Mitochondrial DNA Copy Number and Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Risk in Two Prospective Studies

Mitochondria are the principal organelle in eukaryotic cells responsible for energy production through the generation of adenosine triphosphate (ATP) via the electron transport chain (ETC; ref. 1). Mitochondria have a singular circular mitochondrial DNA (mtDNA) molecule that is approximately 16k-bp long. As mitochondria age, generation of reactive oxygen species (ROS) increases. This intracellular production of ROS increases oxidative stress, causing damage to DNA, inducing modification of the purine and pyrimidine bases, single- and double-strand breaks, and cross-links to other molecules (2). As mitochondria experience more oxidative stress, pro-inflammatory cytokine production increases (3), which has been associated with risk of non-Hodgkin lymphoma (NHL; ref. 4).

Mitochondrial DNA lacks the protective histones and repair capacity of nuclear chromosomal DNA (2). Expression and stability of mtDNA have been suggested to play a critical role in human pathogenesis due to deficiency in maintenance and stability of the ETC (5), and mitochondria may modify genomic copy number as a mechanism to cope with increased genomic instability and damage (6). Measurements in blood samples of subjects have shown that oxidative stress measurements by thiobarbituric acid–reactive substances, 8-hydroxy-2′-deoxyguanosine, and 4,977 bp deletions and mtDNA CN have been positively correlated (7).

Several studies have examined variation in mtDNA copy number (mtDNA CN) and cancer risk; most prospective studies have found a positive association, and retrospective studies have found an inverse association (8–17), including a strong association observed between increased mtDNA CN and risk of NHL, particularly CLL/SLL, in a prospective cohort study of Finnish male smokers (13) and Europeans (18). Liao and colleagues noted an increased risk in gastric cancer cases diagnosed recently in relation to blood draw/entry into study (≤2 years) in a cohort of Chinese women (19), suggesting that mtDNA CN could be influenced by the result of disease, and more prospective data are needed. Contrastingly, low mtDNA CN has been associated with lung tumor (20), breast cancer (21), and ovarian cancer (22) progression.

To confirm previous findings of mtDNA CN and risk of NHL/CLL/SLL, we conducted a nested case–control study in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial and pooled previously published data (13) from the Alpha-Tocopherol, Beta-Carotene cancer prevention study (ATBC). In addition, we utilized the newest case definitions of NHL subtypes from the Pathology Working Group of the International Lymphoma (INTERLYMPH) Epidemiology Consortium (23) to update the previously published ATBC study.

The PLCO screening trial has been described in detail elsewhere (24). Briefly, PLCO is a large randomized trial to determine whether screening for these cancers reduces cause-specific mortality. A total of 154,910 males and females ages 55to 74 years with no prior history of cancer were enrolled during 1993–2001 in 10 different centers around the United States. Self-administered questionnaire inquired information on demographics and risk factors as well blood specimens for intervention-arm participants. Within 2 hours of blood collection, specimens were processed and frozen at −70°C. The PLCO study was reviewed by the institutional review boards of the National Cancer Institute and the 10 study centers, and all participants provided written informed consent in accordance with the Declaration of Helsinki.

Cancer cases in the PLCO trial were identified through annual follow-up questionnaires and telephone calls. First primary cancers of the prostate, lung, colorectal, and ovarian were also identified through screening tests conducted in the first 6 years of the trial. We identified 301 cases of first primary lymphoid malignancies (244 NHL cases, 95 CLL/SLL cases, and 57 multiple myeloma cases) with serum collected at baseline from the screening arm in PLCO. These cases were confirmed by medical record review at each screening center. Controls were individually matched to cases (1:1 ratio) by age at baseline (5-year categories), sex, race, and interview/blood collection date (±3 months) from among subjects who had not been diagnosed with any type of malignancy except nonmelanoma skin cancer at the time of the case diagnosis date.

The ATBC study has been described in detail (25). Briefly, 29,133 eligible male smokers, ages 50 to 69 years, were recruited from southwest Finland and randomized from 1985 to 1988 to determine whether supplementation with α-tocopherol or β-carotene reduced the incidence of lung and other cancers. At the first visit, the males completed interviews with a study nurse on basic demographic characteristics and whole blood was collected at follow-up (1992–1993). All cases of NHL were identified using the Finnish Cancer Registry, which provides virtually 100% of case ascertainment from the ATBC cohort (26). The hospital records of the identified cases were reviewed by an experienced study oncologist for confirmation of the lymphoma diagnosis. Through April 30, 2002, 142 individuals with incident cases of lymphoid neoplasms (122 NHL cases, 47 CLL/SLL cases, and 20 multiple myeloma cases) with an available prediagnostic blood specimen for analysis were identified. Controls were individually matched to cases (1:1) on date of birth (±5 years). In the previously published data from ATBC (13), 107 cases were utilized; updated INTERLYMPH and SEER classifications of neoplasms account for 15 NHL cases (12 CLL/SLL) that were not previously included in the analyses. The ATBC study was approved by institutional review boards at the National Cancer Institute and the National Public Health Institute of Finland. Participants provided written informed consent in accordance with the Declaration of Helsinki.

