Background: We previously reported that higher levels of mitochondrial DNA copy number (mtDNA CN) were associated with lung cancer risk among male heavy smokers (i.e., ≥20 cigarettes per day) in the Alpha-Tocopherol Beta-Carotene (ATBC) study. Here, we present two additional prospective investigations nested in the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial and the Shanghai Women's Health Study (SWHS), and pooled with previously published data from ATBC.

Materials: All DNA were extracted from peripheral whole blood samples using the phenol–chloroform method, and mtDNA CN was assayed by fluorescence-based qPCR. Multivariate unconditional logistic regression models were used to estimate ORs and 95% confidence intervals for the association of mtDNA CN and lung cancer risk.

Results: Overall, mtDNA CN was not associated with lung cancer risk in the PLCO, SWHS, or pooled populations (all P trends > 0.42, P heterogeneity = 0.0001), and mtDNA CN was inversely associated with lung cancer risk among male smokers in PLCO, the opposite direction observed in ATBC. In addition, the mtDNA CN association observed among male heavy smokers in ATBC was the opposite direction in PLCO.

Conclusions: mtDNA CN was not consistently associated with lung cancer risk across three prospective study populations from Europe, Asia, and the United States.

Impact: This pooled study suggests no consistent association between prediagnostic mtDNA CN levels and lung cancer risk across several populations. Cancer Epidemiol Biomarkers Prev; 23(12); 2977–80. ©2014 AACR.

Mitochondria are primarily responsible for energy production in eukaryotic cells (1). Mitochondria have a singular circular mitochondrial DNA molecule. Mitochondria copy number (mtDNA CN) varies to meet energy needs and cope with oxidative stress (2). Oxidative stress disrupts homeostasis and forms reactive oxygen species leading to DNA damage and genomic instability (3). In a prospective study among Finnish male smokers, we observed a positive association between mtDNA CN in peripheral blood leukocytes and lung cancer risk (4).

To replicate the mtDNA CN–lung cancer risk association, nested case–control studies were conducted in two prospective studies, the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial, a population of men and women across the United States, and the Shanghai Women's Health Study (SWHS), a population of mostly nonsmoking Chinese women. These data were then pooled with published data from the Alpha-Tocopherol Beta-Carotene (ATBC) study (4).

Study subjects

The ATBC (5), PLCO (6), and SWHS (7) studies have been described in detail. From ATBC, a total of 229 incident cases with an available blood specimen for analysis were previously identified (4). Controls were matched to cases 1:1 based on date of birth. From PLCO, a total of 442 cases of first primary incident lung cancer with serum at baseline from the screening arm were identified. Controls were matched by age, sex, race, blood collection date, and diagnosing center. From SWHS, 226 incident lung cancer cases with adequate blood for mtDNA analysis were identified, with one control matched to case by age, smoking status, and date of blood collection.

Laboratory analysis

In all three studies, DNA was extracted from peripheral whole blood by the phenol–chloroform method. mtDNA CN was assayed in the same laboratory by qPCR using the ratio of the estimated threshold cycle number of ND1 mitochondrial gene and the B-globin nuclear gene, HBB (8). The coefficient of variation for PLCO, SWHS, and ATBC was 14%, 7%, and 13%, respectively. After excluding bad runs, poor-quality DNA, or subjects with missing demographic variables, 227 cases and 227 controls in ATBC, 426 cases and 436 controls in PLCO, and 221 cases and 222 controls in SWHS were included in the final analysis.

Statistical analysis

Differences between cases and controls for demographic characteristics were tested with the Wilcoxon-signed rank-sum test for continuous variables and the Pearson χ2 test for categorical variables. mtDNA CN was categorized by quartiles among controls in each study. Unconditional logistic regression models generated ORs and 95% confidence intervals (95% CI) to estimate the association of mtDNA CN and risk of lung cancer. Models were adjusted for age, body mass index (BMI), race, pack-years smoking, and date of blood collection. PLCO was additionally adjusted for sex and study center. Pooled analyses were additionally adjusted for study. Additional analyses were stratified by sex, smoking status, and smoking pack-years. P trend modeled mtDNA CN quartiles continuously. Between-study heterogeneity was tested by random effects with the rmeta package [Thomas Lumley (2012). rmeta: Meta-analysis. R package version 2.16. http://CRAN.R-project.org/package=rmeta]. Analyses were performed in SAS 9.3 (SAS Institute).

