Introduction

Oncology Letters

1792-1074 1792-1082

D.A. Spandidos

10.3892/ol.2018.9515

OL-0-0-9515

Articles

Clinical application of plasma mitochondrial DNA content in patients with lung cancer

Chen

Jianhua

1 Zhang

Lemeng

1 Yu

Xun

1 Zhou

Hui

2 Luo

Yongzhong

1 Wang

Wei

1 Wang

Lijing

1Thoracic Medicine Department 1, Hunan Cancer Hospital Affiliated to Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China 2Hematology Department, Hunan Cancer Hospital Affiliated to Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China 3Department of Geriatrics, Respiratory Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China

Correspondence to: Dr Lemeng Zhang, Thoracic Medicine Department 1, Hunan Cancer Hospital Affiliated to Xiangya Medical School, Central South University, 283 Tongzipo Road, Changsha, Hunan 410013, P.R. China, E-mail: 497730787@qq.com

12 2018

27 09 2018

16 6 7074 7081 14012018 29082018

2018

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Alterations of mitochondrial DNA (mtDNA) have been identified in several types of solid tumor. However, to the best of our knowledge, the clinical significance of plasma mtDNA content in lung cancer remains unknown. Thus, the current study explored the diagnostic and prognostic value of plasma mtDNA quantification in patients with lung cancer. Plasma mtDNA copy numbers of patients with lung cancer (n=128) and healthy individuals (n=107) were quantified by quantitative polymerase chain reaction. Plasma mtDNA copy numbers in patients and healthy controls were 0.89×10⁴ and 1.37×10⁴ copies/µl, respectively (P<0.0001). Furthermore, lower plasma mtDNA content was associated with tumor size, lymph node metastases, distant metastases and serum carcinoembryonic antigen levels (P<0.05), but was not associated with pathological type, age, sex or main driver gene mutation status (P>0.05). Plasma mtDNA facilitated the detection of lung cancer at a threshold of 1.19×10⁴ copies/µl with a sensitivity of 71.1% and specificity of 70.1%, as determined by receiver operating characteristic curve analysis. Advanced stage (III and IV) patients with a lower mtDNA copy number (cutoff: 1.02×10⁴ copies/µl) tended to exhibit poorer prognosis (P<0.05). These results indicated that plasma mtDNA content is a promising and complementary candidate with tissue mtDNA for diagnosis and prognostic prediction for lung cancer.

lung cancer mitochondrial DNA quantitative polymerase chain reaction prognosis diagnosis

Introduction

Mitochondria are pivotal organelles responsible for cellular energy metabolism, free radical formation, calcium homeostasis and the intrinsic apoptotic pathway (1). Each mitochondrion has its own extra-chromosomal genome, termed mitochondrial DNA (mtDNA). The number of mtDNA copies is tightly controlled and remains relatively stable, which is important for maintaining homeostasis (2). However, mutations and alterations of mtDNA can lead to abnormalities in energy metabolism and oxidative stress, which may be involved in carcinogenesis (3–5).

Alterations of mtDNA content have been reported in human malignancies, but with contradictory findings. Increased mtDNA copy numbers have been identified in endometrial adenocarcinoma cells (6) and esophageal squamous cell carcinoma (7). By contrast, decreased mtDNA copy numbers have been identified in lung cancer (8,9), colorectal cancer (10), breast cancer (11) and renal cell carcinoma (12). Increased circulating mtDNA could be a compensatory response to decreased respiratory function (13) and may be associated with increased tumor burden, increased mitochondrial damage, or release from apoptotic and/or necrotic cancer cells in cancerous lesions (14,15). Conversely, decreased mtDNA content may be a consequence of exposure to excessive reactive oxygen species (16).

Compared with nuclear DNA, the quantification of mtDNA has several advantages, including short length, simple molecular structure and abundance. However, assessing mtDNA in tumor tissues requires invasive techniques. For dynamic patient management, a non-invasive diagnostic and prognostic biomarker is required (14). The monitoring of circulating mtDNA is non-invasive, convenient and suitable for dynamic observation, which makes it an ideal candidate for clinical application. Circulating mtDNA has been considered a biomarker in numerous types of tumor. As with tissue mtDNA, serum mtDNA values vary among malignancies and are increased in patients with urologic malignancies (17), epithelial ovarian cancer (18), gastric cancer (19) and testicular cancer (20), but are decreased in patients with breast cancer (21) and Ewing's sarcoma (22).