Histologic type was obtained by medical record abstraction or recorded by the tumor registrar at each participating medical center using standard ICD-O-2 codes that were re-coded to ICD-O-3. Subtypes of NHL were categorized by the most recent INTERLYMPH classification update from 2010 (23) utilizing the fifth hierarchical level (ATBC analyses were regrouped to this classification). When an ICD-O-3 code was recorded in the PLCO or ATBC records but not defined in INTERLYMPH, codes were identified using NCI's SEER classification for ICD-O-3 coding materials to classify the remaining subtypes (http://seer.cancer.gov/icd-o-3/sitetype.icdo3.d20121205.pdf). The subtype groupings were classified as follows: multiple myeloma (9731, 9732, 9733, 9734), CLL/SLL/B-PLL/MCL (9670, 9673, 9823, 9832, 9833; all cases were 9670 or 9823, referred to CLL/SLL throughout for simplicity), diffuse large B-cell lymphoma (DLBCL; 9678, 9679, 9680–9689, 9712, 9735, 9737, 9738), follicular (9690–9699) and other/NOS (9590, 9591, 9596, 9673, 9675, 9684, 9687, 9704, 9707, 9710, 9715, 9727, 9728, 9765, 9766, 9811–9812, 9820, 9835, 9836, 9940, 9970). Case histology by cohort is presented in Table 1.

DNA was extracted from the peripheral white blood cells of the whole blood using the phenol–chloroform method (27), and fluorescence-based quantitative polymerase chain reaction determined mtDNA CN using the ratio of the estimation of threshold cycle number of ND1, a mitochondrial gene, and the B-globin gene, HBB, a nuclear gene (7). Samples for both studies were processed and assayed by the same laboratory. Cases and controls were blindly assayed consecutively within each batch. Blinded quality control duplicate samples (8 samples from 2 subjects) were interspersed in each batch to evaluate assay reproducibility. In the PLCO analysis, the overall coefficient of variation (CV) of this assay was 6.8%; 9 of 301 selected cases and 2 of 303 selected controls had poor quality DNA or the assay malfunctioned, leaving 292 cases and 301 controls in the final analysis. In the ATBC analysis, 142 matched sets of cases and controls were previously assayed, with an overall CV of 13% (13). In total, 434 cases and 443 controls comprised the pooled ATBC and PLCO analyses.

Differences in selected demographic characteristics between cases and controls were assessed using the Wilcoxon rank sum test for continuous variables and Pearson χ² test for categorical variables. Unconditional logistic regression models were used to generate odds ratios (OR) and 95% confidence intervals (95% CI) to estimate the association of mtDNA CN and risk of CLL/SLL. Polytomous logistic regression models were used in place of binary logistic regression in analyses stratified by NHL, subtypes (DLBCL, follicular, other/NOS), multiple myeloma, and time since blood draw. Cohort-specific distribution of mtDNA CN among all controls was used to determine cutoff points for tertiles. In the pooled analysis, tertile groupings were maintained from each cohort to minimize any potential effects of lab drift. Unconditional model results were similar to conditional models, but to maximize statistical stability, unconditional models that utilized all controls were used for all analyses. All models compared the higher versus the lowest tertile of mtDNA. Models were adjusted for sex (male, female-PLCO, and pooled only), age (continuous), tobacco (pack-years), BMI (continuous), race (white, black, other-PLCO and pooled only), date of blood draw (year), and study (PLCO, ATBC). After adjustment for supplementation group in ATBC, results were similar to nonadjusted estimates; thus, supplement group was left out of the final models. We also conducted analyses stratifying by sex (male, female) and smoking status (ever, never). In addition, we conducted stratified analyses in males by time since blood draw to diagnosis (≤6 years, >6–8 years, >8 years). P_trend utilized the tertiles of mtDNA modeled continuously. Significance tests were two-sided at the 0.05 level. All analyses were conducted in SAS 9.22 (SAS Institute).

