Low copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in early-stage laryngeal cancer patients
- Siwen Dang†1,
- Yiping Qu†1,
- Jing Wei1,
- Yuan Shao2,
- Qi Yang1,
- Meiju Ji3,
- Bingyin Shi1 and
- Peng Hou1Email author
© Dang et al.; licensee BioMed Central Ltd. 2014
Received: 13 December 2013
Accepted: 31 December 2013
Published: 5 February 2014
Alterations in mitochondrial DNA (mtDNA) copy number have been widely reported in various human cancers, and been considered to be an important hallmark of cancers. However, little is known about the value of copy number variations of mtDNA in the prognostic evaluation of laryngeal cancer.
Design and methods
Using real-time quantitative PCR method, we investigated mtDNA copy number in a cohort of laryngeal cancers (n =204) and normal laryngeal tissues (n =40), and explored the association of variable mtDNA copy number with clinical outcomes of laryngeal cancer patients.
Our data showed that the relative mean mtDNA content was higher in the laryngeal cancer patients (11.91 ± 4.35 copies) than the control subjects (4.72 ± 0.70 copies). Moreover, we found that mtDNA content was negatively associated with cigarette smoking (pack-years), tumor invasion, and TNM stage. Notably, variable mtDNA content did not affect overall survival of laryngeal cancer patients. However, when the patients were categorized into early-stage and late-stage tumor groups according to TNM stage, we found that low mtDNA content was strongly associated with poor survival in the former, but not in the latter.
The present study demonstrated that low mtDNA content was strongly correlated with some of clinicopathological characteristics, such as cigarette smoking, tumor invasion and TNM stage. In addition, we found a strong link between low mtDNA content and worse survival of the patients with early-stage tumors. Taken together, low copy number of mtDNA may be a useful poor prognostic factor for early-stage laryngeal cancer patients.
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1841771572115955
KeywordsLaryngeal cancer Mitochondrial DNA (mtDNA) Copy number Real-time quantitative PCR Clinical outcomes
Laryngeal cancer represents the second most common malignancy of the head and neck worldwide . Given the fundamental role the larynx plays in human speech and communication, determining the optimal management of laryngeal cancer is critical. Despite multiple and aggressive therapeutic interventions, there has been no fundamental improvement in the 5-year survival rates of the patients over the past decades [1, 2]. Current methods used to predict the outcome of laryngeal cancer patients include some clinicopathological factors, including TNM stage, differentiation grade and metastasis, as well as several biomarkers, such as hTERC amplification and VEGF expression [3–11].
In recent years, copy number variations of mitochondrial DNA (mtDNA) have been reported in various human cancers, including head and neck cancer [12, 13]. The major role of mitochondria, which are organelles found in all nucleated cells, is to generate cellular adenosine triphosphate (ATP) through oxidative phosphorylation . Human mitochondrial DNA (mtDNA) is a 16.5-kb double-stranded DNA molecule, which contains genes coding for 13 polypeptides of the respiratory chain, 22 tRNAs and 2 rRNAs . Mutations in the displacement loop (D-loop), a noncoding region essential for the replication and transcription of mtDNA, can cause a reduction in mtDNA copy number or altered mtDNA gene expression [16, 17]. Cellular mtDNA content typically ranges from hundreds to more than 10 000 copies per cell, varying across different cell types. Because of lack of introns, inability to bind to histones, and inefficient mtDNA proofreading and DNA repair systems, mtDNA is more susceptible to oxidative damage than nuclear DNA (nDNA) . In general, mtDNA copy number in the cells is not under stringent control; and various internal or external factors associated with ATP demand may influence its level, such as hypoxia (a strict microenvironment that carcinoma cells can proliferate fast and survive in). Until now, there have been only a few studies suggesting an increase in mtDNA copy number in laryngeal cancers as compared with normal laryngeal tissues . However, the association of mtDNA content with clinical outcomes of laryngeal cancer patients remains largely unknown.
In this study, using real-time quantitative PCR method, we investigated mtDNA copy number in a large cohort of laryngeal cancer tissues, and further explored the association of mtDNA content with clinical outcomes of laryngeal cancer patients.