Selected demographic characteristics of the study population are described in Table 1. Statistically significant differences between cases and controls in BMI and years of smoking were observed in the ATBC and PLCO populations but not in SWHS. Age, sex, race (all Caucasian in ATBC, Asian in SWHS), and mtDNA CN levels were not different between cases and controls across the three populations.

Table 1.

Demographic and mtDNA CN characteristics of ATBC, PLCO, and SWHS populations

CasesControls
Study (Ca/Co)CharacteristicMean/NSD/%Mean/NSD/%P valuea
ATBC BMI (kg/m2) 25.59 3.49 26.35 3.87 0.045 
(227/227) Age 56.67 5.00 58.41 4.79 0.68 
 Years smoking 38.47 7.11 35.83 9.22 0.005 
 Ever smoker 227 100 227 100 1 
 mtDNA CN 134.23 45.27 127.85 36.07 0.26 
  median, IQR 125.86 (102.04–160.18) 124.79 (100.63–151.51)  
PLCO BMI (kg/m2) 26.84 4.39 27.43 4.36 0.028 
(426/436) Age 64.07 4.95 63.67 4.71 0.22 
 Years smoking 34.89 15.15 15.57 17.47 <0.0001 
 Ever smoker 380 89.20 238 54.59 <0.0001 
 mtDNA CN 128.37 128.26 127.39 59.96 0.079 
  median, IQR 110 (87–140) 114 (90–145)  
 Sex 
  Male 259 60.80 267 61.24 0.89 
  Female 167 39.20 169 38.76  
 Race 
  White 399 93.66 404 92.66 0.81 
  Black 15 3.52 18 4.13  
  Other 12 2.82 14 3.21  
 Center 
  University of Colorado 23 5.40 23 5.28 1 
  Georgetown University 19 4.46 19 4.36  
  Pacific Health Research and Education Institute 9 2.11 9 2.06  
  Henry Ford Health System 66 15.49 63 14.45  
  University of Minnesota 126 29.58 131 30.05  
  Washington University 50 11.74 50 11.47  
  University of Pittsburgh 43 10.09 46 10.55  
  University of Utah 29 6.81 31 7.11  
  Marshfield Clinic Research Foundation 45 10.56 47 10.78  
  University of Alabama Birmingham 16 3.76 17 3.90  
SWHS BMI (kg/m2) 24.58 3.45 25.01 3.53 0.16 
(221/222) Age 59.22 8.20 59.18 8.35 0.93 
 Years smoking 2.97 10.67 1.74 8.19 0.23 
 Ever smoker 17 7.69 11 4.95 0.24 
 mtDNA CN 191.84 315.45 185.88 268.6 0.82 
  median, IQR 118 (94–158) 124 (93–162)  
CasesControls
Study (Ca/Co)CharacteristicMean/NSD/%Mean/NSD/%P valuea
ATBC BMI (kg/m2) 25.59 3.49 26.35 3.87 0.045 
(227/227) Age 56.67 5.00 58.41 4.79 0.68 
 Years smoking 38.47 7.11 35.83 9.22 0.005 
 Ever smoker 227 100 227 100 1 
 mtDNA CN 134.23 45.27 127.85 36.07 0.26 
  median, IQR 125.86 (102.04–160.18) 124.79 (100.63–151.51)  
PLCO BMI (kg/m2) 26.84 4.39 27.43 4.36 0.028 
(426/436) Age 64.07 4.95 63.67 4.71 0.22 
 Years smoking 34.89 15.15 15.57 17.47 <0.0001 
 Ever smoker 380 89.20 238 54.59 <0.0001 
 mtDNA CN 128.37 128.26 127.39 59.96 0.079 
  median, IQR 110 (87–140) 114 (90–145)  
 Sex 
  Male 259 60.80 267 61.24 0.89 
  Female 167 39.20 169 38.76  
 Race 
  White 399 93.66 404 92.66 0.81 
  Black 15 3.52 18 4.13  
  Other 12 2.82 14 3.21  
 Center 
  University of Colorado 23 5.40 23 5.28 1 
  Georgetown University 19 4.46 19 4.36  
  Pacific Health Research and Education Institute 9 2.11 9 2.06  
  Henry Ford Health System 66 15.49 63 14.45  
  University of Minnesota 126 29.58 131 30.05  
  Washington University 50 11.74 50 11.47  
  University of Pittsburgh 43 10.09 46 10.55  
  University of Utah 29 6.81 31 7.11  
  Marshfield Clinic Research Foundation 45 10.56 47 10.78  
  University of Alabama Birmingham 16 3.76 17 3.90  
SWHS BMI (kg/m2) 24.58 3.45 25.01 3.53 0.16 
(221/222) Age 59.22 8.20 59.18 8.35 0.93 
 Years smoking 2.97 10.67 1.74 8.19 0.23 
 Ever smoker 17 7.69 11 4.95 0.24 
 mtDNA CN 191.84 315.45 185.88 268.6 0.82 
  median, IQR 118 (94–158) 124 (93–162)  