The high morbidity and mortality of lung cancer (23) has prompted interest in the study of circulating mtDNA. Hou et al (24) have reported that serum mtDNA is increased in patients with lung cancer. However, associations between mtDNA content and tumor size, lymph node metastases, distant metastases, driver gene mutation status, chemo-sensitivity and prognosis are still largely unknown. Given the numerous potential interactions between mtDNA and carcinogenesis, the current study hypothesized that alterations of plasma mtDNA content may be a biomarker for the presence and development of lung cancer. Thus, using quantitative polymerase chain reaction (qPCR) assays, the current study measured the copy number of plasma mtDNA in patients with lung cancer. Understanding the associations between plasma mtDNA content and the clinicopathological characteristics and prognosis of lung cancer may allow for a non-invasive and dynamic means of evaluating the disease.

Materials and methods <sec> <title>Patients, study design and sample collection

A total of 128 patients with lung cancer and 107 well-matched healthy controls from Medical Examination Center were included in the current study. In the lung cancer group, there are 72 males and 56 female patients with a median age of 55.2±7.8 years. In the healthy control group, there are 57 male and 50 female individuals with a median age of 53.5±8.8 years. The patients with lung cancer were diagnosed from January to December 2015 at the Hunan Cancer Hospital of China (Changsha, China). The present study was approved by the Ethics Committee of Hunan Cancer Hospital. Informed consent was obtained from healthy controls and each patient according to protocols approved by the hospital's Ethics Committees. None of the patients had received pre-operative radiotherapy or chemotherapy. The exclusion criteria included autoimmune diseases, inherited diseases, mitochondrial-related diseases, or patients who refused to provide informed consent. Whole blood was collected in EDTA anticoagulant tubes and samples were centrifuged at 400 × g at 4°C for 2 h. The plasma samples were collected and stored at −80°C until use. A case-control study was conducted to evaluate the associations between mtDNA copy number and clinical characteristics, and to retrospectively explore the diagnostic and prognostic value of mtDNA content. The central laboratory of Hunan Cancer Hospital measured serum tumor biomarkers. The TNM classification was according to National Comprehensive Cancer Network Classification Standard; Seventh Edition (25). The following categories of patients were enrolled for analysis of progression-free survival (PFS): Advanced lung cancer (stage III and IV); Eastern Cooperative Oncology Group performance-status score of 0 or 1 (on a 5-point scale) (26); and measurable disease according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (27). Enrolled patients had received no primary systemic chemotherapy for advanced or metastatic disease. Epidermal growth factor receptor (EGFR) mutation was detected by direct sequencing and anaplastic lymphoma kinase (ALK) mutation was detected by immunohistochemistry as described previously (28). Patients with driver gene mutations who had received EGFR-TKI or crizotinib were excluded from analysis of PFS. PFS was defined as the length of time during and following primary treatment of lung cancer when a patient lived with the disease but without progression, as demonstrated by radiological and clinical examinations.

DNA isolation and qPCR

Total plasma DNA was isolated with a QIAamp DNA Blood Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. The mtDNA content was measured using qPCR as described previously (29). The mtDNA plasmid was constructed by YRBIO (Changsha, China) and a standard curve was generated using six dilutions of DNA (10¹−10⁶ copies/µl). The ratio of mitochondrial ND1 to human 36B4 was used. Forward ND1 primer: 5′-CCCTAAAACCCGCCACATCT-3′; reverse ND1 primer: 5′-GAGCGATGGTGAGAGCTAAGGT-3′; forward human 36B4 primer: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; reverse human 36B4 primer: 5′-CCCATTCTATCATCAACGGGTACAA-3′. qPCR was applied to 20-µl reaction volumes containing 2 µl DNA, 10 µl PCR Master Mix (Toyobo Life Science, Osaka, Japan), 4 µl primers, 3.6 µl double-distilled water and 0.4 µl ROX dye (Qiagen, Inc., Valencia, CA, USA). PCR was performed as follows: 95°C for 60 sec, followed by 95°C for 15 sec and 60°C for 45 sec, repeated for 40 cycles. Relative gene expression was calculated using the comparative 2^=ΔΔCq method (30).

Statistical analysis

The SPSS statistical package was used for all analyses (version 22.0, IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation. The Kolomogorov-Smirnov test was used to check whether the samples independent exhibited a normal distribution. For normal distributions, a paired Student's t-test was used for two groups, and one-way analysis of variance followed by Tukey's post-hoc test was used for comparison of multiple groups. For variables not in a normal distribution, unpaired samples were compared by use of the Mann-Whitney U test, and multiple independent samples were compared with the Kruskall-Wallis H test with Tukey's post-hoc test. A receiver operating characteristic (ROC) curve was used to analyze the diagnostic applicability of plasma mtDNA with the Youden index for identification of the optimal cut-off point. The Kaplan-Meier method was used for prognostic analysis. The log-rank test was used. Spearman's correlation coefficient was used to calculate the correlation between carcinoembryonic antigen (CEA) and mtDNA copy number. It also was used to analyze the correlation with age. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Characteristics of the study population

The characteristics of the study population are listed in Table I. A total of 128 patients with lung cancer and 107 healthy controls were enrolled, and they were well matched in terms of sex, age, and smoking history. The distributions of tumor size, lymph node metastases and distant metastases are listed. Of the cancer cases, 32 were TNM stage I, 20 were stage II, 34 were stage III and 42 were stage IV. Of the cancer cases, 54 were adenocarcinoma, 44 were squamous cell carcinoma and 30 were small-cell lung cancer. A total of 29 patients had epidermal growth factor receptor (EGFR) mutations and 9 had an anaplastic lymphoma kinase (ALK) fusion.