Material and methods
Clinicopathological characteristics of laryngeal cancer patients
No. of patients (%)
Lymph node metastasis (LNM)
Genomic DNA was extracted from paraffin-embedded tissues as previously described . Briefly, after a treatment for 12 h at room temperature with xylene to remove paraffin, the tissues were then subjected to digestion with 1% sodium dodecyl sulfate (SDS) and proteinase K at 48°C for 48 h, with addition of several spiking aliquots of concentrated proteinase K to facilitate digestion. DNA was subsequently isolated using a 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
Reverse primer sequence
(5′ → 3′)
(5′ → 3′)
(5′ → 3′)
The copy number of mtDNA between laryngeal cancer and normal laryngeal tissues were compared by the Mann–Whitney U test. Association of mtDNA copy number with clinicopathological characteristics was univariately assessed using the SPSS statistical package (version 11.5, Chicago, IL). Multivariate models were then developed that adjusted for the most important covariates, including gender, age, smoking history, and TNM stage. The day of primary tumor surgery to the day of death or last clinical follow-up was used to determine the survival length. The Kaplan–Meier method was used for survival analysis grouping with mtDNA copy number. Differences between curves were analyzed using the log-rank test. Multivariate Cox regression analysis was employed to evaluate the impact of variable 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 laryngeal cancer
Association of variable mtDNA copy number with clinicopathological characteristics of laryngeal cancer patients
Copy number variations of mtDNA in laryngeal cancer — univariate associations with clinicopathological characteristics
Copy number <4.02
Copy number >5.42
OR* (95% CI)
OR* (95% CI)
Male vs. Female
Copy number variations of mtDNA in early-stage laryngeal cancer — univariate associations with clinicopathological characteristics
Copy number <4.02
Copy number >5.42
Copy number variations of mtDNA in late-stage laryngeal cancer — univariate associations with clinicopathological characteristics
Copy number <4.02
Copy number >5.42
Male vs. Female
Copy number variations in laryngeal cancer — multivariable models assessing gender, age, smoking history, and TNM stage
Copy number <4.02
Copy number >5.42
Impact of variable mtDNA content on poor survival of laryngeal cancer patients
Prognostic value of clinicopathological factors and copy number variation of mtDNA in univariate and multivariate Cox regression analysis (n =204)
Hazard Ratio (95% CI)
Hazard Ratio (95% CI)
4.02 ~ 5.42
Lymph node metastasis
Given the essential involvement of mitovhondria in cellular bioenergetic and in many important physiological processes, including metabolism, signaling, apoptosis, cell cycle, and differentiation, it is not surprising that mitochondria dysfunction can contribute to the development of various human disease, inculding cancers [25, 26]. Unlike nuclear DNA, mtDNA is present at a consistently high level in normal cells , and the mitochondrial genome lacks introns and protective histones. As a consequence, the mutation rate of mtDNA is substantially greater than that of nuclear genomic DNA [26, 28]. It has been well documented that excess reactive oxygen species (ROS) acts not only mutagens and initiators of oxidative stress, but are also significant inter- and intra-cellular signaling molecules, contribute to a number of nuclear and mitochondrial changes in gene expression [29, 30]. Moreover, mitochondrial has been reported to be highly susceptible to ROS . ROS is thus often considered as an important determinant of cancer risk.
Mitochondrial aberrants, including mtDNA somatic mutations and copy number variations, have been frequently reported in various human cancers [13, 21, 24, 32–35], including laryngeal cancer . However, the prognostic value of copy number variations of mtDNA in laryngeal cancer patients remains to be explored. In this study, we investigated relative mtDNA copy number in a cohort of laryngeal cancers and normal larygeal tissues (control subjects) by using real-time quantitative PCR method. Our data showed that relative mean mtDNA content was higher in laryngeal cancer patients than control subjects. In line with this finding, a previous study has reported the increased mtDNA copy number in laryngeal cancer tissues as compared with paracancerous normal tissues, and demonstrated that mtDNA copy number in the cases which carried D-loop mutations was significantly higher than that of the negative cases . These observatiosns suggest that the increase in mtDNA copy number, together with a high frequence of mtDNA mutations or polymorphisms in D-loop region, may play a key role in larynx carcinogenesis. Moreover, we did not find the associations of mtDNA content with gender, age, differentiation, and survival status. However, our data demonstrated that mtDNA copy number was significantly negatively associated with cigarette smoking, as supported by a previous study that cigarette smoking could modulate the mtDNA content in a negative manner in the lung tissues of the smokers . We also found that low copy number of mtDNA was significantly positively associated with tumor invasion depth. In addition, our data showed that mtDNA copy number was closely associated with TNM stage. The patients with late-stage tumors had a significant lower mtDNA copy number than those with early-stage tumors. These findings suggest that variable mtDNA content, particularly low mtDNA content, may contribute to poor prognosis of laryngeal cancer patients.