Abbreviations: Ca, cases; Co, controls; IQR, interquartile range.

aWilcoxon rank-sum test for continuous variables/Pearson χ2 for categorical variables.

View Large

mtDNA CN was not associated with lung cancer risk in the PLCO or SWHS data; pooled analysis was also null with evidence of heterogeneity across studies (P heterogeneity = 0.0001; Table 2). mtDNA CN and lung cancer risk associations were inverse in male smokers in PLCO, the opposite direction of ATBC. Similarly, comparing heavy smokers to non/light smokers, associations in PLCO were the opposite direction of ATBC (data not shown). No differential associations were observed by histology or follow-up time (data not shown).

Table 2.

mtDNA copy number and risk of lung cancer across ATBC, PLCO, and SWHS studies by sex and smoking status

ATBC (Ca 227/Co 227)PLCO (Ca 426/Co 436)SWHS (Ca 221/Co 222)Pooled
mtDNA%Ca/CoORa95% CICa/CoORa95% CICa/CoORa95% CICa/CoORa95% CI
Overall 
 Q1 52/57 1.00 (ref.) 117/107 1 (ref.) 54/56 1.00 (ref.) 223/220 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 119/110 0.98 (0.64–1.50) 72/58 1.27 (0.48–1.47) 250/224 1.08 (0.82–1.42) 
 Q3 43/57 1.15 (0.61–2.17) 96/108 0.84 (0.55–1.29) 44/53 0.84 (0.48–1.47) 183/218 0.83 (0.62–1.11) 
 Q4 73/57 2.38 (1.12–5.06) 94/111 0.98 (0.63–1.52) 51/55 0.91 (0.53–1.57) 218/223 1.04 (0.79–1.39) 
 P trend  0.049 P trend  0.74 P trend  0.42 P trend  0.78 
Female 
 Q1  — — 32/40 1 (ref.) 54/56 1.00 (ref.) 86/96 1.00 (ref.) 
 Q2  — — 38/41 1.51 (0.74–3.11) 72/58 1.27 (0.48–1.47) 110/99 1.34 (0.89–2.02) 
 Q3  — — 41/44 1.47 (0.73–2.98) 44/53 0.84 (0.48–1.47) 85/97 1.03 (0.67–1.58) 
 Q4  — — 56/44 2.12 (1.07–4.22) 51/55 0.91 (0.53–1.57) 107/99 1.24 (0.82–1.89) 
    P trend  0.043 P trend  0.42 P trend  0.59 
Male 
 Q1 52/57 1.00 (ref.) 85/67 1 (ref.)  — — 137/125 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 81/69 0.76 (0.44–1.28)  — — 140/125 0.89 (0.61–1.29) 
 Q3 43/57 1.15 (0.61–2.17) 55/64 0.59 (0.34–1.05)  — — 98/121 0.69 (0.46–1.02) 
 Q4 73/57 2.38 (1.12–5.06) 38/67 0.55 (0.29–0.99)  — — 111/124 0.92 (0.62–1.35) 
 P trend  0.049 P trend  0.028    P trend  0.39 
Never smoker 
 Q1  — — 9/51 1 (ref.) 52/53 1.00 (ref.) 61/104 1.00 (ref.) 
 Q2  — — 11/51 1.35 (0.49–3.72) 66/58 1.15 (0.68–1.94) 77/109 1.17 (0.74–1.85) 
 Q3  — — 12/42 1.23 (0.43–3.57) 40/51 0.79 (0.44–1.40) 51/93 0.86 (0.52–1.41) 
 Q4  — — 14/54 1.49 (0.55–4.08) 46/49 0.94 (0.54–1.65) 60/103 1.03 (0.64–1.68) 
    P trend  0.49 P trend  0.54 P trend  0.79 
Ever smoker 
 Q1 52/57 1.00 (ref.) 108/56 1 (ref.) 2/3 1.00 (ref.) 162/116 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 108/59 0.91 (0.56–1.47) 6/0 — — 173/115 1.03 (0.73–1.47) 
 Q3 43/57 1.15 (0.61–2.17) 84/66 0.72 (0.44–1.17) 4/2 4.33 (0.24–79.49) 132/125 0.78 (0.54–1.12) 
 Q4 73/57 2.38 (1.12–5.06) 80/57 0.84 (0.50–1.39) 5/6 1.05 (0.087–12.81) 158/120 1.05 (0.73–1.49) 
