Cancer Risk Diagnosis Research Articles

Abstract 6014: Cancer risk diagnosis by epimutation burden: a multicenter prospective study

Abstract Aberrant DNA methylation, induced by aging and accelerated by chronic inflammation, accumulates in normal tissue. Accumulation of DNA methylation in multiple genes, namely epimutation burden, is closely associated with cancer risk. Indeed, we demonstrated that the measurement of the methylation level of a marker gene in normal tissue is useful for cancer risk prediction of metachronous cancers, by a multicenter prospective study with gastric cancer patients (Asada, Gut, 64:388, 2015: Maeda, Gut, 66:1721, 2017). In this study, we attempted to predict the risk of primary gastric cancer by performing a new multicenter prospective study. We recruited 1,880 healthy people, after Helicobacter pylori eradication, with a clinically high risk of gastric cancer. Biopsy specimens were obtained from the antrum and body, and the methylation level of RIMS1, which is a preselected epigenomic risk marker for this study, was measured in 1,757 participants. The participants are annually being followed-up to detect primary gastric cancer, for 5-8 years. Currently, the median follow-up is 4.05 years, and 27 people have developed primary gastric cancers (as of September 2022). The results of statistical analysis are being determined. This study will establish the clinical utility of a cancer risk diagnosis by epimutation burden in normal tissue, and is promising for application in various inflammation-associated cancers. Citation Format: Harumi Yamada, Masahiro Maeda, Seiichiro Abe, Taichi Shimazu, Takayuki Ando, Kazunari Murakami, Shinji Tanaka, Takao Maekita, Osamu Inatomi, Ken Sugimoto, Tetsuro Kawagoe, Nobutake Yamamichi, Toshikazu Ushijima. Cancer risk diagnosis by epimutation burden: a multicenter prospective study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6014.

Immunoassay of antibodies to benzo[a]pyrene for lung cancer risk diagnosis among coal-mining workers

Th e authors detected association of G-class antibodies specifi c to chemical carcinogen benzo[a]pyrene (IgG-Bp) with lung cancer in healthy men not working at coal mines. Objective — to reveal possibilities of immunoassay of IgG-Bp in evaluating risk of lung cancer in coal mine workers. Th e study covered serum IgG-Bp in 501 miners aged under 60 (161 healthy, not working in mine; 239 miners; 101 patients with lung cancer), by means of non-concurrent immune-enzyme analysis using benzo[a]pyrene conjugates with bovine serum albumin as an antigen. Findings are that increased levels of IgG-Bp in lung cancer patients are more frequent (50.5%) than in healthy men not working in mine (37.2%, p=0.047, OR=1.7) and in miners (30.5%, p=0.0008, OR=2.3). Length of service in mines did not infl uence serum levels of IgG-Bp in coal industry workers. In the miners, with age adjustment consideration, OR for lung cancer equaled 2.0 (p=0.01), and with adjustment for smoking OR=2.3 (p=0.001). Occupational (work in mine), lifestyle (smoking) and endogenous (age) factors do not have signifi cant infl uence on intensity of individual specifi c reaction to benzo[a]pyrene in males. Immunoassay of IgG-Bp is recommended for lung canc er diagnosis in coal mine workers.

Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues

Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69-89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52-23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.

1406P - Increased mutation burden in high-risk lung tissues: Toward precision cancer risk diagnosis