The demographic characteristics were fairly similar between the lymphoid cases, CLL/SLL cases, and controls for both studies (Table 1). Sex, tobacco, race, age, BMI, and pack-years of tobacco smoked were not statistically different between CLL/SLL cases and controls (P > 0.05). mtDNA CN was significantly higher in cases than in controls in the ATBC study (P < 0.05). Being male and pack-years of smoking were negatively associated with mtDNA CN among controls (pooled: linear regression, β: −37.97, −0.26, respectively; P < 0.05). The range of mtDNA CN was wider in PLCO than in ATBC, and the median mtDNA CN was greater in PLCO (median of controls, 121) than in ATBC (median of controls, 109). There was no significant difference (P = 0.11) in mtDNA CN comparing male smoker controls in PLCO (mean, 128.3) versus controls in ATBC (mean, 117.7; data not shown).

Mitochondrial DNA copy number was not associated with risk of NHL in PLCO (Supplementary Table S1). Mitochondrial DNA copy number was elevated but not significantly associated with risk of CLL/SLL in PLCO (highest vs. lowest tertile: OR, 1.53; 95% CI, 0.84–2.81), but was significantly associated in the pooled population (OR, 2.25; 95% CI, 1.35–3.74; Table 2). In males, the elevated risk of CLL/SLL was observed in both studies individually and the pooled analysis (P_trend <0.05, P_interaction by sex < 0.05), especially among smokers (highest vs. lowest tertile: OR, 4.28; 95% CI, 2.06–8.88). When PLCO analyses were restricted to the characteristics of the ATBC population (males and ever smokers), CLL/SLL risk was elevated (OR, 2.93; 95% CI, 1.01–8.51). Compared with the PLCO population, the effect estimates in the ATBC population were greater, although not statistically different (P, 0.13). There was suggestion of no association in nonsmokers, and there was no association in females. Additional stratified analyses by time to diagnosis from blood draw among males showed a consistent elevation of risk at ≤6, 6–8, and >8 years in the pooled population (P_interaction: 0.65, Table 3). Analyses with non-whites removed were not substantially different, and stratified analyses by age and BMI were not dramatically different or statistically unstable (data not shown).

When the analyses were conducted for NHL, NHL subtypes, or multiple myeloma, no significant associations were observed in the pooled data (P_trend > 0.05; Supplementary Table S1). Analyses for overall NHL and mtDNA CN associations stratified by sex, smoking status, and time from blood draw to diagnosis were unremarkable (Supplementary Tables S2 and S3).

We pooled data from two prospective studies that consisted of new data from PLCO and previously published data from ATBC (13). We found consistent evidence of an association between increased mtDNA CN and CLL/SLL risk among males and male smokers in the PLCO study and the pooled study. By pooling data, we were able to observe the mtDNA-CLL/SLL risk association in cases diagnosed as long as 8+ years after baseline blood draw. Similar to data previously published (13, 18), these results suggest mtDNA CN could reflect a process that reflects CLL/SLL development. There was no evidence of an association between mtDNA CN and CLL/SLL in nonsmokers; similarly, there was no observed association in females, although limited by few cases. We observed no association with mtDNA CN and risk of overall NHL, other NHL subtypes, or multiple myeloma risk.

In general, studies of mtDNA CN and risk of cancer have observed a positive association at various sites in prospective studies: lung (9, 16), cervix (11), colorectal (14, 17), and pancreas (15). One prospective study did not observe an overall association with gastric cancer risk in females, but cases diagnosed within two years of blood draw had an increased risk, suggesting that mtDNA CN elevation was an effect of the disease (19). Consistent with other prospective studies, but unlike the aforementioned gastric cancer study (19), this study observed an mtDNA CN association with CLL/SLL risk in male smokers, similar to a previously published study (13). However, the strength of the association was slightly weaker in the new PLCO data and more similar to a study in Europeans (18). As leukocytes are from the same target tissue as CLL/SLL, mtDNA measured leukocytes could be a direct indication of mitochondrial activity and regulation, which could be a reflection of a disease process that results from genomic damage, oxidative stress, and inflammation occurring in the lymphatic system. Studies where mtDNA was measured in subjects with disease, inverse associations were observed in oral cancer (8), soft-tissue sarcoma (10), and renal cell carcinoma (12) and tumor progression (20–22), suggesting mtDNA CN levels decrease as cancer progresses. One study of colorectal cancer was retrospective, but found a positive association with mtDNA CN (13).