 P trend  0.049 P trend  0.33 P trend  0.46 P trend  0.81 
Male smoker 
 Q1 52/57 1.00 (ref.) 78/38 1 (ref.)  — — 130/95 1 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 78/40 0.8 (0.45–1.45)  — — 137/96 0.94 (0.63–1.39) 
 Q3 43/57 1.15 (0.61–2.17) 53/45 0.58 (0.32–1.06)  — — 96/102 0.69 (0.46–1.03) 
 Q4 73/57 2.38 (1.12–5.06) 33/37 0.50 (0.25–0.98)  — — 106/94 0.95 (0.63–1.43) 
 P trend  0.049 P trend  0.021    P trend  0.45 
ATBC (Ca 227/Co 227)PLCO (Ca 426/Co 436)SWHS (Ca 221/Co 222)Pooled
mtDNA%Ca/CoORa95% CICa/CoORa95% CICa/CoORa95% CICa/CoORa95% CI
Overall 
 Q1 52/57 1.00 (ref.) 117/107 1 (ref.) 54/56 1.00 (ref.) 223/220 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 119/110 0.98 (0.64–1.50) 72/58 1.27 (0.48–1.47) 250/224 1.08 (0.82–1.42) 
 Q3 43/57 1.15 (0.61–2.17) 96/108 0.84 (0.55–1.29) 44/53 0.84 (0.48–1.47) 183/218 0.83 (0.62–1.11) 
 Q4 73/57 2.38 (1.12–5.06) 94/111 0.98 (0.63–1.52) 51/55 0.91 (0.53–1.57) 218/223 1.04 (0.79–1.39) 
 P trend  0.049 P trend  0.74 P trend  0.42 P trend  0.78 
Female 
 Q1  — — 32/40 1 (ref.) 54/56 1.00 (ref.) 86/96 1.00 (ref.) 
 Q2  — — 38/41 1.51 (0.74–3.11) 72/58 1.27 (0.48–1.47) 110/99 1.34 (0.89–2.02) 
 Q3  — — 41/44 1.47 (0.73–2.98) 44/53 0.84 (0.48–1.47) 85/97 1.03 (0.67–1.58) 
 Q4  — — 56/44 2.12 (1.07–4.22) 51/55 0.91 (0.53–1.57) 107/99 1.24 (0.82–1.89) 
    P trend  0.043 P trend  0.42 P trend  0.59 
Male 
 Q1 52/57 1.00 (ref.) 85/67 1 (ref.)  — — 137/125 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 81/69 0.76 (0.44–1.28)  — — 140/125 0.89 (0.61–1.29) 
 Q3 43/57 1.15 (0.61–2.17) 55/64 0.59 (0.34–1.05)  — — 98/121 0.69 (0.46–1.02) 
 Q4 73/57 2.38 (1.12–5.06) 38/67 0.55 (0.29–0.99)  — — 111/124 0.92 (0.62–1.35) 
 P trend  0.049 P trend  0.028    P trend  0.39 
Never smoker 
 Q1  — — 9/51 1 (ref.) 52/53 1.00 (ref.) 61/104 1.00 (ref.) 
 Q2  — — 11/51 1.35 (0.49–3.72) 66/58 1.15 (0.68–1.94) 77/109 1.17 (0.74–1.85) 
 Q3  — — 12/42 1.23 (0.43–3.57) 40/51 0.79 (0.44–1.40) 51/93 0.86 (0.52–1.41) 
 Q4  — — 14/54 1.49 (0.55–4.08) 46/49 0.94 (0.54–1.65) 60/103 1.03 (0.64–1.68) 
    P trend  0.49 P trend  0.54 P trend  0.79 
Ever smoker 
 Q1 52/57 1.00 (ref.) 108/56 1 (ref.) 2/3 1.00 (ref.) 162/116 1.00 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 108/59 0.91 (0.56–1.47) 6/0 — — 173/115 1.03 (0.73–1.47) 
 Q3 43/57 1.15 (0.61–2.17) 84/66 0.72 (0.44–1.17) 4/2 4.33 (0.24–79.49) 132/125 0.78 (0.54–1.12) 
 Q4 73/57 2.38 (1.12–5.06) 80/57 0.84 (0.50–1.39) 5/6 1.05 (0.087–12.81) 158/120 1.05 (0.73–1.49) 
 P trend  0.049 P trend  0.33 P trend  0.46 P trend  0.81 
Male smoker 
 Q1 52/57 1.00 (ref.) 78/38 1 (ref.)  — — 130/95 1 (ref.) 
 Q2 59/56 1.34 (0.71–2.37) 78/40 0.8 (0.45–1.45)  — — 137/96 0.94 (0.63–1.39) 
 Q3 43/57 1.15 (0.61–2.17) 53/45 0.58 (0.32–1.06)  — — 96/102 0.69 (0.46–1.03) 
 Q4 73/57 2.38 (1.12–5.06) 33/37 0.50 (0.25–0.98)  — — 106/94 0.95 (0.63–1.43) 
 P trend  0.049 P trend  0.021    P trend  0.45 