Background: Mutations are believed to accumulate in normal tissues at extremely low levels as a result of exposure to various carcinogenic factors. The degree of accumulation, namely mutation burden, is likely to be associated with cancer risk. However, owing to the limits of current detection methods for such extremely low frequency mutations, the mutation burden present in normal human lung tissues has been unclear. To overcome this limitation, we established a novel method for the quantification of extremely low frequency mutations in DNA samples. Using this method, we aimed to reveal the presence of mutation burden in normal lung tissues and its association with cancer risk. Methods: Somatic mutations were quantified in normal lung tissues without smoking history (n = 11) (“entirely normal lung tissues”:G1), normal lung tissues with smoking history (n = 11) (“smoking-exposed normal tissues”:G2), and non-cancerous lung tissues of patients with lung cancer and smoking history (n = 11) (“smoking-exposed non-cancerous tissues”:G3). A sequence library (15,724 bases of 291 regions of 55 cancer-related genes) was prepared by multiplex PCR using 100 DNA molecules. Libraries were sequenced using a next generation sequencer. Results: The mutation burden in G3 (2.7 ± 0.8 × 10−5 mutations/base) was significantly higher than that in G1 (1.8 ± 0.5 × 10−5 mutations/base) (p = 0.0189). Accumulation of somatic mutations tended to be associated with increased cancer risk (OR = 3.75; 95% CI = 0.54–26.046). C>T mutations were significantly more frequent in G2 and G3 than in G1, which is in accordance with reported mutation signatures in cancer tissues [Alexandrov et al., Science, 354:2016]. GCC>GTC and CCC>CTC mutations, signatures of exposure to the nitrosamines contained in tobacco smoke, were significantly enriched in G2 and G3. Conclusions: To the best of our knowledge, this is the first study showing that mutations accumulate in high-risk lung tissues due to exposure to tobacco smoking. This will lead to a novel approach to precision cancer risk diagnosis. Legal entity responsible for the study: Toshikazu Ushijima Funding: None Disclosure: All authors have declared no conflicts of interest.

Abstract LB-092: High impact of methylation accumulation on cancer risk: 5-year follow-up of a multicenter prospective cohort study

Abstract Aberrant DNA methylation can be accumulated in normal-appearing tissues, and its degree can be correlated with cancer risk [Hattori, Genome Med, 8:10, 2016]. Such accumulation is especially prominent in inflammation-associated cancers, including gastric cancer. To bring the findings into cancer risk diagnosis, we conducted a multicenter prospective cohort study with 826 patients [Asada, Gut, 64:388, 2015]. Between 2008 and 2010, 826 patients after endoscopic treatment of gastric cancer and without current H. pylori infection were enrolled, and a gastric biopsy was collected. Methylation levels of pre-defined three marker genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were classified into quartiles according to methylation levels of individual genes (Q1, the lowest; Q4, the highest). Among the 826 enrolled patients, 795 patients received annual follow-ups by endoscopy with a median period of 5.46 years (interquartile range: 3.95 - 6.09). By the end, 133 patients developed a metachronous gastric cancer, and 116 of them developed a cancer after 1 year of the enrollment (authentic metachronous cancer). Kaplan-Meier analysis showed that Q4 had a higher cumulative incidence of metachronous gastric cancer than Q1 with p-values of <0.001 to 0.004 (log-rank test). Multivariate analyses adjusted for hospital, gender and age, pepsinogen index, history of endoscopic treatment, smoking and green vegetable intake also showed that Q4 had significantly high HRs compared with Q1, being 3.0 (95%CI: 1.58 to 5.72, p=0.0017) for miR-124a-3 [Maeda, Gut, online]. The results clearly showed that accumulation levels of aberrant DNA methylation can be used for cancer risk diagnosis. Even among the patients who carried a high risk of metachronous gastric cancer, cancer risk stratification was achieved. The strong influence of methylation accumulation on gastric cancer risk indicated a large contribution of aberrant DNA methylation to H. pylori infection-induced gastric carcinogenesis. Epigenetic cancer risk diagnosis is now becoming real. Citation Format: Masahiro Maeda, Takeshi Nakajima, Ichiro Oda, Taichi Shimazu, Nobutake Yamamichi, Takao Maekita, Kiyoshi Asada, Chizu Yokoi, Takayuki Ando, Takeichi Yoshida, Sohachi Nanjo, Mitsuhiro Fujishiro, Takuji Gotoda, Masao Ichinose, Toshikazu Ushijima. High impact of methylation accumulation on cancer risk: 5-year follow-up of a multicenter prospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-092. doi:10.1158/1538-7445.AM2017-LB-092

Mechanisms for the induction of gastric cancer by Helicobacter pylori infection: aberrant DNA methylation pathway.

