Background
Breast cancer (BC) is the most common cancer worldwide affecting women, accounting for 20% of all malignancies in females
Because age is a principal risk factor for cancer, studies on aging have provided additional understanding of DNA repair processes
With respect to DNA repair in cases of BC, the Nucleotide Excision Repair (NER) pathway is receiving increasing attention
The aim of our study was to to assess the statistical association of DRC level and breast cancer (BC) using a case–control epidemiological study in a Hispanic community. In contrast to previously published studies, including our (Ramos
Methods
Epidemiologic design
Over a 6-year period, we recruited 824 women of Hispanic origin, age 21 or older: 285 women newly diagnosed with BC and 539 without BC. Calculations of sample size done initially revealed that a sample size of 824 participants (312 women with BC, 515 women without BC) would allow us to have a statistically significant odds ratio as low as 1.7 when the percent exposed to a low DRC among controls is 15% or higher (e.g., 15% controls are 21 to 30 years of age) with 5% significant level and 80% of statistical power.
The population represents a genetically diverse population that is an admixture of European, African, and Amerindian ethnic groups (per studies from 106 ancestry markers—Dr. Julie Dutil, unpublished observations). Women who were obtaining mammograms and other routine gynecological and primary care/screening services at the same medical offices where patients with BC were being treated were recruited consecutively at the following locations: Ponce School of Medicine and Health Sciences Outpatient Clinic, Auxilio Mutuo Hospital (San Juan), Damas Hospital (Ponce), and St. Luke’s Hospital (Ponce), as well as Yauco and other selected collaborating cities throughout Puerto Rico, representing 65 (83%) of the 78 municipalities (counties) on the island. Because Puerto Rico offers universal health insurance coverage, any healthy women who might develop BC would be treated in the same facilities where BC patients were recruited. This selection procedure minimized selection bias due that could have otherwise been a factor (site, screening/treatment modalities) if healthy women were recruited from the general population by other means (e.g., through random-digit dialing, as noted by Rothman
This study was approved by the IRB of the Ponce School of Medicine and Health Sciences (Ponce, Puerto Rico) and participating hospitals. Informed consent was obtained from all participants before interviewing them, drawing blood samples, collecting tumor material, and obtaining pathology reports.
The two main inclusion criteria for selecting women without BC were 1) a normal breast examination done by a primary care physician and 2) a normal mammogram, both within the last six months. These criteria reduced the likelihood of the existence of undiagnosed BC in this cohort.
We studied only recently diagnosed, treatment-naïve BC patients with primary tumors. Exclusion criteria included those with metastatic BC, secondary BC, breast metastases from another type of cancer, or any acquired or genetic immunodeficiency. Because blood transfusions, chemotherapy, and radiotherapy can significantly affect DRC
Pathology reports from all patients were obtained to confirm the 1) diagnosis, 2) tumor grade, 3) tumor size, 4) presence or absence of axillary lymph node metastasis, and 5) other clinically relevant information. An epidemiological questionnaire soliciting information and variables related to BC risk was provided to each participant.
Blood collection and isolation of lymphocytes from women
Approximately 30 mL of peripheral blood was obtained from each participant and stored in heparinized tubes. The lymphocytes were then isolated by the Ficoll gradient technique and suspended in 2 mL of freezing media containing 10% dimethyl sulfoxide, 40% RPMI 1640 medium, 50% fetal bovine serum, and 1% antibiotic/antimycotic. Aliquots were stored in a −80°C freezer for 1–3 weeks. The lymphocytes were later thawed in batches of 5–7 samples for the host-cell reactivation (HCR) assays (details follow). Collection periods were approximately the same for patients and women without BC because recruitment was conducted concurrently.
Plasmid preparation for host cell reactivation (HCR) assay for measuring DNA repair capacity
The late Dr. Lawrence Grossman (Johns Hopkins School of Public Health, Baltimore, MD) provided the luciferase plasmid for the HCR assay and the protocol for its use. A non-replicating plasmid expression vector (pCMVl
HCR assay to measure DRC
The HCR assay that we utilized to measure DRC levels in lymphocytes has been described in previously published molecular epidemiological studies of cancer
To assess this assay’s precision, we used a data subset that involved duplicates of 50 women with BC, 50 women without BC, and 90 samples of three commercial cell lines. A correlation of 0.97 (95% overall confidence interval: 0.95–1.00) was found (
The measurement of DRC has a coefficient of variation of 23% in the data presented in this study. A batch effect associated with inter-technician variability in performance of the DRC assay was found; this was corrected by excluding 36 samples (35 BC cases, 1 control). Grossman and Wei
Validation of stable transfection
To confirm achievement of stable transfection, we utilized the Dual-GloR® Luciferase Assay System (Promega; Madison, WI), which is based on the combined use of the Firefly and
Preparation of samples and controls
Previously frozen peripheral blood lymphocytes from patients and women without BC were assayed in batches, as described by Ramos
Calculation of DRC
The assay for the gene expression of luciferase activity was measured using a luminometer (Turner Designs, model TD-20/20, Sunnyvale, CA). The percentage of DRC was calculated as the percentage of luciferase activity present after the repair of damaged plasmid DNA, compared to the DRC of undamaged plasmid DNA (100%). This method produced a range of DRC values from nearly 0 to 19% DRC.