Although stratified analyses in other reports have been limited, several studies of mtDNA CN and cancer risk have observed significant associations among smokers and males but not in females. Two studies found the association was strongest among smokers for oral cancer and pancreatic cancer (8, 15). Two studies conducted in male-only populations observed significant associations with lung cancer and pancreatic cancer (9, 15). Three studies did not find significant associations among females for gastric cancer (entire study population was female; ref. 19), renal cell carcinoma (∼40% of study was female; ref. (12), and colorectal cancer (∼40% cases and ∼60% controls of study were female; ref. 17). However, due to relatively few females in our study, the analyses by sex were somewhat underpowered. Thus, we cannot state definitively that there was no association in females—only that there was limited suggestion of an association.

Male smokers had increased risk of CLL/SLL when mtDNA CN was elevated. Tobacco smoking (PLCO: ever, 133; never, 155) and being male (PLCO: males, 115; females, 137) were negatively associated with mtDNA CN in controls in this study. Similar trends by sex and smoking status were also observed in a population of Koreans (28). Because male smokers had a lower baseline mtDNA CN, and male smokers with higher mtDNA CN were at increased risk of CLL/SLL, these individuals may have genomic characteristics that are ideal for CLL/SLL development. Mitochondrial DNA genomes from CLL/SLL patients contain high levels of mtDNA content (29), are very stable, and can create genomic copies with deletions and mutations (30) without undergoing apoptosis or senescence.

In the time-based analyses, the mtDNA CN association was similar across several time points up to 8 years after blood draw until diagnosis, which suggests that mtDNA CN could reflect a risk factor for CLL/SLL development. In addition, approximately 20% (n = 27 of 142) of cases were in the first tertile, a disproportionately small proportion of cases. Although these analyses suggest mtDNA CN could be an etiologic biomarker in CLL/SLL risk, the number of cases available for these stratified analyses were somewhat sparse, so these results should be interpreted with caution.

Individuals with asymptomatic CLL/SLL, mtDNA CN could be an indicator of disease development. Precursor condition to CLL/SLL, monoclonal B-cell lymphocytosis, is detectable in CLL/SLL patients up to 77 months before diagnosis (31). But in healthy individuals, mtDNA CN and lymphocytes were not correlated (r = 0.029, P = 0.72; n = 140), suggesting that mtDNA CN is not just a surrogate marker of lymphocytosis.

The primary strengths of this study were due to the prospective designs of the PLCO and ATBC studies. All subjects in this study were recruited before diagnosis of any cancer, permitting analyses of associations stratified by the time of blood collection in relation to diagnosis. We assessed mtDNA in white blood cells, the target tissue for CLL/SLL. We utilized the latest revision of the INTERLYMPH working group definitions for histologic subtype classifications of NHL (23), which allowed for more specific analyses of lymphoma subtypes. These revised classifications allowed for more accurate analyses of what are heterogeneous diseases. 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. Exactly how mtDNA changes as disease development progresses is currently unclear. In addition, there may be residual confounding that may alter mtDNA CN. There was limited evidence of an association in women and nonsmokers in PLCO, but only 39% (n = 230) of the study subjects were female and 52% (n = 303) were nonsmokers. Smaller sample size of females and nonsmokers compared with males and smokers (particularly when combined with ATBC study subjects) restricted the statistical stability of stratified analyses.

In conclusion, we observed that mtDNA CN was associated with risk of developing CLL/SLL in males and male smokers in PLCO, which replicated the results previously found in ATBC. After pooling PLCO and ATBC data, the association was observable as long as 8 years after entry into the study, suggesting that mtDNA CN may reflect a process that contributes to CLL/SLL development.

No potential conflicts of interest were disclosed.

Conception and design: M.R. Bonner, Q. Lan, N. Rothman

Development of methodology: C.-S. Liu, W.-L. Cheng, N. Rothman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Kim, M.P. Purdue, W.-Y. Huang, S. Männistö, S.J. Weinstein, D. Albanes, Q. Lan, N. Rothman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Kim, W.-Y. Huang, R. Vermeulen, H.D. Hosgood, Q. Lan, N. Rothman

Writing, review, and/or revision of the manuscript: C. Kim, B.A. Bassig, W.J. Seow, W. Hu, M.P. Purdue, W.-Y. Huang, S. Männistö, R. Vermeulen, S.J. Weinstein, U. Lim, H.D. Hosgood, N.E. Caporaso, D. Albanes, Q. Lan, N. Rothman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Kim, W. Hu, W.-Y. Huang

Study supervision: Q. Lan, N. Rothman

This study was supported by the NIH intramural research program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.