NOTE: %Quartiles: ATBC: <100.6, ≥100.6–124.8, ≥124.8–151.5, ≥151.5; PLCO: <90, ≥90–114, ≥114–145, ≥145; SWHS: <93, ≥93–124, ≥124–162, ≥162; Median: ATBC: ≤124.8; PLCO: ≤114, SWHS: <124.

Abbreviations: Ca, cases; Co, controls; Q1–Q4, quartiles.

aAdjusted for age, BMI, pack-years, race, sex, date of enrollment, center (if applicable), and study.

View Large

The mtDNA CN and lung cancer risk association observed in ATBC did not replicate in PLCO, SWHS, and pooled study populations. There was no consistent evidence of an association across populations by sex or smoking status/intensity. This pooled study suggests no consistent association between prediagnostic mtDNA CN levels and lung cancer risk across several populations.

Our study included diverse study populations. Additional strengths included the combined large sample size and the standardized specimen processing and mtDNA CN assay across the three studies. The primary weakness of this study was the single measurement of mtDNA CN. Copy number could change over time, and this study was unable to determine intrapersonal variation over time.

No potential conflicts of interest were disclosed.

Conception and design: C. Kim, B.-T. Ji, H.D. Hosgood, N. Rothman, Q. Lan

Development of methodology: C. Kim, C.-S. Liu, W.-L. Cheng, H.D. Hosgood

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.P. Purdue, X.-O. Shu, W.-Y. Huang, Y.-B. Xiang, Y.-T. Gao, S. Männistö, S.J. Weinstein, D. Albanes, W. Zheng, N. Rothman, Q. Lan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Kim, M.P. Purdue, W.-Y. Huang, B.-T. Ji, N. Rothman, Q. Lan

Writing, review, and/or revision of the manuscript: C. Kim, B.A. Bassig, W.J. Seow, M.P. Purdue, X.-O. Shu, W.-Y. Huang, Y.-B. Xiang, B.-T. Ji, W.-H. Chow, S. Männistö, S.J. Weinstein, D. Albanes, W. Zheng, U. Lim, N. Rothman, Q. Lan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Hu, T.-T. Lin, Y.-B. Xiang, B.-T. Ji, Y.-T. Gao, S.J. Weinstein, D. Albanes, W. Zheng

Study supervision: X.-O. Shu, Y.-T. Gao, N. Rothman, Q. Lan

Other (former co-PI of one of the prospective studies): W.-H. Chow

This study was supported by NIH intramural research program.

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©2014 American Association for Cancer Research.
2014