Statistical analyses
The association of DRC levels was based on the accuracy detecting BC when a woman has a high versus low DRC level. Data analyses were conducted by using the IBM SPSS statistical package version 17.0 (IBM; Armonk, N.Y.). The Wilcoxon or Mann Whitney
During crude analysis, variables such as DRC, age, weight, and body mass index, were first analyzed as continuous variables. The mean difference was used to assess DRC and anthropometric difference between patients with BC and women without BC using the logistic regression model
Results
Association between DNA Repair capacity levels and population variables in women with and without breast cancer
Women with BC (n = 285) had a mean DRC of 2.40%; this was 60% lower than the average DRC level (
% DRC of female cohorts in Puerto Rico with breast cancer (n = 385) and without breast cancer (n = 539).
% DRC of female cohorts in Puerto Rico with breast cancer (n = 385) and without breast cancer (n = 539). DRC was measured in lymphocytes by a host reactivation assay with a luciferase reporter gene. Women with breast cancer have a DRC that, on average, is 60% lower than women without breast cancer (
DRC detecting the presence of breast cancer ROC Curve (285 cases and 539 Controls).
DRC detecting the presence of breast cancer ROC Curve (285 cases and 539 Controls).
Table
Table
Discussion
While the HCR assay as described in this study assesses the overall DNA repair capacity, it preferentially measures repairs made by the nucleotide excision repair (NER) pathway. NER repairs bulky adducts caused by UV irradiation
The HCR assay, developed in the early 1990s, provided a way to obtain a quantitative, phenotypic measurement of DRC in lymphocytes
Studies published to date of HCR assays of lymphocytes have frequently, but not always
However, when DRC was analyzed either as a dichotomous or as a categorical variable (low, medium, high), the ORs are much higher. Such high ORs are unusual and have been reported only a few studies. For example, Latimer
In this study, we observed an unusually strong increment on the OR after adjustment. Therefore, we repeated the analysis entering, first entering DRC alone in the model; then the covariates were entered one by one. In doing that, we found that the adjusted OR increased gradually as we added each covariate to the model.
No evidence of interaction terms were found in the multivariate logistic model (p>0.05), so DRC can be considered as a potential independent risk factor for DRC because it is biologically plausible, shows a strong association, and exhibits a dose–response relationship after adjusting for all confounders simultaneously.
Despite the usefulness of the HCR assay to estimate DRC and detect BC risk, future prospective studies are needed to further validate the use of this approach. Specifically, further studies are needed to ascertain 1) whether women without BC with a low DRC have a higher incidence of BC compared to those who have a high DRC and 2) whether women with BC and a low DRC are at an increased risk of recurrent BC compared to those with high DRC.
In this study, patients were evaluated for DRC after BC was diagnosed; thus, reverse causation could be present if BC lowers DRC, which would explain why we found a low DRC associated with BC. Nevertheless, reverse causation is an unlikely explanation of our findings because experimental evidence demonstrates that a low DRC or impairments in DNA repair pathways and/or genes is a mechanistic component involved in BC carcinogenesis
Given that newly diagnosed patients are not always truly incident patients, we compared DRC results to tumor size (data not shown) as a surrogate for the length of time period between disease onset and diagnosis to address the potential changes that DRC in lymphocytes might have had after a woman develops BC. With these analyses, we did not find any statistically significant DRC level differences by tumor size that could interfere with our comparisons.
DRC at its cutoff point selected based on its median value (4.3%) was found in this study to have 83.2% sensitivity and 77.6% specificity for detecting BC. ROC analysis showed a high accuracy detecting BC (88.4% of the area under the curve), which was statistically significant. Because any given specific value of DRC level is associated with a quantitative BC risk estimate, DRC has the potential to be a biomarker to rule out BC risk in low-prevalence or low-risk populations or confirm its presence in high-prevalence or high-risk populations. However, a higher specificity and sensitivity would increase the value of DRC as a detector of BC. In addition, these findings need to be validated in other populations and with future prospective studies.
Conclusions
Our findings confirm the hypothesis that a low DRC is an important risk factor for BC. Thus, measuring DRC could be a useful tool to assess BC risk, especially in women from low-prevalence or low-risk populations, such as the population that we studied. For every percent unit of decrease in DRC, there is 64% more likelihood of having BC. Although our results support the validity of using DRC level to estimate BC risk, further studies (including prospective studies) are needed to verify whether such an appraisal would be a useful addition to existing screening and risk-assessment tests for BC, as other covariates (for example, family history, age at first pregnancy) could modify its predictive value.
Abbreviations
BC: Breast cancer; DRC: DNA repair capacity; HCR: Host cell reactivation assay; LUC: Luciferase; NER: Nucleotide Excision Repair.
Competing interest
Two of the authors (JM, MB) were awarded have received a utility patent award (US 8,163,473 B2) from the United States Patent Trade Office for some findings presented in this manuscript.
Authors' contributions
The experiments were performed by EN, LM. Participant recruitment and administration of epidemiological questionnaire was done by WV. JD explained DRC results to some participants, performed ancestry marker analysis and helped with DRC assay internal validation. Participant clinical samples were provided by ME, FSG, ERL, AT, JOR, GB, JGC, JL, RB, SSM, AR and RM. Study design, supervision and preparation of manuscript was done by JM. Epidemiological and biostatistical analysis was done by MB, ES and CA. Manuscript preparation was done by JM and MB. All authors read and approved the final manuscript.
Disclaimer
The opinions and assertions presented in this article are the private views of the authors and are not to be construed as conveying either official endorsement or criticism by the US Department of Health and Human Services, the Public Health Service, or the US Food and Drug Administration.