To further explore the association of mtDNA content with clinical outcomes of laryngeal cancer patients, we categorized the patients into three groups by using two cutoff points (the lower and upper limit of 95% confidence interval for all control subjects), including low mtDNA content (<4.02 copies), medium mtDNA content or reference (4.02-5.42 copies) and high mtDNA content (>5.42 copies). Our data showed that mtDNA content was significantly negatively associated with TNM stage in laryngeal cancer patients, as supported by a previous study that positive correlation was found with decrease in mtDNA content with the increase in tumor stages in oral cancer . Moreover, our data also showed that low mtDNA content was closely associated with an increased risk of lymph node metastasis for laryngeal cancer patients as compared to reference. When the patients were further categorized into early-stage and late-stage groups based on TNM stage, a significantly negative relationship between mtDNA content and smoking history was only found in the patient with early-stage tumors, but not in those with late-stage tumors. These observations suggest that copy number variations of mtDNA may be invloved in laryngeal cancer progression. Next, we investigated the effect of variable mtDNA content on poor survival of laryngeal cancer patients. The data showed that both low and high mtDNA content were not associated with overall survival of cancer patients. However, further analysis revealed that low mtDNA content was closely associated with poor survival only in the patients with early-stage tumors, but not in those with late-stage tumors. These data implicate that low mtDNA content can predict worse survival in the early-stage laryngeal cancer patients, as supported by a previous study that low mtDNA copy number may result in a stronger tolerance to hypoxia, and make tumor cells reduce the dependence of mitochondrial oxidative phosphorylation and get the energy for tumor progression mainly from anaerobic metabolism, further contribute to tumor cell invasion and survival under hypoxic conditions .
In conclusion, we investigated relative mtDNA content in a large cohort of laryngeal cancers, and demonstrated that mtDNA content was negatively associated with cigarette smoking, tumor invasion and TNM stage. In addition, low mtDNA content predicts worse survival for the patients with early-stage tumors. Therefore, variable mtDNA content may be used as a valuable biomarker to evaluate clinical outcomes of early-stage laryngeal cancer patients.
World Health Organization
Sodium dodecyl sulfate
Mitochondrially encoded NADH dehydrogenase 1
Reactive oxygen species.
This work was supported by the National Natural Science Foundation of China (No. 81171969 and 81272933), 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.PubMedView ArticleGoogle Scholar
- Choong N, Vokes E: Expanding role of the medical oncologist in the management of head and neck cancer. CA Cancer J Clin. 2008, 58: 32-53. 10.3322/CA.2007.0004.PubMedView ArticleGoogle Scholar
- Almadori G, Bussu F, Paludettii G: Predictive factors of neck metastases in laryngeal squamous cell carcinoma. Towards an integrated clinico-molecular classification. Acta Otorhinolaryngol Ital. 2006, 26: 326-334.PubMedPubMed CentralGoogle Scholar
- Gourin CG, Conger BT, Sheils WC, Bilodeau PA, Coleman TA, Porubsky ES: The effect of treatment on survival in patients with advanced laryngeal carcinoma. Laryngoscope. 2009, 119: 1312-1317. 10.1002/lary.20477.PubMedView ArticleGoogle Scholar