Multiple pathogenic mechanisms by which Helicobacter pylori infection induces gastric cancer have been established in the last two decades. In particular, aberrant DNA methylation is induced in multiple driver genes, which inactivates them. Methylation profiles in gastric cancer are associated with specific subtypes, such as microsatellite instability. Recent comprehensive and integrated analyses showed that many cancer-related pathways are more frequently altered by aberrant DNA methylation than by mutations. Aberrant DNA methylation can even be present in noncancerous gastric mucosae, producing an "epigenetic field for cancerization." Mechanistically, H. pylori-induced chronic inflammation, but not H. pylori itself, plays a direct role in the induction of aberrant DNA methylation. The expression of three inflammation-related genes, Il1b, Nos2, and Tnf, is highly associated with the induction of aberrant DNA methylation. Importantly, the degree of accumulated aberrant DNA methylation is strongly correlated with gastric cancer risk. A recent multicenter prospective cohort study demonstrated the utility of epigenetic cancer risk diagnosis for metachronous gastric cancer. Suppression of aberrant DNA methylation by a demethylating agent was shown to inhibit gastric cancer development in an animal model. Induction of aberrant DNA methylation is the major pathway by which H. pylori infection induces gastric cancer, and this can be utilized for translational opportunities.

Abstract 4460: Precision cancer risk diagnosis by accumulation of epigenetic alterations

Abstract Background and Aim: Aberrant DNA methylation is induced by chronic inflammation, and can be accumulated in normal-appearing tissues [reviewed in Ushijima and Hattori, Clin Cancer Res, 2:143, 2012]. Cross-sectional studies have shown that the degree of accumulation of aberrant DNA methylation was correlated with cancer risk, especially inflammation-associated cancers. In this study, we aimed to demonstrate that cancer risk can be predicted by an epigenetic cancer risk marker by a multicenter prospective cohort study. Methods: We enrolled 826 patients with early gastric cancer, aged 40-80 years, who had undergone endoscopic resection. If a patient had active Helicobacter pylori (H. pylori) infection, the patient was enrolled after eradication of H. pylori. At the time of enrollment, one gastric biopsy was taken, and methylation levels of three preselected genes, miR-124a-3, EMX1 and NKX6-1, were measured by quantitative methylation-specific PCR. The patients were followed up annually by endoscopy to detect metachronous gastric cancer. To exclude cancers undetected at the time of enrollment, “authentic” metachronous gastric cancers were defined as those developed 1 year after enrollment. Results: With a median follow-up of 2.97 years, 782 of 826 patients had at least one follow-up. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1-2, 2-3 and ≥3 years after the enrollment, respectively. The highest quartile of the miR-124a-3 methylation level had a significantly high HR in univariate analysis (95% CI) [2.17 (1.07 - 4.41); p = 0.032] and adjusted HR in multivariate analysis [2.30 (1.03 - 5.10); p = 0.042] [Asada et al, Gut, 64:388, 2015]. More importantly, with a median follow-up of 4.84 years, a multivariate analysis yielded a larger HR [2.79 (1.34 - 5.81)] with a smaller p value (0.0061). Conclusions: This study, for the first time, demonstrated the usefulness of an epigenetic cancer risk marker by a multicenter prospective cohort study. DNA methylation accumulated in normal-appearing tissues was considered to be highly useful for precision cancer risk diagnosis. Citation Format: Toshikazu Ushijima, Kiyoshi Asada, Masahiro Maeda, Takeshi Nakajima, Taichi Shimazu. Precision cancer risk diagnosis by accumulation of epigenetic alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4460.

Molecular Pathways: Involvement of Helicobacter pylori–Triggered Inflammation in the Formation of an Epigenetic Field Defect, and Its Usefulness as Cancer Risk and Exposure Markers

Infection-associated cancers account for a large proportion of human cancers, and gastric cancer, the vast majority of which is associated with Helicobacter pylori infection, is a typical example of such cancers. Epigenetic alterations are known to occur frequently in gastric cancers, and H. pylori infection has now been shown to induce aberrant DNA methylation in gastric mucosae. Accumulation of aberrant methylation in gastric mucosae produces a field for cancerization, and methylation levels correlate with gastric cancer risk. H. pylori infection induces methylation of specific genes, and such specificity is determined by the epigenetic status in normal cells, including the presence of H3K27me3 and RNA polymerase II (active or stalled). Specific types of inflammation, such as that induced by H. pylori infection, are important for methylation induction, and infiltration of monocytes appears to be involved. The presence of an epigenetic field defect is not limited to gastric cancers and is observed in various types of cancers. It provides translational opportunities for cancer risk diagnosis incorporating life history, assessment of past exposure to carcinogenic factors, and cancer prevention.