Published OnlineFirst August 26, 2014; DOI: 10.1158/1055-9965.EPI-14-0471 Cancer Epidemiology, Research Article Biomarkers & Prevention Circulating Mitochondrial DNA Level, a Noninvasive Biomarker for the Early Detection of Gastric Cancer Julien Fernandes1,2, Valerie Michel1,2, Margarita Camorlinga-Ponce3, Alejandro Gomez3, Carmen Maldonado4, Hilde De Reuse1,2, Javier Torres3, and Eliette Touati1,2 Abstract Background: Gastric cancer represents a major health burden worldwide and is often diagnosed at an advanced stage. Biomarkers for screening and prevention of gastric cancer are missing. Changes in peripheral blood mitochondrial DNA (mtDNA) have emerged as a potential preventive/diagnosis biomarker for cancer risk. We aimed to determine whether peripheral leukocytes mtDNA levels are associated with stages of the gastric carcinogenesis cascade. Methods: We measured mtDNA by quantitative real-time PCR assay in peripheral leukocytes of 28 patients with non-atrophic gastritis (NAG), 74 patients with gastric cancer, and 48 matched asymptomatic controls. In parallel, the serologic level of IL8 was determined. Results: Mean mtDNA level was higher in patients with gastric cancer (P ¼ 0.0095) than in controls, with values >8.46 significantly associated with gastric cancer (OR, 3.93). Three ranges of mtDNA values were identified: interval I, <2.0; interval II, 2.0–20; and interval III, >20. Interval I included mainly NAG cases, and few gastric cancer samples and interval III corresponded almost exclusively to patients with gastric cancer. All controls fell in interval II, together with some NAG and gastric cancer cases. IL8 levels were significantly higher in patients with gastric cancer (P < 0.05), with levels >50 pg/mL observed exclusively in patients with gastric cancer, allowing to distinguish them within interval II. We validated mtDNA results in a second cohort of patients, confirming that mtDNA was significantly higher in gastric cancer than in patients with preneoplasia. Conclusions: Circulating levels of mtDNA and IL8 constitute a potential biomarker for the early detection of gastric cancer. Impact: Our findings lead us to propose a new noninvasive method to detect patients with gastric cancer risk. Cancer Epidemiol Biomarkers Prev; 23(11); 2430–8. Ó2014 AACR. Introduction highest incidence of gastric cancer, nation-wide strategies Gastric cancer represents a major health burden world- based on improved tests for detection of early gastric wide, affecting about 1 million people per year (1, 2). cancer or precancerous lesions have decreased the inci- Gastric cancer is often diagnosed at an advanced stage dence of gastric cancer and increased survival rate (5). and consequently carries a poor prognosis (3). Important- Although gastric cancer arises from the complex inter- play of environmental and host genetic factors (6, 7), the ly, if it is detected at an early asymptomatic stage, it can be Helicobacter pylori curable (4); for example, in Japan, a country with the major risk factor is infection, which is associated with more than 80% of all distal gastric cancer cases (8). The prevalence of H. pylori infection is high, with 1Institut Pasteur, Department of Microbiology, Helicobacter Pathogenesis 80% to 95% of the population infected in developing Unit. CNRS, Paris, France. 2CNRS, ERL3526, Paris, France. 3Unidad de countries and up to 30% to 40% of adults in industrialized Investigacion en Enfermedades Infecciosas, UMAE Pediatria, IMSS, Mex- countries (6). All infected individuals develop a gastritis ico City, Mexico. 4Hospital Infantil de Mexico, SS, Mexico City, Mexico. that evolve to peptic ulcer diseases in about 10% of the Note: Supplementary data for this article are available at Cancer Epide- cases, whereas gastric adenocarcinoma and mucosa-asso- miology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). ciated lymphoid tissue (MALT) lymphoma develop in Current address for J. Fernandes: Unite U1152, Physiopathologie et <3% and 0.3% of infected subjects, respectively (9, 10). Epidemiologie des Maladies Respiratoires, Universite Paris Diderot, FacultedeM edecine, site Bichat, 16 Rue Henri Huchard, 75870 Paris Two types of gastric cancer can be distinguished, the cedex 18, France. intestinal and diffuse types. The intestinal type develops Corresponding Author: Eliette Touati, Institut Pasteur, Helicobacter Path- through progressive changes in the gastric mucosa from ogenesis Unit, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France. non-atrophic gastritis (NAG), atrophic gastritis, intestinal Phone: 33140613785; Fax: 3314061364; E-mail: [email protected] metaplasia, dysplasia, and gastric cancer (11). It has been doi: 10.1158/1055-9965.EPI-14-0471 shown that eradication of the infection at an early stage Ó2014 American Association for Cancer Research. can reverse gastric lesions and, more importantly, prevent 2430 Cancer Epidemiol Biomarkers Prev; 23(11) November 2014 Downloaded from cebp.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 26, 2014; DOI: 10.1158/1055-9965.EPI-14-0471 Circulating Mitochondrial DNA as a Gastric Cancer Biomarker the development of preneoplasia (7, 12, 13). A clinical Materials and Methods H. study conducted in Japan demonstrated the efficacy of Study population pylori eradication to reduce the incidence of gastric cancer Two cohorts of Mexican adult patients were studied. (14) and confirmed that it was not enough to prevent all Cohort 1 included 48 healthy asymptomatic H. pylori– gastric cancer cases. These data raise the need for the negative blood donors, 28 patients with NAG, and 74 development of biomarkers to detect precancerous lesions patients with gastric cancer, for a total of 150 adults or early gastric cancer; the test should be a simple and recruited during the period 2009 to 2011. The blood noninvasive method, applicable in large-scale screening donors were recruited at the blood bank of the Instituto programs. In the case of intestinal-type gastric cancer, the Mexicano del Seguro Social (IMSS), Medical Center SXXI measure of pepsinogen levels has been shown to be useful in Mexico City. Cohort 2 was meant to validate results in to detect gastric atrophy, although its use in non–Asian cohort 1 and included 46 patients with NAG, 31 patients countries is controversial (15). Thus, despite several with intestinal metaplasia, and 49 patients with gastric efforts to develop biomarkers to identify patients at risk cancer, for a total of 126 patients recruited during the for distal gastric cancer (16), no efficacious screening test is period 1999 to 2002. Patients from both cohorts were yet available. adults who were attended for gastroduodenal diseases Mitochondria are essential organelles of eukaryotic cells at the IMSS. We selected patients who were not under that possess their own genome. Mitochondrial DNA treatment for cancer and who had not been treated with (mtDNA) is a circular molecule present at 2 to 10 copies antibiotics, bismuth compounds, proton pump inhibitors, per organelle (17). Both mutations and alterations of and NSAIDs for at least 2 preceding weeks. Diagnosis was mtDNA content have been described in many different based on endoscopic examination and histopathologic cancer types (18–20). mtDNA mutations have been analysis (36). All patients and asymptomatic controls detected at early stages of gastric carcinogenesis (21, 22). H. pylori– were informed and asked to sign a consent letter. The In infected patients, mtDNA mutations are sig- study was approved by the ethical committee from the nificantly more frequent in patients with gastric cancer National Council for Research on Health, IMSS. than in cancer-free patients (23). According to our previous in vitro H. pylori studies, mtDNA mutations are induced in – Collection of clinical samples and histologic analysis infected gastric epithelial cells and in the gastric mucosa of For each patient, 10 mL of blood was collected, and chronically infected mice (24, 25). A decrease in mtDNA gastric tissue specimens were isolated. For patients with content has also been described in most tumor tissues of NAG or intestinal metaplasia, gastric biopsies collected advanced gastric cancer, compared with nearby nontumor from both the antrum and the corpus were taken. For control tissue (26–28). Changes in peripheral blood patients with gastric cancer, one fraction of tumoral and mtDNA levels have recently emerged as a potential pre- adjacent tissues were collected during surgery. Biopsies ventive/diagnosis biomarker associated with cancer risk were immersed in formalin and processed for hematox- (18). Circulating mtDNA levels were significantly higher ylin and eosin (H&E) staining for histologic analysis and in patients with urologic malignancies (29), breast (30), diagnosis of gastric lesions. The presence of H. pylori was colorectal (31), and lung cancers (32, 33). These studies confirmed by Giemsa staining and serology. indicated that increased mtDNA content in peripheral blood is associated with elevated cancer risk. However, Circulating mtDNA contrasting reports have found mtDNA depletion in the Peripheral blood (10 mL) was taken from each patient, blood of patients with stage I breast tumors (34). A recent and mononuclear cells were purified by centrifugation large prospective cohort study in women from Shanghai through a Ficoll-Hypaque density gradient. DNA was found no association between the leukocyte mtDNA copy isolated from these cells using the