- Johansen LV, Grau C, Overgaard J: Laryngeal carcinoma–multivariate analysis of prognostic factors in 1252 consecutive patients treated with primary radiotherapy. Acta Oncol. 2003, 42: 771-778. 10.1080/02841860310017595.PubMedView ArticleGoogle Scholar
- Lohynska R, Slavicek A, Bahanan A, Novakova P: Predictors of local failure in early laryngeal cancer. Neoplasma. 2005, 52: 483-488.PubMedGoogle Scholar
- Marioni G, Marchese-Ragona R, Cartei G, Marchese F, Staffieri A: Current opinion in diagnosis and treatment of laryngeal carcinoma. Cancer Treat Rev. 2006, 32: 504-515. 10.1016/j.ctrv.2006.07.002.PubMedView ArticleGoogle Scholar
- Nguyen-Tan PF, Le QT, Quivey JM, Singer M, Terris DJ, Goffinet DR, Fu KK: Treatment results and prognostic factors of advanced T3–4 laryngeal carcinoma: the University of California, San Francisco (UCSF) and Stanford University Hospital (SUH) experience. Int J Radiat Oncol Biol Phys. 2001, 50: 1172-1180. 10.1016/S0360-3016(01)01538-3.PubMedView ArticleGoogle Scholar
- Yu L, Xiao-li D, Cheng T, Hong-gang L: Human telomerase RNA component (hTERC) gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx. Diagn Pathol. 2002, 7: 34-Google Scholar
- Yurdanur S, Seda G, Sinan A, Filiz K, Bedri K: Poor prognostic clinicopathologic features correlate with VEGF expression but not with PTEN expression in squamous cell carcinoma of the larynx. Diagn Pathol. 2010, 5: 35-10.1186/1746-1596-5-35.View ArticleGoogle Scholar
- Hans-Ullrich V, Matthias S, Sylvia H, Philipp S, Rudolf H, Hans M-H, Matthias E: Differential diagnosis of laryngeal spindle cell carcinoma and inflammatory myofibroblastic tumor ? report of two cases with similar morphology. Diagn Pathol. 2007, 2: 1-10.1186/1746-1596-2-1.View ArticleGoogle Scholar
- Jiang WW, Masayesva B, Zahurak M, Carvalho AL, Rosenbaum E, Mambo E, Zhou S, Minhas K, Benoit N, Westra WH, Alberg A, Sidransky D, Koch W, Califano J: Increased mitochondrial DNA content in saliva associated with head and neck cancer. Clin Cancer Res. 2005, 11: 2486-2491. 10.1158/1078-0432.CCR-04-2147.PubMedView ArticleGoogle Scholar
- Jiang WW, Rosenbaum E, Mambo E, Zahurak M, Masayesva B, Carvalho AL, Zhou S, Westra WH, Alberg AJ, Sidransky D, Koch W, Califano JA: Decreased mitochondrial DNA content in posttreatment salivary rinses from head and neck cancer patients. Clin Cancer Res. 2006, 12: 1564-1569. 10.1158/1078-0432.CCR-05-1471.PubMedView ArticleGoogle 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.PubMedView ArticleGoogle Scholar
- Fernández-Silva P, Enriquez JA, Montoya J: Replication and transcription of mammalian mitochondrial DNA. Exp Physiol. 2003, 88: 41-56. 10.1113/eph8802514.PubMedView ArticleGoogle 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.PubMedPubMed CentralView ArticleGoogle Scholar
- Chinnery PF, Hudson G: Mitochondrial genetics. Br Med Bull. 2013, 106: 135-159. 10.1093/bmb/ldt017.PubMedPubMed CentralView ArticleGoogle Scholar
- Gredilla R, Bohr VA, Stevnsner T: Mitochondrial DNA repair and association with aging–an update. Exp Gerontol. 2010, 45: 478-488. 10.1016/j.exger.2010.01.017.PubMedPubMed CentralView ArticleGoogle Scholar
- Guo W, Yang D, Xu H, Zhang Y, Huang J, Yang Z, Chen X, Huang Z: Mutations in the D-loop region and increased copy number of mitochondrial DNA in human laryngeal squamous cell carcinoma. Mol Biol Rep. 2013, 40: 13-20. 10.1007/s11033-012-1939-7.PubMedView ArticleGoogle Scholar
- Shi J, Zhang G, Yao D, Liu W, Wang N, Ji M, He N, Shi B, Hou P: Prognostic significance of aberrant gene methylation in gastric cancer. Am J Cancer Res. 2012, 2: 116-129.PubMedPubMed CentralGoogle Scholar