Distribution of plasma mtDNA content in patients with lung cancer and healthy controls

A qPCR assay was performed to explore the distribution of plasma mtDNA content in patients with lung cancer and healthy control subjects. As illustrated in Fig. 1, plasma mtDNA content in patients with lung cancer was significantly lower compared with that in healthy controls (0.89×10⁴ and 1.37×10⁴ copies/µl, respectively; P<0.0001).

Associations between plasma mtDNA content and clinicopathological characteristics of lung cancer

The associations between plasma mtDNA content and clinicopathological characteristics of lung cancers were also evaluated. As illustrated in Fig. 2A-E, plasma mtDNA content was negatively associated with tumor size (T3 and T4 vs. T1, P<0.05), lymph node metastases (N1-3 vs. N0, P<0.05), distant metastases (M1 vs. M0, P<0.0001) and TNM stage (III and IV vs. I, P<0.01; III/IV vs. I/II, P<00001). As illustrated in Fig. 2F, mtDNA contents were significantly higher in patients with smoking history compared with in non-smokers (P=0.0029). However, mtDNA contents were not associated with pathological type (Fig. 3A) or sex (Fig. 3B), and were not correlated with age (Fig. 3C) (P>0.05).

Associations between plasma mtDNA content and driver gene mutation status and serum tumor biomarkers

There is a strong association between driver gene mutations and the progression of lung cancer (31). The current study analyzed plasma mtDNA content in patients with or without epidermal growth factor receptor/anaplastic lymphoma kinase (EGFR/ALK) mutations. The results revealed that plasma mtDNA content in patients with EGFR/ALK mutations was not significantly different compared with patients without EGFR/ALK mutations (P=0.0777). As demonstrated in Fig. 4B, CEA levels were negatively correlated with plasma mtDNA copy number (r=−0.3820, P<0.0001). Serum tumor biomarkers, neuron-specific enolase, carbohydrate antigen-125 and carbohydrate antigen-199, were not correlated with plasma mtDNA content (data not shown).

Diagnostic value of mtDNA in patients with lung cancer

An ROC curve was used to analyze the diagnostic applicability of plasma mtDNA. As demonstrated in Fig. 5, plasma mtDNA facilitated the detection of lung cancer at a cutoff value of 1.19×10⁴ copies/µl with a sensitivity of 71.1% and specificity of 70.1%. With this cutoff value, the diagnostic accuracy of plasma mtDNA is 71.1%, the positive predictive value is 74.2%, and the negative predictive value is 67.6%. This finding suggests promise for mtDNA as a reliable test for lung cancer.

Prognostic value of mtDNA in advanced lung cancer patients

mtDNA content has previously been associated with prognosis in patients with colon cancer and gastric cancer at an advanced stage (32,33). To explore the prognostic value of mtDNA in patients with lung cancer, the current study determined the PFS in patients with advanced stage (III and IV) cancer divided into two groups based on mtDNA cutoff point of 1.02×10⁴ copies/µl. As illustrated in Fig. 6, PFS was longer in patients with mtDNA content higher than the cutoff point compared with patients with mtDNA content lower than the cutoff point, as assessed by Kaplan-Meier curve analysis (7.8 vs. 6.9 months; P<0.05).

Discussion

The morbidity and mortality of lung cancer are still extremely high for patients with malignant tumors. There were an estimated 222,500 new cases and 155,870 deaths in the United States in 2017 (34). Lack of methods for early diagnosis and effective treatment are at least partly responsible for the low 5-year survival rate (35). Although quantitative analyses have focused on the identification of biomarkers for lung cancer, there is still a need for novel, specific biomarkers for early detection, prognosis and dynamic observation.

