Recent studies show there is an increasing trend in the incidence of female EOGC.From clinicopathological and endoscopic point of view,it is different from traditional old GC.Because of less environmental toxins exposure,hereditary factors may take an important place in its carcinogenesis.Familial gastric cancer is GC with family aggregation,while hereditary gastric cancer is a special type of FGC,caused by germline mutated genes.EOGC have more FGC,so we need to figure out the genetic background of it to achieve early diagnosis and early treatment.Besides,hereditary diffuse gastric cancer(HDGC)is a well-studied hereditary gastric cancer syndrome with CDH1 germline mutation,however,it has different mutation frequency between GC high incidence country and low incidence one.So far the role of genetic factors and estrogen signaling in EOGC remains unclear.Here,we aimed to analyze clinicopathological features of EOGC in China through involving EOGC from our hospital since 2004 to 2014,and choose two FGC families to conduct whole-genome sequencing and a series ofin vitro studies to confirm the role of estrogen in GC.Methods1?Searching database of our pathological department from Jan 2004 to Dec 2014,we included all EOGCs and 250 old GC cases as control.We collected all the clinicopathological,endoscopic information and follow up those patients with their living status and detailed family history.2?Immunohistochemistry and Western Blotting were applied to assess E-cadherin?ER??ER? and p53 location in EOGC,and analyzed expression with clinicopathological characteristics and prognosis.3?Choose two FGC families(all four patients)to conduct whole-genome sequencing,and use bioinformatics tools to analyze all genetic alterations,as well as adopting TCGA database and referring to previous studies.4?ER??ER? and Tff1 expression were analyzed in AGS?HGC27?MKN45?NCI-N87?KATO-? and GES-1 with western blotting.And ER??ER?and Tff1 location was analyzed with immunofluorescence.After transfecting siRNA against ESR2 in gastric cancer cell lines,colony formation assay and soft agar colony were used to detect proliferation ability,Annexin V/PI was applied to assess apoptosis status as well as western blotting to detect apoptosis associated protein expression level.P53 null gastric cancer cell line KATO-? was also transfected with siESR2,Annexin V/PI,western blotting of apoptosis associated protein were used.Next,transmission electron microscope,fluorescence and western blotting were used to assess autophagy flux.Gadd45a and MAPK signaling proteins were detected by western blotting.ResultsCompared with traditional GCs,EOGC has more female patients(65.1%VS 28.8%,P<0.05)and diffuse GC(79.4%VS 29.2%,P<0.05).FGC accounts for 25%of all EOGCs,however,in terms of clinicopathological features,no significant difference was seen between FGC and SGC.51.5%EOGCs were positive for Hp infection and it was strongly associated with Lauren's intestinal type and less invasion depth(pT1 and pT2).Independent prognostic factors for EOGCs were higher CA72-4 and CA125,positive resection margin and advanced GC.Immunohistochemistry and western blotting indicated that tumor tissue had less E-cadherin expression,more ERa and ERp expression.P53 staining was positive in 31 percent of EOGC and was associated with worse survival.Positive ERp expression was seen in younger patients(33.6 VS 35.3,P=0.005)and advanced stage(p?,P?,P?,P=0.009).Meanwhile,positive p53 expression was also seen in younger patients(32.2 VS 34.6,P<0.01)and advanced stage(P<0.01).Whole-genome sequencing revealed that no CDH1 or p53 germline mutation were seen.But one SNP was considered to have high protein impact(LILRA4 c.953-2A>G).Finally,in vitro studies indicated that under low estrogen level,ERK1/2 can be activated.ER? and ER? were detected in gastric cancer cell lines and ER? was more predominant.siESR2 can lead to apoptosis and autophagy,while inhibition of autophagy decreased apoptosis.siESR2 can also activate MAPK,then increase Gadd45a transcription level.ConclusionsEOGC is different from traditional GCs,mainly exhibited with more female patients and diffuse type,aggressive biological behavior.Patients with higher CA72-4 and CA125 had poorer survival.If patients are diagnosed at a resectable stage,the survival is better.Regarding molecular pathology,EOGC has less E-cadherin and more p53 expression.ERa and ERp act as oncogenes in gastric cancer tumorigenesis,with predominant ER? expression.Compared with p53 negative patients,those with positive expression have poorer survival.Low estrogen level can promote proliferation of gastric cancer cells.Transfecting siRNA against ESR2 can inhibit growth of GC cells and lead to both apoptosis and autophagy.All in all,early diagnosis and surgical procedure is the best management for EOGC and estrogen receptor modulators can be a therapy for ER? positive EOGCs.