Recent innovations in the next-generation sequencing technologies have unveiled that the accumulation of genetic alterations results in the transformation of normal cells into cancer cells. Accurate and timely repair of DNA is, therefore, essential for maintaining genetic stability. Among various DNA repair pathways, the mismatch repair (MMR) pathway plays a pivotal role. MMR deficiency leads to a molecular feature of microsatellite instability (MSI) and predisposes to cancer. Recent studies revealed that MSI-high (MSI-H) or mismatch repair-deficient (dMMR) tumors, regardless of their primary site, have a promising response to immune checkpoint inhibitors (ICIs), leading to the approval of the anti-programmed cell death protein 1 monoclonal antibody pembrolizumab for the treatment of advanced or recurrent MSI-H/dMMR solid tumors that continue to progress after conventional chemotherapies. This new indication marks a paradigm shift in the therapeutic strategy of cancers; however, when considering the optimum indication for ICIs and their safe and effective usage, it is important for clinicians to understand the genetic and immunologic features of each tumor. In this review, we describe the molecular basis of the MMR pathway, diagnostics of MSI status, and the clinical importance of MSI status and the tumor mutation burden in developing therapeutic strategies against gastrointestinal and hepatobiliary malignancies.

Background & Aims Helicobacter pylori infection is a risk factor for gastric cancer. To explore the genetic basis of gastric cancer that develops in inflamed gastric mucosa, we investigated genetic aberrations that latently accumulate in nontumorous gastric epithelium with H pylori infection. Methods We performed whole-exome sequencing of gastric tumors, noncancerous tissues with gastritis, and peripheral lymphocytes from 5 patients. We performed additional deep-sequencing analyses of selected tumor-related genes using 34 gastritis mucosal samples from patients with or without gastric cancer. We also performed deep sequencing analyses of gastric mucosal tissues from mice that express transgenic human TP53 and constitutively express activation-induced cytidine deaminase (AICDA or AID) (human TP53 knock-in/AID-transgenic mice). Results Whole-exome sequencing revealed that somatic mutations accumulated in various genes in inflamed gastric tissues. Additional deep-sequencing analyses of tissues from regions of gastritis confirmed nonsynonymous low-abundance mutations in TP53 in 15 cases (44.1%) and ARID1A in 5 cases (14.7%). The mutations that accumulated in gastric mucosal tissues with H pylori–induced gastritis, as well as gastric tumors, were predominantly C:G>T:A transitions in GpCpX motifs—a marker of cytidine deamination induced by AID. Constitutive expression of AID in the gastric mucosa of mice led to mutations in the human TP53, at amino acid coding positions identical to those detected in human gastric cancers. Conclusions Studies of gastric tumors and tissues from humans and mice indicate that somatic mutations accumulate in various genes in gastric mucosal tissues with H pylori infection. Increased cytidine deaminase activity in these tissues appears to promote the accumulation of these mutations and might promote gastric carcinogenesis in patients with H pylori infection.

Background & Aims: Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutations in the immunoglobulin gene. We recently revealed that ectopic AID expression serves as a link between the cellular editing machinery and high mutation frequencies, leading to human cancer development. In the current study, we investigated whether AID might contribute to the development of colitis-associated colorectal cancers. Methods: The expression and regulation of AID in association with proinflammatory cytokine stimulation were investigated in cultured colonic cells. Genotoxic activity of AID in colonic cells was analyzed using retroviral system. Immunohistochemistry for AID was carried out on various human colonic tissues specimens. Results: Tumor necrosis factor-α induced aberrant AID expression via IκB kinase-dependent nuclear factor (NF)-κB-signaling pathways in human colonic epithelial cells. Moreover, AID expression was also induced in response to the T helper cell 2-driven cytokines interleukin-4 and interleukin-13, which are activated in human inflammatory bowel disease. Aberrant activation of AID in colonic cells preferentially induced genetic mutations in the TP53 gene, whereas there were no nucleotide alterations of the APC gene. Immunohistochemistry revealed enhanced expression of endogenous AID protein not only in the inflamed colonic mucosa of ulcerative colitis patients but also in tumor lesions of colitis-associated colorectal cancers. Conclusions: Our findings indicate that proinflammatory cytokine-mediated aberrant expression of AID in colonic epithelial cells is a genotoxic factor linking inflammation, somatic mutations, and colorectal cancer development.

Chronic inflammation is a strong risk factor for the development of cancer. Many previous studies have demonstrated that a transcriptional factor, nuclear factor (NF)‐κB, plays an important role in the association between inflammation and cancer development, particularly tumor promotion and tumor progression. Although it is well recognized that cancer develops via stepwise accumulation of genetic aberrations, the mechanisms underlying the generation of these genetic alterations in normal epithelial cells under inflammatory conditions are not known. We recently demonstrated that pathogenic bacterial or viral factors and the subsequent inflammatory reactions lead to the aberrant expression of a DNA mutator enzyme, activation‐induced cytidine deaminase (AID), in various epithelial cells via NF‐κB activation, which causes the accumulation of genetic alterations in tumor‐related genes. AID activation is widely observed in gastrointestinal tissues with cancer‐associated inflammation, such as chronic viral hepatitis, Helicobacter pylori‐related gastritis, Barrett's esophagus and inflammatory bowel disease. Furthermore, a deficiency of endogenous AID expression reduces both accumulation of somatic mutations in tumor‐related genes and tumor incidence in a mouse model of inflammation‐associated cancer development. These findings strongly suggest that AID plays an integral role in inflammation‐associated carcinogenesis and is therefore a potential target molecule for the prevention and treatment of cancers. (Cancer Sci 2012; 103: 1201–1206)

The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail. Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy. Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents. Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.

Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.