Energy metabolism reprogramming is an important characteristic of cancer (36). One of the underlying mechanisms is mitochondrial dysfunction caused by disorders of structure and of the mitochondrial genome (36). Abnormalities of mtDNA may lead to increased reactive oxygen species, disrupted calcium homeostasis, reprogramed metabolism and resistance to apoptosis (37). Changes in mtDNA content have been reported in malignancies, with both down- and upregulation of mtDNA content in solid tumors. Decreased mtDNA content has been associated with renal cell carcinoma (38), hepatocellular carcinoma (39) and esophageal adenocarcinoma (40). By contrast, increased mtDNA content in pancreatic cancer (41), colorectal cancer (42) and breast cancer (43) has been reported. Since mtDNA may be involved in carcinogenesis, oncologists and pulmonologists have been motivated to monitor its dynamic alterations in patients with lung cancer. Hou et al (24) reported increased serum mtDNA content, while others have revealed significantly reduced mtDNA content in lung cancer tissues (8,9). In the current study, it was identified that circulating mtDNA content was significantly lower in patients with lung cancer compared with healthy subjects. The lower mtDNA content may reflect the reduced capacity of compensatory responses to oxidative stress damage (44). The decrease in mtDNA may also be a consequence of mutation or depletion in the mtDNA D-loop caused by reactive oxygen species (45).

Alterations in somatic mtDNA copy number have been revealed to strongly correlate with clinicopathological characteristics, early diagnosis, progression, and radiotherapy and chemotherapy efficacy in malignancies (14). The current study identified that lower plasma mtDNA contents were negatively associated with tumor size, lymph node metastases, distant metastases and serum CEA level, but were not associated with age, sex, pathological type or main driver gene mutation status. The incidence of D-loop mutations of mtDNA in advanced cancer stages has been revealed to be higher when compared with patients in the early stage of disease (8), which may lead to lower mtDNA copy numbers in patients with late-stage cancer. These observations may account for the current findings that plasma mtDNA content was negatively associated with tumor size, lymph node metastases and distant metastases.

Serum tumor biomarkers including CEA have been widely used to monitor the progression of tumors (46). In the current study, a negative correlation between plasma mtDNA content and serum CEA level was revealed, which to the best of our knowledge, is the first time this correlation has been recorded. Both serum CEA levels and plasma mtDNA can be dynamically monitored for chemotherapeutic effects and disease progression (46). Thus, the combined determination of CEA and mtDNA content may improve the efficacy and prognostic ability of these tests in lung cancer. In related work, no relationship between mtDNA content and serum prostate-specific antigen levels has been identified in prostate cancer and epithelial ovarian cancer (18,47). Further studies should be performed to explore the predictive value of the combination of dynamic monitoring of CEA level and circulating mtDNA alteration during chemotherapy. However, the efficacy of chemotherapy in multiple solid tumors is affected by a number of factors (48). A perspective study, with these relevant factors well matched, is required to explore the predictive value of dynamic circulating mtDNA content.

The quantification of plasma mtDNA may be used to recognize patients with poor prognosis, similar to the lower mtDNA copy number in patients who have advanced breast cancer, glioma and prostate cancer (32,33,49,50). Lower mtDNA content has been identified to be associated with tumor progression in lung cancer tissues following chemotherapy (51). Variations in mtDNA have been reported to be associated with radiation-induced toxicity (52). The change of mtDNA content can also improve the cytotoxicity of chemotherapeutic agents. DNA damage in mitochondria alters the mitochondrial apoptotic signaling pathway, thereby promoting the survival of cancer cells and even changing their resistance to anticancer drugs (53). Radiotherapy and chemotherapy are the most important treatments for advanced lung cancer. Clinically, sensitivity to radiotherapy and chemotherapy are determining factors for prognosis. Given that mtDNA depletion affects radiotherapy and chemotherapy sensitivity (5), the current study further explored the prognostic value of lower plasma mtDNA content in advanced lung cancer patients. As previously reported (54), the current study revealed that a lower mtDNA copy number was associated with poor prognosis in patients with advanced stage cancer, which suggests that plasma mtDNA copy number is a promising prognostic candidate for lung cancer.

Further studies are required to elucidate the mechanisms and consequences of decreased mtDNA content in lung cancer carcinogenesis. The lower mtDNA copy number may alter mitochondrial gene expression and lead to decreased mitochondrial function and energy metabolism and cause increased generation of reactive oxygen species (55,56), enhanced glycolysis, and even alterations in mitochondrial bioenergetic and biosynthetic states (57). Decreased mtDNA may also promote epithelial-mesenchymal transition (58–60) and apoptosis-resistant cancer cells through the phosphoinositide 3-kinase/Akt signaling pathway (61). Changes in mtDNA copy number may explain the persistent deficiency in mitochondrial activity. Thus, the mtDNA-depleted ρ0 cell line may be used to further explore the consequences and mechanism of mtDNA deficiency in lung cancer carcinogenesis.

To the best of our knowledge, the current study is the largest that has explored the diagnostic, predictive and prognostic clinical application of plasma mtDNA content in patients with lung cancer. The current study is also the most comprehensive study of plasma mtDNA alteration in patients with lung cancer. However, further studies of specific subgroups with large sample sizes are still required to further confirm the subgroup conclusions made by the current study.