- Zhang G, Qu Y, Dang S, Yang Q, Shi B, Hou P: Variable copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in advanced gastric cancer patients. Diagn Pathol. 2013, 8: 173-10.1186/1746-1596-8-173.PubMedPubMed CentralView 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.PubMedPubMed CentralView ArticleGoogle Scholar
- Xing J, Chen M, Wood CG, Lin J, Spitz MR, Ma J, Amos C, 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.PubMedPubMed CentralView ArticleGoogle 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.PubMedPubMed CentralView ArticleGoogle 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.PubMedPubMed CentralView ArticleGoogle Scholar
- Wallace DC: Mitochondria and cancer. Nat Rev Cancer. 2012, 12: 685-698. 10.1038/nrc3365.PubMedPubMed CentralView ArticleGoogle 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.PubMedView ArticleGoogle Scholar
- Copeland WC, Wachsman JT, Johnson FM, Penta JS: Mitochondrial DNA alterations in cancer. Cancer Invest. 2002, 20: 557-569. 10.1081/CNV-120002155.PubMedView ArticleGoogle Scholar
- Verschoor ML, Wilson LA, Singh G: Mechanisms associated with mitochondrial-generated reactive oxygen species in cancer. Can J Physiol Pharmacol. 2010, 88: 204-219. 10.1139/Y09-135.PubMedView ArticleGoogle 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-PubMedPubMed CentralView ArticleGoogle Scholar
- Kroemer G: Mitochondrial control of apoptosis: an introduction. Biochem Biophys Res Commun. 2003, 304: 433-435. 10.1016/S0006-291X(03)00614-4.PubMedView ArticleGoogle Scholar
- Zheng S, Qian P, Li F, Qian G, Wang C, Wu G, Li Q, Chen Y, Li J, Li H, He B, Ji F: Association of mitochondrial DNA variations with lung cancer risk in a Han Chinese population from southwestern China. PLoS One. 2012, 7: e31322-10.1371/journal.pone.0031322.PubMedPubMed CentralView ArticleGoogle Scholar
- Masuda S, Kadowaki T, Kumaki N, Tang X, Tokuda Y, Yoshimura S, Takekoshi S, Osamura RY: Analysis of gene alterations of mitochondrial DNA D-loop regions to determine breast cancer clonality. Br J Cancer. 2012, 107: 2016-2023. 10.1038/bjc.2012.505.PubMedPubMed CentralView ArticleGoogle Scholar
- Xu E, Sun W, Gu J, Chow WH, Ajani JA, Wu X: Association of mitochondrial DNA copy number in peripheral blood leukocytes with risk of esophageal adenocarcinoma. Carcinogenesis. 2013, 34: 2521-2524. 10.1093/carcin/bgt230.PubMedPubMed CentralView ArticleGoogle Scholar
- Warowicka A, Kwasniewska A, Gozdzicka-Jozefiak A: Alterations in mtDNA: a qualitative and quantitative study associated with cervical cancer development. Gynecol Oncol. 2013, 129: 193-198. 10.1016/j.ygyno.2013.01.001.PubMedView ArticleGoogle Scholar
- Lee HC, Lu CY, Fahn HJ, Wei YH: Aging- and smoking-associated alteration in the relative content of mitochondrial DNA in human lung. FEBS Lett. 1998, 441: 292-296. 10.1016/S0014-5793(98)01564-6.PubMedView ArticleGoogle Scholar
- Mondal R, Ghosh SK, Choudhury JH, Seram A, Sinha K, Hussain M, Laskar RS, Rabha B, Dey P, Ganguli S, Nathchoudhury M, Talukdar FR, Chaudhuri B, Dhar B: Mitochondrial DNA copy number and risk of oral cancer: a report from Northeast India. PLoS One. 2013, 8: e57771-10.1371/journal.pone.0057771.PubMedPubMed CentralView ArticleGoogle Scholar
- Stoeltzing O, McCarty MF, Wey JS, Fan F, Liu W, Belcheva A, Bucana CD, Semenza GL, Ellis LM: Role of hypoxia-inducible factor 1alpha in gastric cancer cell growth, angiogenesis, and vessel maturation. J Natl Cancer Inst. 2004, 96: 946-956. 10.1093/jnci/djh168.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.