Helicobacter Pylori, EBV And The Risk Of Adenocarcinomas Of The Gastric Cardiac And Distal Stomach In North China

Objective: Gastric adenocarcinoma remains the second most common cause of cancer-related mortality in the world.Especially the areas of high gastric cancer incidence in North China had a mortality rate of77.67/100,000/year.In recent decades,dramatic changes have been witnessed in both the location and lethality of gastric cancer in western countries.Adenocarcinomas at the esophagogastric junction(EGJ;joining the proximal stomach and distal esophagus)have increased remarkably.Since the 1990 s,a similarly increasing incidence of gastric cardia adenocarcinoma(GCA)has been found in the area of high esophageal and gastric cancer incidence in North China.In 1996,Siewert defined this series of tumors as adenocarcinoma of EGJ(AEG)and further classified them into three types(types I,II,and III)according to the location of the tumor center.These tumors seem to constitute a distinct clinicopathological entity,but problems regarding in etiology,immunophenotype,and invasion remain controversial.Etiologically,AEG is mainly associated with gastroesophageal reflux disease,obesity and a Caucasian background,whereas distal gastric adenocarcinoma(DGA)generally has close relationships with Helicobacter pylori(H.pylori)infection and dietary factors.Now,multiple studies have widely demonstrated the close link between H.pylori infection and DGA,but the results have remained quite controversial in the development of AEG.Some papers have shown a null association between H.pylori seropositivity and gastric cardia cancer risk,whereas others have proven the existence of positive links between them.Epstein-Barr virus(EBV)is another risk factor involved in gastric carcinogenesis.According to comprehensive reports,approximately 1.3 %-20.1 % of gastric cancer cases present EBV.EBV-associated gastric carcinoma tends to be proximally located and often has a diffuse histologicalsubtype.However,another study supports an opposing view with EBV detected more frequency in intestinal type gastric cancer.To our knowledge,there is no directly comparative study about EBV and the risk of gastric cancer by different subsites in the high gastric cancer incidence area of China.The close link between H.pylori infection and distal gastric adenocarcinoma(DGA)has widely been demonstrated,but the results have remained quite controversial in adenocarcinomas of the esophagogastric junction(AEG).The aim was to compare H.pylori,EBV each infection and the risk of gastric cancer by subsites in North China.Method:1 Traditional staining for H.pyloriBiopsy specimens from the stump tissues of GCA and DGA were formalin-fixed and stained with traditional staining for H.pylori,including modified Giemsa staining,methylene blue staining,Warthin-Starry silver staining,and hematoxylin-eosin staining(HE).2 Immunohistochemical analyses(IHC)Expressions of TNF-? and NF-?B were undertaken on paraffin-embedded specimens.According to the retrieval protocol,after deparaffinization and rehydration,high-pressure antigen retrieval was performed in citrate buffer(p H 6.0)for 5 min.Endogenous peroxidase activity was measured in 3% hydrogen peroxidase-methanol for 10 min.Then,the sections were incubated overnight at 4°C in a humidified chamber with antibodies for TNF-?,NF-?B,and H.pylori antigen(1:100).The following assay was performed as described by the PowerVisionTM-9000 kit(PV-9000).The primary antibody was replaced with PBS as a negative control.3 Total DNA extraction and Real-time PCRGenomic DNA was extracted from paraffin-embedded tissue.According to our previous paper,admixtures of tumorous and non-tumorous tissue were evaluated after staining with hematoxylin and eosin.Eight 10-mm-thick sections were cut from each specimen and placed in a 2 ml polypropylene tube.Briefly,the DNA was isolated using a Genomic DNA Kit.Real-time PCR was performed according to GoTaq?qPCR Master Mix.Ten microlitersof GoTaq?qPCR Master Mix,2?L DNA,and 0.4?L sense and antisense primers were used in a final of reaction volume of 20?L.The final concentration of the primers was 0.2 ?M.The amplification was performed with anMx3005 p instrument.The optimal cutoff points were calculated by defining a positive result as CT?35.4 Statistical analysisSPSS 21.0 for Windows was used for the statistical analysis,and the clinical variables were analyzed with the chi-squared test and Fisher's exact test.All of the tests were two-tailed.P<0.05 was considered to be statistically significant.Results:1 Hp and EBV infection in patients with gastric cancerHp infection was evaluated by histologic examination using HE,modified Giemsa,Warthin-Starry silver,methylene blue and IHC staining(Figure.1-5).Among 113 gastric cancers,85 cases(75.2%)showed H.pylori infection.H.pylori strain CagA+,VacA+,and CagA+/VacA+ were present in34(30.1%),44(38.9%)and 27(21.2%)samples,respectively.EBV was detected in 15(15.6%)among 96 cases of gastric cancer.2 Different H.pylori and EBV infection rates between GCA and DGAThe results of comparing H.pylori infection between the two entities were shown in table 2.The rate of H.pylori 16 s rRNA detection was 73.8%among 61 cases of GCA and 76.9% among 52 cases of DGA.A significant difference was hardly detected between the two groups(P>0.05).However,the detection rate of H.pylori strain VacA+ was higher in GCA than in DGA(49.2% vs 26.9%,P<0.05),and the odds ratio(OR)was 2.627(95%confidence interval [CI]: 1.190-5.800;P<0.05).Furthermore,among the gastric cancer with H.pylori 16 s rRNA+,CagA+ and/or VacA+ H.pylori differed significantly between GCA and DGA(73.3% vs 45.0%,P<0.05,Table 3).Moreover,the rate of EBV was 15.6%(10/47)in GCA,beingsignificantly higher than in DGA(10.2%)with OR of 2.378(95% CI:0.746-7.580,Table 1).3 Double infection of H.pylori and EBV between GCA and DGARegarding double infection with H.pylori and EBV(H.pylori+/EBV+),the detailed statistical data are shown in Table 4.Interestingly,a difference was again detected between the two entities.Four of 47(8.5%)GCA cases showed H.pylori VacA+/EBV+,significantly higher than in the two of 49 DGA cases(4.1%,P<0.05).4 Relationships of H.pylori infection with pathological parameters in GCA and DGAAs shown in Table 5,H.pylori CagA+ was detected more frequently in people ?60 years old regardless of GCA or DGA(P<0.05).Interestingly,H.pylori VacA+ was manifestly associated with lymph node metastasis and tumor stage in DGA(P<0.05)but not in GCA(P>0.05).Moreover,16 of 34(47.1%)GCA cases with lymph node metastasis presented H.pylori VacA+,whereas only three of 30(10.0%)such DGA cases did so(P<0.05).5 Expression of TNF-? and NF-?B in GCA and DGANF-?B and TNF-? were investigated in the two entities of gastric cancer(Figure 2).These two antigens were detected more frequently in GCA as shown in Table 6.In the H.pylori infection group,the positive rates of TNF-?and NF-?B were also higher in GCA than in DGA(TNF-?: 95.6% vs 57.5%;NF-?B: 75.0% vs 48.5%;P<0.05).Conclusions:1 There is no different infection of Hp between two entities,which suggested that Hp is a risk factor for not only DGA but also GCA.2 GCA cases showed higher incidence of Hp VacA+ and CagA+/VacA+infection than DGA,but displayed no differences of Hp CagA+ from DGA.It may indicate that Hp VacA+ strain is a more important risk factor for GCA than DGA.3 The expressions of TNF-? and NF-?B were higher in GCA with or without Hp infection,showing that Hp infection played an important roles inGCA.4 gastirc adenocarcinomas from two subsites showed no differences of EBV infection which suggested that EBV may be a risk factor for gastric adenocarcinoma,but it had no relationships with subsite.