In conclusion, decreased plasma mtDNA content was associated with tumor metastatic potential and unfavorable prognosis in patients with lung cancer. Monitoring circulating mtDNA content is a promising approach for diagnosis and prognosis of lung cancer. Further studies of the epigenetic alterations of mtDNA are required to understand its downstream effectors and role in lung cancer pathogenesis. Although translating plasma mtDNA quantification into routine clinical practice may take several steps, knowledge regarding the potential applicability of circulating mtDNA quantification in the diagnosis and prognosis of lung cancer has progressed considerably.

Acknowledgements

Not applicable.

Funding

The present study was supported by grant from the National Natural Science Foundation of China (grant no. 81401631) and by grant from Key Research and Develop Program of Hunan Province, China (grant no. 2017WK2061).

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

Authors' contributions

JC and LZ conceived and designed the experiments; LZ, XY, HZ and YL performed the experiments and contributed to molecular analysis; WW and LW analyzed the data and LZ wrote the manuscript.

Ethics approval and consent to participate

The present study was approved by Department of Ethics Committee of Hunan Cancer Hospital (Changsha, China), and written informed consent was obtained from all participants.

Patient consent for publication

Informed consent for the publication or any associated images was obtained from healthy controls and patients according to protocols approved by the hospital's Ethics Committee.

Competing interests

The authors declare that they have no competing interests.

References 1

Wallace

Mitochondria and cancer

Nat Rev Cancer126856982012

10.1038/nrc3365

23001348

D'Erchia

Atlante

Gadaleta

Pavesi

Chiara

De Virgilio

Manzari

Mastropasqua

Prazzoli

Picardi

Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity

Mitochondrion2013212015

10.1016/j.mito.2014.10.005

25446395

Akhmedov

Marin-Garcia

Mitochondrial DNA maintenance: An appraisal

Mol Cell Biochem4092833052015

10.1007/s11010-015-2532-x

26286847

Hsu

Tseng

Lee

Role of mitochondrial dysfunction in cancer progression

Exp Biol Med (Maywood)241128112952016

10.1177/1535370216641787

27022139

van Gisbergen

Voets

Starmans

de Coo

Yadak

Hoffmann

Boutros

Smeets

Dubois

Lambin

How do changes in the mtDNA and mitochondrial dysfunction influence cancer and cancer therapy? Challenges, opportunities and models

Mutat Res Rev Mutat Res76416302015

10.1016/j.mrrev.2015.01.001

26041263

Wang

Liu

Xue

Tsang

Cheung

Ngan

The increase of mitochondrial DNA content in endometrial adenocarcinoma cells: A quantitative study using laser-captured microdissected tissues

Gynecol Oncol981041102005

10.1016/j.ygyno.2005.04.015

15921730

Lin

Lee

Shen

Wang

Chen

Wei

High mitochondrial DNA copy number and bioenergetic function are associated with tumor invasion of esophageal squamous cell carcinoma cell lines

Int J Mol Sci1311228112462012

10.3390/ijms130911228

23109849

Lee

Yin

Lin

Chen

Chi

Tam

Wei

Mitochondrial genome instability and mtDNA depletion in human cancers

Ann N Y Acad Sci10421091222005

10.1196/annals.1338.011

15965052

Dai

Zhang

Liu

Min

Mitochondrial genome microsatellite instability and copy number alteration in lung carcinomas

Asian Pac J Cancer Prev14239323992013

10.7314/APJCP.2013.14.4.2393

23725147

Lin

Yang

Wang

Chang

Expression of beta-F1-ATPase and mitochondrial transcription factor A and the change in mitochondrial DNA content in colorectal cancer: Clinical data analysis and evidence from an in vitro study

Int J Colorectal Dis23122312322008

10.1007/s00384-008-0539-4

18769884

Fan

Radpour

Haghighi

Kohler

Xia

Hahn

Holzgreve

Zhong

Mitochondrial DNA content in paired normal and cancerous breast tissue samples from patients with breast cancer

J Cancer Res Clin Oncol1359839892009

10.1007/s00432-008-0533-9

19125299

Meierhofer

Mayr

Foetschl

Berger

Fink

Schmeller

Hacker

Hauser-Kronberger

Kofler

Sperl

Decrease of mitochondrial DNA content and energy metabolism in renal cell carcinoma

Carcinogenesis25100510102004

10.1093/carcin/bgh104

14764459

Hosgood

IIILiu

Rothman

Weinstein

Bonner

Shen

Lim

Virtamo

Cheng

Albanes

Lan

Mitochondrial DNA copy number and lung cancer risk in a prospective cohort study

Carcinogenesis318478492010

10.1093/carcin/bgq045

20176654

Circulating cell-free mitochondrial DNA as a novel cancer biomarker: Opportunities and challenges

Mitochondrial DNA233293322012

10.3109/19401736.2012.696625

22775429

Strelkova

Abdullaev

Snigireva

Bezlepkin

Gaziev

Share of extracellular mutated mitochondrial DNA increases in plasma of lung cancer patients following radiotherapy

