Association of Fusobacterium nucleatum with immunity andmolecular alterations in colorectal cancer

The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum(F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6%(44/511), which was lower than that in United States cohort studies(13%). Similar to the United States studies, F. nucleatum positivityin Japanese colorectal cancers was significantly associated with microsatellite instability(MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets(i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain micro RNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. Micro RNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in colorectal cancer cells. Thus, emerging evidence may provide insights for strategies to target microbiota, immune cells and tumor molecular alterations for colorectal cancer prevention and treatment. Further investigation is needed to clarify the association of Fusobacterium with T-cells and micro RNA expressions in colorectal cancer.

Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).There were 7 cases of lymphoepithelioma-like carcinomas(LELC) and 6 of those cases were EBV-positive(percent/EBV:6/18,33%;percent/all LELC:6/7,86%).pAkt expression was positive in 119(53%) cases but showed no correlation with clinicopathological characteristics.pAkt expression was significantly correlated with HER2 overexpression(16/20 vs 103/211,P < 0.01) but not with PIK3CA mutations(12/20 vs 107/211,P = 0.37) or EBV infection(8/18 vs 103/211,P = 0.69).The frequency of pAkt expression was higher in cancers with exon20 mutations(100%) than in those with exon1(40%) or exon9(56%) mutations.One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection.However,no cases showed both PIK3CA mutations and HER2 overexpression.One EBVpositive cancer with PIK3CA mutation(H1047R) was MSI-positive.Three of these 4 cases were positive for pAkt expression.In survival analysis,pAkt expression significantly correlated with a poor prognosis(hazard ratio 1.75;95%CI:1.12-2.80,P = 0.02).CONCLUSION:HER2 expression,PIK3CA mutations and EBV infection in gastric cancer were characterized.pAkt expression significantly correlates with HER2 expression and with a poor prognosis.

IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions

AIM:To investigate insulin-like growth factor 2(IGF2)differentially methylated region(DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions.METHODS:To accurately analyze the association between the histological types and molecular features of each type of serrated lesion,we consecutively collected1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types[hyperplastic polyps(HPs,n=121),sessile serrated adenomas(SSAs,n=132),traditional serrated adenomas(TSAs,n=111),non-serrated adenomas(n=195),and colorectal cancers(n=827)].We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1(LINE-1)in HPs(n=115),SSAs(n=120),SSAs with cytological dysplasia(n=10),TSAs(n=91),TSAs with high-grade dysplasia(HGD)(n=15),non-serrated adenomas(n=80),non-serrated adenomas with HGD(n=105),and CRCs(n=794).For the accurate quantification of the relative methylation levels(scale 0%-100%)of IGF2 DMR0 and LINE-1,we used bisulfite pyrosequencing method.Tumor specimens were analyzed for microsatellite instability,KRAS(codons 12 and 13),BRAF(V600E),and PIK3CA(exons 9and 20)mutations;MLH1 and MGMT methylation;and IGF2 expression by immunohistochemistry.RESULTS:The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas(with or without HGD)was as follows:mean61.7,median 62.5,SD 18.0,range 5.0-99.0,interquartile range 49.5-74.4.The IGF2 DMR0 methylation level was divided into quartiles(Q1≥74.5,Q2 62.6-74.4,Q3 49.6-62.5,Q4≤49.5)for further analysis.With regard to the histological type,the IGF2 DMR0 methylation levels of SSAs(mean±SD,73.1±12.3)were significantly higher than those of HPs(61.9±20.5),TSAs(61.6±19.6),and non-serrated adenomas(59.0±15.8)(P<0.0001).The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level(r=-0.21,P=0.0051).IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs,TSAs,and non-serrated adenomas(P<0.0001).Multivariate logistic regression analysis also showed that IGF2 DMR0 hypomethylation was inversely associated with SSAs(P<0.0001).The methylation levels of IGF2 DMR0 and LINE-1 in TSAs with HGD(50.2±18.7and 55.7±5.4,respectively)were significantly lower than those in TSAs(61.6±19.6 and 58.8±4.7,respectively)(IGF2 DMR0,P=0.038;LINE-1,P=0.024).CONCLUSION:IGF2 DMR0 hypomethylation may be an infrequent epigenetic alteration in the SSA pathway.Hypomethylation of IGF2 DMR0 and LINE-1 may play a role in TSA pathway progression.