Variable copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in advanced gastric cancer patients
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 16 July 2013
Accepted: 11 October 2013
Published: 21 October 2013
Change of mitochondrial DNA (mtDNA) copy number is widely reported in various human cancers, including gastric cancer, and is considered to be an important hallmark of cancers. However, there is remarkably little consensus on the value of variable mtDNA content in the prognostic evaluation of this cancer.
Using real-time quantitative PCR approach, we examined mtDNA copy number in a cohort of gastric cancers and normal gastric tissues, and explored the association of variable mtDNA content with clinical outcomes of gastric cancer patients.
Our data showed that the majority of gastric cancer patients had low mtDNA content as compared to control subjects although the relative mean mtDNA content was higher in the former than the latter. Moreover, we found that variable mtDNA content was strongly associated with lymph node metastasis and cancer-related death of the patients with late-stage tumors. Notably, variable mtDNA content did not affect overall survival of gastric cancer patients, however, we found that increased mtDNA content was associated with poor survival in the patients with late-stage tumors.
In this study, we demonstrated that variable mtDNA content markedly increased the risk of lymph node metastasis and high mortality of the patients with late-stage tumors. Additionally, we found a strong link between increased mtDNA content and worse survival of the patients with late-stage tumors. Taken together, variable mtDNA content may be a valuable poor prognostic factor for advanced gastric cancer patients.
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Gastric cancer is the second cause of cancer deaths after lung cancer, and is a major health burden worldwide . Despite advances in therapeutic modalities during the past decades, the prognosis at the advanced stage is still dismal, with an average 5-year survival rate of less than 20% [2, 3]. The cause of gastric cancer is multifactorial, and the prognosis varies widely in gastric cancer patients due to yet undetermined biologic factors . Thus, there is increasing need to develop reliable biomarkers for predicting clinical outcomes and establishing new therapeutic and preventive strategies to this disease.
Although a number of biomarkers have been demonstrated to be closely associated with poor prognosis of gastric cancer patients [5–9], most of them are concerned with the roles of nuclear DNA (nDNA) alterations in gastric tumorigenesis [10–14]. These genetic or epigenetic alterations cause gain-of-function in oncogenes and loss-of-function in tumor suppressor genes [15, 16], however, relatively less attention has been paid to mitochondrial DNA (mtDNA) alterations. Mitochondria are organelles found in all nucleated cells. The major role of mitochondria is to generate cellular adenosine triphosphate (ATP) through oxidative phosphorylation . Human mtDNA is a 16,569 base-pair, double-stranded, closed-circular DNA molecule that encodes 13 polypeptides, 2 rRNAs, and a set of 22 tRNAs required for protein synthesis in mitochondria . The displacement loop (D-loop) is a noncoding region essential for the replication and transcription of mtDNA. Mutations in the D-loop may cause a reduction in mtDNA copy number or altered mtDNA gene expression [19, 20]. Generally, each human cell contains several hundred to 1000 mitochondria, and each mitochondrion has 2 to 10 copies of mtDNA. The mitochondrial genome is more vulnerable to oxidative damage and undergoes a higher rate of mutation than does the nDNA [21, 22]. Increasing evidences have demonstrated the association of increased mtDNA content in peripheral blood with increased risk of non-Hodgkin lymphoma , lung cancer , pancreatic cancer , breast cancer , and colorectal cancer , whereas increased risk of renal cancer is associated with decreased mtDNA content . Although several studies have reported depletion in mtDNA copy number in gastric cancers as compared with normal gastric tissues [28, 29], there is no relationship between leukocyte mtDNA content and the risk of developing gastric cancer . Until now, the association of mtDNA content with clinical outcomes of gastric cancer patients remains largely unknown.
In the present study, we investigated mtDNA copy number in a cohort of gastric cancers and normal gastric tissues using real-time quantitative PCR approach, and explored the effect of mtDNA content on clinical outcomes of gastric cancer patients.
Clinicopathological characteristics of gastric cancer patients
No. of patients (%)
Tumor size (cm3)
Lymph node metastasis (LNM)
No. of LNM
Serial sections from each tumor sample were cut. One section was stained using hematoxylin and eosin (H&E) and was marked as a tumor representative tissue by an expert surgical pathologist for gastric cancer. Tumor tissues were then isolated by manual microdissection under an inverted microscope using the marked H&E section for target tissue identification. DNA was extracted from isolated tumor tissues as previously described . Briefly, the tissues were first treated with xylene for 12 h at room temperature to remove the paraffin, and were then subjected to digestion with 1% sodium dodecylsulfate (SDS) and proteinase K at 48°C for 48 to 72 h with addition of several spiking aliquots of concentrated proteinase K to facilitate digestion. Genomic DNA was isolated from the digested tissues followed by standard phenol-chloroform extraction and ethanol precipitation protocol, and stored at −80°C until use.
mtDNA copy number analysis
The primer and TaqMan probe sequences used in this study
Forward primer sequence (5′→3′)
Probe sequence (5′→3′)
Reverse primer sequence (5′→3′)
Amplification efficiency (%)
The Mann–Whitney U test was used to compare mtDNA copy number between gastric cancer and normal gastric tissues. Association of mtDNA copy number with clinicopathological characteristics was assessed univariately using the SPSS statistical package (version 11.5, Chicago, IL). Multivariate models were then developed that adjusted for the most important covariates, including age, tumor size, differentiation, and lymph node metastasis. Survival length was determined from the day of primary tumor surgery to the day of death or last clinical follow-up. The Kaplan–Meier method was used for survival analysis grouping with copy number variations of mtDNA. Differences between curves were analyzed using the log-rank test. Multivariate Cox regression analysis was used to evaluate the effect of mtDNA copy number on survival of independently of the number of lymph node metastasis, tumor invasion and differentiation. All statistical analyses were performed using the SPSS statistical package (version 11.5, Chicago, IL). P values < 0.05 were considered significant.
Relative mtDNA copy number in gastric cancer
Association of variable mtDNA content with clinicopathological characteristics of gastric cancer patients
Copy number variations of mtDNA in gastric cancer — univariate associations with clinicopathological characteristics
Copy number <3.61
Copy number >5.35
Male vs. Female
Lymph node metastasis
Copy number variations of mtDNA in early-stage gastric cancer — univariate associations with clinicopathological characteristics
Copy number <3.61
Copy number >5.35
Male vs. Female
Lymph node metastasis
Copy number variations of mtDNA in late-stage gastric cancer — univariate associations with clinicopathological characteristics
Copy number <3.61
Copy number >5.35
Male vs. Female
Lymph node metastasis
Copy number variations in gastric cancer — multivariable models assessing age, tumor size, differentiation and lymph node metastasis
Copy number <3.61
Copy number >5.35
Lymph node metastasis
Effect of variable mtDNA content on poor survival of gastric cancer patients
Prognostic value of clinicopathological factors and copy number variation of mtDNA in univariate and multivariate Cox regression analysis (n=103)
Hazard ratio (95% CI)
Hazard ratio (95% CI)
The number of lymph node metastasis
Although much of the current funding is aligned to continuing to further understand the functional details of the nuclear genome, the mitochondrion and its modest complement of DNA and protein is emerging as a crucial component of the biological networking of nuclear pathways . Mitochondria are eukaryotic organelles involved in many important physiological processes, including metabolism, signaling, apoptosis, cell cycle, differentiation and responsible for energy production . It has been well documented that the enhanced production of mitochondrial reactive oxygen species (ROS), most notably superoxide, hydroxyl radicals, and hydrogen peroxide is a prominent byproduct of cancer cell metabolism . Within various cells, tissues and organs, mtDNA copy number is different, and this difference can also occur in a given type of cell under different conditions or internal or external microenvironments [34, 35]. Unlike nuclear DNA, mtDNA is present at a consistently high level in each cell , and mtDNA mutation rate is much higher than that of nuclear DNA [18, 21]. Mitochondrial aberrants, including mtDNA mutations and copy number variations, have been frequently identified in different types of human cancers, including gastric cancer [28–30, 36, 37], suggesting that mtDNA aberrations play a critical role in gastric tumorigenesis. However, the prognostic values of mtDNA aberrants, particularly copy number variations, in gastric cancer patients ramain largely unclear.
In this study, we investigated relative mtDNA copy number in a cohort of gastric cancers and normal gastric tissues (control subjects) using real-time quantitative PCR approach. Our data showed that the majority of the cancer patients had low levels of mtDNA copy number as compared to control subjects, although mean mtDNA content was a little bit higher in gastric cancer patients than control subjects. In line with this study, the previous studies have demonstrated that mtDNA depletion is frequently found in gastric cancers as compared with normal gastric tissues [28, 29], implicating that low mtDNA content is involved in the formation and progression of gastric cancer. Moreover, we did not find the association of mtDNA content with most of clinicopathological features, such as gender, age, tumor localization, tumor size, differentiation, tumor invasion, TNM stage and survival status. However, we found that the patients with lymph node metastasis had a lower mtDNA copy number than the patients without lymph node metastasis, although the difference between two groups was not statistically significant.
To further explore the association of mtDNA content with clinicopathological characteristics and poor survival of gastric cancer patients, we categorized the patients into three groups based on two cutoff points (the lower and upper limit of 95% confidence interval for all control subjects), such as decreased mtDNA content (<3.61 copies), normal mtDNA content or reference (3.61-5.35 copies) and increased mtDNA content (>5.35 copies). Our findings showed that variable mtDNA content (whatever decreased or increased mtDNA content) was closely associated with an increased risk of lymph node metastasis for gastric cancer patients as compared to reference. Strikingly, when gastric cancer patients were further categorized into early-stage and late-stage groups based on TNM stage, variable mtDNA content was not asscoiated with lymph node metastasis for the patients with early-stage tumors. However, both decreased and increased mtDNA content significantly increased the risk of of lymph node metastasis for the patients with late-stage tumors. These observations suggest that copy number variations of mtDNA may be invloved in gastric cancer progression. Similar to our findings in the present study, a previous study showed that mtDNA content was increased gradually from the non-cancerous esophageal mucosa to esophageal squamous cell carcinoma (ESCC) and then the metastatic lymph nodes . Moreover, our data showed that variable mtDNA content was associated with cancer-related death of the patients with late-stage tumors. Collectively, our findings suggest that variable mtDNA content may contribute to poor clinical outcomes of gastric cancer patients, particularly the patients with advanced tumors. Next, we evaluated the effect of variable mtDNA content on poor survival of gastric cancer patients. Our data showed that both decreased and increased mtDNA content were not associated with overall survival of gastric cancer patients. However, when the patients were categorized into early-stage and late-stage tumor groups, increased mtDNA content was strongly associated with poor survival in the latter, but not in the former, as supported by a previous study that high mtDNA copy number may contribute to the high bioenergetic function of mitochondria and further confer an advantage for malignant behaviors of cancer cells, such as tumor invasion .
In summary, we investigated relative mtDNA content in a large cohort of gastric cancers, and demonstrated that variable mtDNA content was closely associated with lymph node metastasis and higher mortality of the patients with late-stage tumors. Moreover, increased mtDNA content predicts worse survival for the patients with late-stage tumors. Thus, variable mtDNA content may be a valuable biomarker in evaluating poor prognosis of advanced gastric cancer patients.
This work was supported by the National Key Program for Developing Basic Research (No. 2010CB933903), the National Natural Science Foundation of China (No. 81171969), the Fundamental Research Funds for the Central Universities, and the Program for New Century Excellent Talents in University (No. NCET-10-0674).
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin. 2011, 61: 69-90. 10.3322/caac.20107.View ArticlePubMedGoogle Scholar
- Blakely AM, Miner TJ: Surgical considerations in the treatment of gastric cancer. Gastroenterol Clin North Am. 2013, 42: 337-357. 10.1016/j.gtc.2013.01.010.PubMed CentralView ArticlePubMedGoogle Scholar
- Tan VP, Wong BC: Gastric cancer chemoprevention: the current evidence. Gastroenterol Clin North Am. 2013, 42: 299-316. 10.1016/j.gtc.2013.02.001.View ArticlePubMedGoogle Scholar
- Allgayer H, Heiss MM, Schildberg FW: Prognostic factors in gastric cancer. Br J Surg. 1997, 84: 1651-1664. 10.1002/bjs.1800841206.View ArticlePubMedGoogle Scholar
- Sotoudeh K, Hashemi F, Madjd Z, Sadeghipour A, Molanaei S, Kalantary E: The clinicopathologic association of c-MET overexpression in Iranian gastric carcinomas; an immunohistochemical study of tissue microarrays. Diagn Pathol. 2012, 7: 57-10.1186/1746-1596-7-57.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamaguchi T, Fujimori T, Tomita S, Ichikawa K, Mitomi H, Ohno K, Shida Y, Kato H: Clinical validation of the gastrointestinal NET grading system: Ki67 index criteria of the WHO 2010 classification is appropriate to predict metastasis or recurrence. Diagn Pathol. 2013, 8: 65-10.1186/1746-1596-8-65.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu X, Xiong H, Li J, He Y, Yuan X: Correlation of hK6 expression with tumor recurrence and prognosis in advanced gastric cancer. Diagn Pathol. 2013, 8: 62-10.1186/1746-1596-8-62.PubMed CentralView ArticlePubMedGoogle Scholar
- Shan L, Ying J, Lu N: HER2 expression and relevant clinicopathological features in gastric and gastroesophageal junction adenocarcinoma in a Chinese population. Diagn Pathol. 2013, 8: 76-10.1186/1746-1596-8-76.PubMed CentralView ArticlePubMedGoogle Scholar
- Yasui W, Oue N, Aung PP, Matsumura S, Shutoh M, Nakayama H: Molecular-pathological prognostic factors of gastric cancer: a review. Gastric Cancer. 2005, 8: 86-94. 10.1007/s10120-005-0320-0.View ArticlePubMedGoogle Scholar
- Corso G, Velho S, Paredes J, Pedrazzani C, Martins D, Milanezi F, Pascale V, Vindigni C, Pinheiro H, Leite M, Marrelli D, Sousa S, Carneiro F, Oliveira C, Roviello F, Seruca R: Oncogenic mutations in gastric cancer with microsatellite instability. Eur J Cancer. 2011, 47: 443-451. 10.1016/j.ejca.2010.09.008.View ArticlePubMedGoogle Scholar
- Yao D, Shi J, Shi B, Wang N, Liu W, Zhang G, Ji M, Xu L, He N, Hou P: Quantitative assessment of gene methylation and their impact on clinical outcome in gastric cancer. Clin Chim Acta. 2012, 413: 787-794. 10.1016/j.cca.2012.01.013.View ArticlePubMedGoogle Scholar
- Qu Y, Dang S, Hou P: Gene methylation in gastric cancer. Clin Chim Acta. 2013, 424C: 53-65.View ArticleGoogle Scholar
- Shi J, Yao D, Liu W, Wang N, Lv H, Zhang G, Ji M, Xu L, He N, Shi B, Hou P: Highly frequent PIK3CA amplification is associated with poor prognosis in gastric cancer. BMC Cancer. 2012, 12: 50-10.1186/1471-2407-12-50.PubMed CentralView ArticlePubMedGoogle Scholar
- Shi J, Yao D, Liu W, Wang N, Lv H, He N, Shi B, Hou P, Ji M: Frequent gene amplification predicts poor prognosis in gastric cancer. Int J Mol Sci. 2012, 13: 4714-4726. 10.3390/ijms13044714.PubMed CentralView ArticlePubMedGoogle Scholar
- Yasui W, Oue N, Kuniyasu H, Ito R, Tahara E, Yokozaki H: Molecular diagnosis of gastric cancer: present and future. Gastric Cancer. 2001, 4: 113-121. 10.1007/PL00011733.View ArticlePubMedGoogle Scholar
- Yokozaki H, Yasui W, Tahara E: Genetic and epigenetic changes in stomach cancer. Int Rev Cytol. 2001, 204: 49-95.View ArticlePubMedGoogle Scholar
- Hatefi Y: The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem. 1985, 54: 1015-1069. 10.1146/annurev.bi.54.070185.005055.View ArticlePubMedGoogle Scholar
- Wallace DC: Mitochondria and cancer. Nat Rev Cancer. 2012, 12: 685-698.PubMed CentralView ArticlePubMedGoogle Scholar
- Shadel GS: Expression and maintenance of mitochondrial DNA: new insights into human disease pathology. Am J Pathol. 2008, 172: 1445-1456. 10.2353/ajpath.2008.071163.PubMed CentralView ArticlePubMedGoogle Scholar
- Chinnery PF, Hudson G: Mitochondrial genetics. Br Med Bull. 2013, 106: 135-159. 10.1093/bmb/ldt017.PubMed CentralView ArticlePubMedGoogle Scholar
- Copeland WC, Wachsman JT, Johnson FM, Penta JS: Mitochondrial DNA alterations in cancer. Cancer Invest. 2002, 20: 557-569. 10.1081/CNV-120002155.View ArticlePubMedGoogle Scholar
- Lan Q, Lim U, Liu CS, Weinstein SJ, Chanock S, Bonner MR, Virtamo J, Albanes D, Rothman N: A prospective study of mitochondrial DNA copy number and risk of non-Hodgkin lymphoma. Blood. 2008, 112: 4247-4249. 10.1182/blood-2008-05-157974.PubMed CentralView ArticlePubMedGoogle Scholar
- Hosgood HD, Liu CS, Rothman N, Weinstein SJ, Bonner MR, Shen M, Lim U, Virtamo J, Cheng WL, Albanes D, Lan Q: Mitochondrial DNA copy number and lung cancer risk in a prospective cohort study. Carcinogenesis. 2010, 31: 847-849. 10.1093/carcin/bgq045.PubMed CentralView ArticlePubMedGoogle Scholar
- Lynch SM, Weinstein SJ, Virtamo J, Lan Q, Liu CS, Cheng WL, Rothman N, Albanes D, Stolzenberg-Solomon RZ: Mitochondrial DNA copy number and pancreatic cancer in the alpha-tocopherol beta-arotene cancer prevention study. Cancer Prev Res (Phila). 2011, 4: 1912-1919. 10.1158/1940-6207.CAPR-11-0002.View ArticleGoogle Scholar
- Shen J, Platek M, Mahasneh A, Ambrosone CB, Zhao H: Mitochondrial copy number and risk of breast cancer: a pilot study. Mitochondrion. 2010, 10: 62-68. 10.1016/j.mito.2009.09.004.View ArticlePubMedGoogle Scholar
- Qu F, Liu X, Zhou F, Yang H, Bao G, He X, Xing J: Association between mitochondrial DNA content in leukocytes and colorectal cancer risk: a case–control analysis. Cancer. 2011, 117: 3148-3155. 10.1002/cncr.25906.View ArticlePubMedGoogle Scholar
- Xing J, Chen M, Wood CG, Lin J, Spitz MR, Ma J, Amos CI, Shields PG, Benowitz NL, Gu J, de Andrade M, Swan GE, Wu X: Mitochondrial DNA content: its genetic heritability and association with renal cell carcinoma. J Natl Cancer Inst. 2008, 100: 1104-1112. 10.1093/jnci/djn213.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu CW, Yin PH, Hung WY, Li AF, Li SH, Chi CW, Wei YH, Lee HC: Mitochondrial DNA mutations and mitochondrial DNA depletion in gastric cancer. Genes, Chromosomes Cancer. 2005, 44: 19-28. 10.1002/gcc.20213.View ArticlePubMedGoogle Scholar
- Lee HC, Yin PH, Lin JC, Wu CC, Chen CY, Wu CW, Chi CW, Tam TN, Wei YH: Mitochondrial genome instability and mtDNA depletion in human cancers. Ann N Y Acad Sci. 2005, 1042: 109-122. 10.1196/annals.1338.011.View ArticlePubMedGoogle Scholar
- Liao LM, Baccarelli A, Shu XO, Gao YT, Ji BT, Yang G, Li HL, Hoxha M, Dioni L, Rothman N, Zheng W, Chow WH: Mitochondrial DNA copy number and risk of gastric cancer: a report from the Shanghai Women’s Health Study. Cancer Epidemiol Biomarkers Prev. 2011, 20: 1944-1949. 10.1158/1055-9965.EPI-11-0379.PubMed CentralView ArticlePubMedGoogle Scholar
- Hou P, Liu D, Shan Y, Hu S, Studeman K, Condouris S, Wang Y, Trink A, El-Naggar AK, Tallini G, Vasko V, Xing M: Genetic alterations and their relationship in the phosphatidylinositol 3-kinase/Akt pathway in thyroid cancer. Clin Cancer Res. 2007, 13: 1161-1170. 10.1158/1078-0432.CCR-06-1125.View ArticlePubMedGoogle Scholar
- Wallace DC: A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet. 2005, 39: 359-407. 10.1146/annurev.genet.39.110304.095751.PubMed CentralView ArticlePubMedGoogle Scholar
- Verschoor ML, Ungard R, Harbottle A, Jakupciak JP, Parr RL, Singh G: Mitochondria and cancer: past, present, and future. Biomed Res Int. 2013, 2013: 612369-PubMed CentralView ArticlePubMedGoogle Scholar
- Robin ED, Wong R: Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J Cell Physiol. 1988, 136: 507-513. 10.1002/jcp.1041360316.View ArticlePubMedGoogle Scholar
- Clay Montier LL, Deng JJ, Bai Y: Number matters: control of mammalian mitochondrial DNA copy number. J Genet Genomics. 2009, 36: 125-131. 10.1016/S1673-8527(08)60099-5.View ArticlePubMedGoogle Scholar
- Burgart LJ, Zheng J, Shu Q, Strickler JG, Shibata D: Somatic mitochondrial mutation in gastric cancer. Am J Pathol. 1995, 147: 1105-1111.PubMed CentralPubMedGoogle Scholar
- Tamura G, Nishizuka S, Maesawa C, Suzuki Y, Iwaya T, Sakata K, Endoh Y, Motoyama T: Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients. Eur J Cancer. 1999, 35: 316-319. 10.1016/S0959-8049(98)00360-8.View ArticlePubMedGoogle Scholar
- Lin CS, Chang SC, Wang LS, Chou TY, Hsu WH, Wu YC, Wei YH: The role of mitochondrial DNA alterations in esophageal squamous cell carcinomas. J Thorac Cardiovasc Surg. 2010, 139: 189-197. 10.1016/j.jtcvs.2009.04.007.View ArticlePubMedGoogle Scholar
- Lin CS, Lee HT, Lee SY, Shen YA, Wang LS, Chen YJ, Wei YH: High mitochondrial DNA copy number and bioenergetic function are associated with tumor invasion of esophageal squamous cell carcinoma cell lines. Int J Mol Sci. 2012, 13: 11228-11246. 10.3390/ijms130911228.PubMed CentralView ArticlePubMedGoogle Scholar
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