Biomed Khim565175252010

10.18097/pbmc20105604517

21032903

Shimura

Kunugita

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1

Cell Cycle15141014142016

10.1080/15384101.2016.1170271

27078622

Ellinger

Muller

Hauser

Heukamp

von Ruecker

Bastian

Walgenbach-Brunagel

Circulating mitochondrial DNA in serum: A universal diagnostic biomarker for patients with urological malignancies

Urol Oncol305095152012

10.1016/j.urolonc.2010.03.004

20870429

Zachariah

Schmid

Buerki

Radpour

Holzgreve

Zhong

Levels of circulating cell-free nuclear and mitochondrial DNA in benign and malignant ovarian tumors

Obstet Gynecol1128438502008

10.1097/AOG.0b013e3181867bc0

18827127

Fernandes

Michel

Camorlinga-Ponce

Gomez

Maldonado

De Reuse

Torres

Touati

Circulating mitochondrial DNA level, a noninvasive biomarker for the early detection of gastric cancer

Cancer Epidemiol Biomarkers Prev23243024382014

10.1158/1055-9965.EPI-14-0471

25159292

Ellinger

Albers

Muller

von Ruecker

Bastian

Circulating mitochondrial DNA in the serum of patients with testicular germ cell cancer as a novel noninvasive diagnostic biomarker

BJU Int10448522009

10.1111/j.1464-410X.2008.08289.x

19154496

Kohler

Radpour

Barekati

Asadollahi

Bitzer

Wight

Bürki

Diesch

Holzgreve

Zhong

Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors

Mol Cancer81052009

10.1186/1476-4598-8-105

19922604

Wan

Zou

Cell-free circulating mitochondrial DNA in the serum: A potential non-invasive biomarker for Ewing's sarcoma

Arch Med Res433893942012

10.1016/j.arcmed.2012.06.007

22728238

Siegel

Miller

Jemal

Cancer statistics, 2016

CA Cancer J Clin667302016

10.3322/caac.21332

26742998

Hou

Chen

Xue

Zheng

Chen

Clinical significance of serum mitochondrial DNA in lung cancer

Clin Biochem46147414772013

10.1016/j.clinbiochem.2013.04.009

23611926

Rusch

Asamura

Watanabe

Giroux

Rami-Porta

Goldstraw

and Members of IASLC Staging Committee

The IASLC lung cancer staging project: A proposal for a new international lymph node map in the forthcoming seventh edition of the TNM classification for lung cancer

J Thorac Oncol45685772009

10.1097/JTO.0b013e3181a0d82e

19357537

Prigerson

Bao

Shah

Paulk

LeBlanc

Schneider

Garrido

Reid

Berlin

Adelson

Chemotherapy use, performance status, and quality of life at the end of life

JAMA Oncol17787842015

10.1001/jamaoncol.2015.2378

26203912

Eisenhauer

Therasse

Bogaerts

Schwartz

Sargent

Ford

Dancey

Arbuck

Gwyther

Mooney

New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)

Eur J Cancer452282472009

10.1016/j.ejca.2008.10.026

19097774

Garassino

Cho

Kim

Mazières

Vansteenkiste

Lena

Jaime

Corral J

Gray

Powderly

Chouaid

Durvalumab as third-line or later treatment for advanced non-small-cell lung cancer (ATLANTIC): An open-label, single-arm, phase 2 study

Lancet Oncol195215362018

10.1016/S1470-2045(18)30144-X

29545095

Hann

Wan

Hann

Wang

Lai

Evans

Myers

Cell-free circulating mitochondrial DNA content and risk of hepatocellular carcinoma in patients with chronic HBV infection

Sci Rep6239922016

10.1038/srep23992

27063412

Livak

Schmittgen

Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method

Methods254024082001

10.1006/meth.2001.1262

11846609

Campbell

Alexandrov

Kim

Wala

Berger

Pedamallu

Shukla

Guo

Brooks

Murray

Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas

Nat Genet486076162016

10.1038/ng.3564

27158780

Wang

Zhu

Qiao

Zhang

High copy number of mitochondrial DNA predicts poor prognosis in patients with advanced stage colon cancer

Int J Biol Markers31e382e3882016

10.5301/jbm.5000211

27197581

Zhang

Dang

Yang

Shi

Hou

Variable copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in advanced gastric cancer patients

Diagn Pathol81732013

10.1186/1746-1596-8-173

24144008

Siegel

Miller

Jemal

Cancer Statistics, 2017

CA Cancer J Clin677302017

10.3322/caac.21387

28055103

Chen

Zheng

Baade

Zhang

Zeng

Bray

Jemal

Cancer statistics in China, 2015

CA Cancer J Clin661151322016

10.3322/caac.21338

26808342

Hanahan

Weinberg

Hallmarks of cancer: The next generation

Cell1446466742011

10.1016/j.cell.2011.02.013

21376230

Sabharwal

Schumacker

Mitochondrial ROS in cancer: Initiators, amplifiers or an Achilles' heel?

Nat Rev Cancer147097212014

10.1038/nrc3803

25342630

Hofmann

Hosgood

IIILiu

Chow

Shuch

Cheng

Lin

Moore

Lan

Rothman

Purdue

A nested case-control study of leukocyte mitochondrial DNA copy number and renal cell carcinoma in the prostate, lung, colorectal and ovarian cancer screening trial

Carcinogenesis35102810312014

10.1093/carcin/bgt495

24398668

Zhao

Yang

Liu

Yang

Zhang

Xing

Association of mitochondrial DNA content in peripheral blood leukocyte with hepatitis B virus-related hepatocellular carcinoma in a Chinese Han population

Cancer science102155315582011

10.1111/j.1349-7006.2011.01968.x

21521418

Sun

Chow

Ajani

Association of mitochondrial DNA copy number in peripheral blood leukocytes with risk of esophageal adenocarcinoma

Carcinogenesis34252125242013

10.1093/carcin/bgt230

23803692

Lynch

Weinstein

Virtamo

Lan

Liu

Cheng

Rothman

Albanes

Stolzenberg-Solomon

Mitochondrial DNA copy number and pancreatic cancer in the alpha-tocopherol beta-carotene cancer prevention study

Cancer Prev Res (Phila)4191219192011

10.1158/1940-6207.CAPR-11-0002

21859925

Liu

Zhou

Yang

Bao

Xing

Association between mitochondrial DNA content in leukocytes and colorectal cancer risk: A case-control analysis

Cancer117314831552011

10.1002/cncr.25906

21246538

Shen

Platek

Mahasneh

Ambrosone

Zhao

Mitochondrial copy number and risk of breast cancer: A pilot study

Mitochondrion1062682010

10.1016/j.mito.2009.09.004

19788937

von Kleist-Retzow

Hornig-Do

Schauen

Eckertz

Dinh

Stassen

Lottmann

Bust

Galunska

Wielckens

Impaired mitochondrial Ca2+ homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

Exp Cell Res313307630892007

10.1016/j.yexcr.2007.04.015

17509565

Lee

Hsu

Yin

Lee

Chi

Heteroplasmic mutation of mitochondrial DNA D-loop and 4977-bp deletion in human cancer cells during mitochondrial DNA depletion

Mitochondrion71571632007

10.1016/j.mito.2006.11.016

17280876

Grunnet

Sorensen

Carcinoembryonic antigen (CEA) as tumor marker in lung cancer

Lung Cancer761381432012

10.1016/j.lungcan.2011.11.012

22153832

Zhou

Zhu

Gui

Huang

Long

Wang

Chen

Yin

Jiang

Dai

Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden

PLoS One9e1094702014

10.1371/journal.pone.0109470

25279731

Lissoni

Brivio

Fumagalli

Messina

Ghezzi

Frontini

Giani

Vaghi

Ardizzoia

Gardani

Efficacy of cancer chemotherapy in relation to the pretreatment number of lymphocytes in patients with metastatic solid tumors

Int J Biol Markers191351402004

10.1177/172460080401900208

15255546

Weerts

Sieuwerts

Smid

Look

Foekens

Sleijfer

Martens

Mitochondrial DNA content in breast cancer: Impact on in vitro and in vivo phenotype and patient prognosis

Oncotarget729166291762016

10.18632/oncotarget.8688

27081694

Meng

Kim

Davis

Wagar

Thompson

Logothetis

Mitochondrial DNA copy number in peripheral blood leukocytes and the aggressiveness of localized prostate cancer

Oncotarget641988419962015

10.18632/oncotarget.5889

26515605

Lin

Wang

Tsai

Wei

Low copy number and low oxidative damage of mitochondrial DNA are associated with tumor progression in lung cancer tissues after neoadjuvant chemotherapy

Interact Cardiovasc Thorac Surg79549582008

10.1510/icvts.2008.177006

18685121

Fachal

Mosquera-Miguel

Gomez-Caamano

Sánchez-García

Calvo

Lobato-Busto

Salas

Vega

Evaluating the role of mitochondrial DNA variation to the genetic predisposition to radiation-induced toxicity

Radiother Oncol1111992052014

10.1016/j.radonc.2014.03.012

24746576

Yeung

Hurren

Nemr

Wang

Hershenfeld

Gronda

Liyanage

Augustine

Lee

Mitochondrial DNA damage by bleomycin induces AML cell death

Apoptosis208118202015

10.1007/s10495-015-1119-z

25820141

Dang

Wei

Shao

Yang

Shi

Hou

Low copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in early-stage laryngeal cancer patients

Diagn Pathol9282014

10.1186/1746-1596-9-28

24499477

Cook

Kim

Terao

Gotoh

Higuchi

Consumption of oxygen: A mitochondrial-generated progression signal of advanced cancer

Cell death Dis3e2582012

10.1038/cddis.2011.141

22258408

Vermulst

Bielas

Loeb

Quantification of random mutations in the mitochondrial genome

Methods462632682008

10.1016/j.ymeth.2008.10.008

18948200

Lin

Lee

Pan

Chiu

Wei

Role of mitochondrial DNA copy number alteration in human renal cell carcinoma

Int J Mol Sci172016

10.3390/ijms17060814

Guha

Srinivasan

Ruthel

Kashina

Carstens

Mendoza

Khanna

Van Winkle

Avadhani

Mitochondrial retrograde signaling induces epithelial-mesenchymal transition and generates breast cancer stem cells

Oncogene33523852502014

10.1038/onc.2013.467

24186204

Naito

Cook

Mizumachi

Wang

Xie

Evans

Kelly

Higuchi

Progressive tumor features accompany epithelial-mesenchymal transition induced in mitochondrial DNA-depleted cells

Cancer Sci99158415882008

10.1111/j.1349-7006.2008.00879.x

18754870

Adib-Conquy

Cavaillon

Compensatory anti-inflammatory response syndrome

Thromb Haemost10136472009

10.1160/TH08-07-0421

19132187

Moro

Arbini

Yao

di San t'Agnese

Marra

Greco

Mitochondrial DNA depletion in prostate epithelial cells promotes anoikis resistance and invasion through activation of PI3K/Akt2

Cell Death Differ165715832009

10.1038/cdd.2008.178

19079138

Figure 1.

Distribution of plasma mtDNA content in patients with lung cancer and healthy controls. Quantitative polymerase chain reaction was used to determine plasma mtDNA copy number. Each circle represents plasma mtDNA content of each case. The horizontal lines represent the median and interquartile range and were compared with Student's t-test. mtDNA, mitochondrial DNA.

Figure 2.

Associations between plasma mtDNA content and (A) tumor size, (B) lymph node metastases, (C) distant metastases, (D) TNM stage I–IV, (E) TNM stage I/II vs. III/IV, and (F) smoking history in patients with lung cancer. Each circle represents the plasma mtDNA content of each case. The horizontal lines represent the median and interquartile range. Student's t-test was used for two groups and analysis of variance followed by the Tukey's post hoc test was used for comparison of multiple groups. *P<0.05 vs. T1 (A) and vs. N0 (B); **P<0.01 vs. N0 (B) and vs. stage I (D). mtDNA, mitochondrial DNA.

Figure 3.

Associations between plasma mtDNA content and (A) pathological type, (B) sex, and age (C) in patients with lung cancer. Each circle represents the plasma mtDNA content of each case. The horizontal lines represent the median and interquartile range. Student's t-test was used for two groups and analysis of variance followed by the Tukey's post hoc test was used for comparison of multiple groups. Correlation analysis between mtDNA content and age was performed by Spearman's correlation coefficient. mtDNA, mitochondrial DNA.

Figure 4.

Association between plasma mtDNA content and (A) driver gene mutation status and (B) serum tumor biomarkers. Each circle represents the plasma mtDNA content of each case. The horizontal lines represent median and interquartile range and were determined with the Mann-Whitney U test. Spearman's correlation coefficient was used to calculate the correlation between CEA and mtDNA copy number. ALK, anaplastic lymphoma kinase; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; mtDNA, mitochondrial DNA.

Figure 5.

Receiver operating characteristic curve analysis to determine the sensitivity and specificity of mitochondrial DNA copy number in patients with lung cancer. CI, confidence interval; AUC, area under the curve.

Figure 6.

Kaplan-Meier survival analysis of mtDNA copy number for estimating prognosis in patients with advanced lung cancer. The median mtDNA copy number (1.02×10⁴ copies/µl) was set as the cutoff point. PFS, progression-free survival; mtDNA, mitochondrial DNA.

Table I.

Characteristics of study population.

Characteristics	Lung cancer (n=128)	Healthy control (n=107)	P-value
Sex, n			0.694
Male	72	57
Female	56	50
Age, years (mean ± SD)	55.2±7.8	53.5±8.8	0.125
Smoking history, n			0.703
Yes	69	55
No	59	52
Tumor size, n
T1	32
T2	44
T3	22
T4	30
Lymph node metastasis, n
N0	38
N1	22
N2	20
N3	48
TNM stage, n
I	32
II	20
III	34
IV	42
Pathology type, n
Adenocarcinoma	54
Squamous cell carcinoma	44
Small cell lung cancer	30
Main driver gene mutation
EGFR	29
ALK	9

EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase.