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Combined Genetic and Nutritional Risk Models of TripleNegative Breast CancerEunkyung Leea, Edward A. Levineb, Vivian I. Francoa, Glenn O. Allena, Feng Gongc, YanbinZhangc & Jennifer J. Hud

a Department of Public Health Sciences and Sylvester Comprehensive Cancer Center,University of Miami Miller School of Medicine, Miami, Florida, USAb Surgical Oncology, Comprehensive Cancer Center and Department of Surgery, Wake ForestUniversity School of Medicine, Winston-Salem, North Carolina, USAc Department of Biochemistry and Molecular Biology and Sylvester Comprehensive CancerCenter, University of Miami Miller School of Medicine, Miami, Florida, USAd Department of Public Health Sciences, Department of Biochemistry and Molecular Biology,and Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine,Miami, Florida, USAPublished online: 14 Jul 2014.

To cite this article: Eunkyung Lee, Edward A. Levine, Vivian I. Franco, Glenn O. Allen, Feng Gong, Yanbin Zhang & JenniferJ. Hu (2014): Combined Genetic and Nutritional Risk Models of Triple Negative Breast Cancer, Nutrition and Cancer, DOI:10.1080/01635581.2014.932397

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Combined Genetic and Nutritional Risk Models of TripleNegative Breast Cancer

Eunkyung LeeDepartment of Public Health Sciences and Sylvester Comprehensive Cancer Center, University of

Miami Miller School of Medicine, Miami, Florida, USA

Edward A. LevineSurgical Oncology, Comprehensive Cancer Center and Department of Surgery, Wake Forest University

School of Medicine, Winston-Salem, North Carolina, USA

Vivian I. Franco and Glenn O. AllenDepartment of Public Health Sciences and Sylvester Comprehensive Cancer Center, University of

Miami Miller School of Medicine, Miami, Florida, USA

Feng Gong and Yanbin ZhangDepartment of Biochemistry and Molecular Biology and Sylvester Comprehensive Cancer Center,

University of Miami Miller School of Medicine, Miami, Florida, USA

Jennifer J. HuDepartment of Public Health Sciences, Department of Biochemistry and Molecular Biology, and

Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami,

Florida, USA

Triple negative breast cancer (TNBC) presents clinicalchallenges due to unknown etiology, lack of treatment targets, andpoor prognosis. We examined combined genetic and nutritionalrisk models of TNBC in 354 breast cancer cases. We evaluated 18DNA-repair nonsynonymous single nucleotide polymorphisms(nsSNPs) and dietary/nutritional intakes. Multivariate AdaptiveRegression Splines models were used to select nutrients of interestand define cut-off values for logistic regression models. Our resultssuggest that TNBC was associated with 6 DNA-repair nsSNPs,ERCC4 R415Q (rs1800067), MSH3 R940Q (rs184967), MSH6G39E (rs1042821), POLD1 R119H (rs1726801), XRCC1 R194W(rs1799782), and XPC A499V (rs2228000) and/or deficiencies in 3micronutrients (zinc, folate, and b-carotene). Combined analysesof these 6 nsSNPs and 3 micronutrients showed significantassociation with TNBC: odds ratios D 2.77 (95% confidenceinterval D 1.01–7.64) and 10.89 (95% confidence interval D 3.50–33.89) for 2 and at least 3 risk factors, respectively. To the best ofour knowledge, this is the first study to suggest that multiplegenetic and nutritional factors are associated with TNBC,

particularly in combination. Our findings, if validated in largerstudies, will have important clinical implication that dietarymodulations and/or micronutrient supplementations may preventor reverse TNBC phenotype, so tumors can be treated with lesstoxic therapeutic strategies, particularly in genetically susceptiblewomen.

INTRODUCTION

Breast cancer is the most common and second leading

cause of cancer death among women in the United States. In

2013, it is estimated that 232,340 new cases will be diagnosed

and 39,620 women will die of breast cancer (1). Gene expres-

sion profiling has enabled the subclassification of breast cancer

into 6 intrinsic subtypes: luminal A, luminal B, human epider-

mal growth factor receptor 2 (HER2)-enriched, basal-like,

normal breast, and claudin-low (2). The presence of the estro-

gen receptor (ER) or HER2 allows for targeted hormonal

therapy or HER2 receptor-specific drugs, herceptin or lapati-

nib. Therefore, treating patients with basal or triple negative

breast cancer (TNBC) that are ER-/progesterone receptor

(PR)-/HER2- is a challenge; the main options are limited to

Submitted 12 June 2013; accepted in final form 10 March 2014.Address correspondence to Jennifer J. Hu, Sylvester Comprehen-

sive Cancer Center, Department of Public Health Sciences, Universityof Miami Miller School of Medicine, 1120 NW 14th Street, CRB1511, Miami, FL 33136. E-mail: Jhu@med.miami.edu

1

Nutrition and Cancer, 0(0), 1–9

Copyright � 2014, Taylor & Francis Group, LLC

ISSN: 0163-5581 print / 1532-7914 online

DOI: 10.1080/01635581.2014.932397

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cytotoxic chemotherapy that are associated with worse quality

of life due to side effects and/or poor prognosis.

The molecular mechanisms of ER downregulation may

include epigenetic factors (e.g., methylation and chromatin

remodeling), proteosomal degradation (e.g., E6-AP and Src),

and hyperactive mitogen-activated protein kinase (MAPK)

(3). The results from various model systems provided some

evidence that TNBC may be associated with deficient DNA

repair and nutritional factors: 1) TNBC and BRCA1-mutated

breast cancer cells are defective in base excision repair (BER)

(4), 2) mismatch repair (MMR) deficient breast cancers are

more likely to be ER- and PR- (5), 3) more frequent aberra-

tions of the double-strand breaks (DSB) sensor complex in

TNBC (6), 4) blueberry phytochemicals inhibit growth/meta-

static potential of TNBC cells (7), 5) high intake of fruit and

vegetables is associated with a decreased risk of ER- and PR-

(8), and 6) polyphenols and sulforaphane in green tea reacti-

vate ER expression (9).

Rare germline mutations in DNA-damage response genes,

such as BRCA1, BRCA2, ATM, FANC, and CHEK2 are associ-

ated with breast cancer susceptibility and highlight the impor-

tance of DNA damage/repair (10). Inter-individual variations

in DNA damage and repair have been associated with an

increased risk of breast cancer (4,11). The extent to which

common genetic variations in DNA-repair genes contribute to

breast cancer risk or TNBC is unknown (12). Complex expo-

sures to different types of DNA damage require multiple repair

pathways to maintain genomic integrity, including BER,

nucleotide excision repair (NER), MMR, and DSBR. The

BER pathway removes DNA damages caused by ionizing radi-

ation, reactive oxidative species, and methylating agents. The

NER pathway plays a critical role in repairing various forms

of DNA damages: bulky adducts generated from genotoxic

compounds, ultraviolet-induced photo lesions, and intrastrand

cross-links. MMR is a highly conserved repair pathway that

functions in improving replication fidelity by correcting repli-

cation-associated base-base and insertion/deletion mispairs.

DSBs may result in cell death or genetic alterations, including

deletions, loss of heterozygosity, translocations, and chromo-

some loss. Our previous study examined genetic variations in

DNA repair and reported that combined nonsynonymous sin-

gle nucleotide polymorphisms (nsSNPs) in multiple DNA

repair pathways was associated with an increased risk of breast

cancer (11). A recent study in Chinese women reported that

nsSNP in hOGG1 gene in the NER pathway is associated with

an increased risk of TNBC (13).

The study results associating diet/nutrition and breast can-

cer risk have not been consistent. However, diet/nutrition may

play roles in subtypes of breast cancer. For example, higher

intakes of linoleic acid, an essential fatty acid, were a greater

risk factor of ER-negative breast cancer than ER-positive

breast cancer (14), whereas multivitamin use was shown to

reduce the risk of ER-/PR- breast cancer (15). Rowe and col-

leagues (16) reported that a polyphenolic bioactive food

compound curcumin induces DNA damage in TNBC cells and

promotes apoptosis. Micronutrients may play critical roles in

the maintenance of genome stability (17).

Our study was designed to pilot test the association between

TNBC and common nsSNPs in 4 repair pathways, including

BER, DSBR, MMR, and NER. Based on the concept that

nsSNPs lead to amino acid substitutions and may result in

altered function, we hypothesize that nsSNPs from different

repair pathways have additive or multiplicative effects on the

risk of TNBC phenotype in breast cancer. Therefore, this study

evaluated 18 nsSNPs in 4 DNA repair pathways. In addition,

we evaluated micronutrient intake using the NCI/Block food

frequency questionnaire (FFQ). We hypothesize that individu-

als with inherited genetic functional defects in DNA repair in

combination with micronutrient deficiencies are at increased

risk of TNBC.

MATERIALS AND METHODS

Study Population

The study design of the parent case-control study was

described previously (11). Briefly, breast cancer cases were

recruited and consented at the Breast Care Clinic of Wake For-

est University Health Sciences and controls were frequency-

matched to cases by age (§5 yr) and race and recruited from

the clinic population receiving routine mammography at the

Breast Screening and Diagnostic Center during 1998–2004. For

study entry, the eligibility criteria included: 1) English-speak-

ing and able to comprehend informed consent; 2) no personal

history of any cancers, including skin cancer; and 3) female at

least 18 yr of age. Newly diagnosed breast cancer cases prior to

any therapy were consented and enrolled. Histopathology and

medical records were reviewed to confirm cancer diagnosis and

ER/PR/HER2 status. Study participants reviewed a description

of the protocol with a research coordinator and signed informed

consent, as approved by the medical center’s Institutional

Review Board. Whole blood (20 ml) was collected from

enrolled subjects and processed within 2 h after phlebotomy.

Every study participant completed a self-administered study-

entry questionnaire that included information on demographics,

reproductive history, medical conditions, smoking history, and

family history (FH) of breast cancer. Positive FH of breast

cancer was defined as a woman with a mother and/or sister with

breast cancer. Ever smoking history was defined as lifetime

smoking history of at least 100 cigarettes. For the current case-

only study, we had genotype data on the first 399 incident breast

cancer cases and 354 (88.7%) cases had available ER/PR/HER2

status data (42 TNBC and 312 others).

Genotyping Analysis

Genomic DNA was extracted from frozen whole blood

using the QIAamp DNA Blood Mini kit (Qiagen, Inc.,

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Valencia, CA). The genetic polymorphisms of interest were

selected based on three criteria: 1) the SNP resulted in an

amino acid substitution; 2) the variant allele frequency was

approximately equal to or greater than 5% in the general

population; and 3) sequence information was available for

accurate assay development. The selected DNA-repair geno-

types were 1) BER: ADPRT V762A (rs1136410), APE1 D148E

(rs3136820), XRCC1 R194W (rs1799782)/R280H (rs25489)/

R399Q (rs25487), and POLD1 R119H (rs1726801); 2) DSBR:

NBS1 E185Q (rs1805794) and XRCC3 T241M (rs861539); 3)

MMR: MLH1 I219V (rs1799977), MSH3 R940Q (rs184967)/

T1036A (rs26279), andMSH6 G39E (rs1042821); and 4) NER:

ERCC2 D312N (rs1799793)/K751Q (rs13181), ERCC4 R415Q

(rs1800067), ERCC5 D1104H (rs17655), and XPC A499V

(rs2228000)/K939Q (rs2228001). The MassARRAY system

(Sequenom, Inc., San Diego, CA) was used to determine geno-

types. Sequences of forward, reverse, and extension primers

used in the analysis of DNA repair nsSNPs were described pre-

viously (11). As part of the quality control protocol, 4 control

samples were genotyped with 92 patient samples on each 96-

well plate, and study cases and controls were loaded on each

plate to minimize systematic bias. The average call rate was

>95% for the genotype assay. The concordance rate for the

quality control samples was 100%.

Assessment of Dietary/Nutritional Intakes

The 110-item NCI/Block 1998 FFQ was used to evaluate

the usual and customary intake of a wide array of nutrients and

food groups including supplement use. The Block FFQ is a

self-administered questionnaire that takes approximately

30 min to complete and has been widely utilized and validated

among several populations (18,19); the validities of the Block

1998 FFQ were moderate to high (pearson correlation coeffi-

cient r D 0.59 for overall and 0.50 to 0.76 for micronutrients

from supplements and food); specifically for zinc (r D 0.62),

folate (r D 0.76), and b-carotene (r D 0.49) in a sample of

Canadian women. Study participants were asked to estimate

their frequencies of eating various foods and normal portion

sizes during the prior year. The analysis of the questionnaire

was conducted at Nutrition Quest, Berkeley, CA. The daily

average intake of 3 major macronutrients, micronutrients

(from diet and supplementations), and USDA MyPyramid

food group servings were obtained from the analysis.

Statistical Analyses

Student’s t-tests and x2 tests were used to compare demo-

graphic, clinical, and dietary variables between cases with or

without TNBC phenotype. Logistic regression was used to

assess the association between nsSNPs and TNBC phenotype

adjusted for age and race. DNA repair genotypes that demon-

strated putative risk associations with an individual odds ratio

(OR) � 1.8 were considered for further polygenic model

analysis. The optimal cut-off values for dietary factors were

determined using the Multivariate Adaptive Regression

Splines (MARS)-logit model as described previously (20,21).

MARS is one of the nonparametric regression methods used to

select sets of important variables adjusting covariates by

cross-validation.

All the dietary factors (macronutrients, micronutrients, and

food groups) were included in the initial MARS model but

were sequentially eliminated if determined to be nonsignificant

contributors to the model. In MARS, we tested for a maximum

of 40 basis functions, and each dietary factor was designated as

a continuous predictor. Bayesian information criterion was

used to determine model fit. Among the dietary factors and

nutrient intakes considered in the MARS model, the optimal

cut-off values for dietary zinc (2.7 mg/day as the cut-off value),

folate supplementation (114.3 mcg/day as the cut-off value),

and dietary b-carotene (1132 mcg/day as the cut-off value)

were selected. These cut-off values were used in logistic regres-

sion models to calculate OR and 95% confidence intervals (CI)

for the association between TNBC and 1) DNA-repair genetic

variants and 2) each micronutrient deficiency, adjusting for

age, race, and total energy intake (22).

In addition, we constructed polygenic models using logistic

regression by summing the number of risk factors for the

SNPs, 3 nutritional factors (zinc, beta carotene, and folate) in

a separate model, and finally summing the number of risk fac-

tors for both the SNPs and the nutritional factors in the com-

bined model. These sums were used as a categorical predictor

of developing TNBC in binary logistic regression models.

Among 354 breast cancer patients, complete dietary data were

available in 235 patients. Therefore we used a sample size of

354 for the association between genotypes and TNBC and a

sample size of 235 for any analysis that involved dietary/nutri-

ent intakes. Age and race adjusted OR and 95% CI were calcu-

lated and linear trend was tested. For dietary/nutrient intakes,

we also included daily energy intake for adjustment. Statistical

analyses were performed using MARS 2.0 software (Salford

Systems, San Diego, CA) and SAS Version 9.3 for Windows

(Cary, NC). Significance level was set at 2-sided alpha D 0.05.

RESULTS

As shown in Table 1, there were 354 breast cancer cases, of

which 42 (11.8%) were TNBC. TNBC cases had a signifi-

cantly higher proportion of younger cases (�40 yr) (21.4% vs.

8.0%, P D 0.007) and African-American women (31.0% vs.

13.5%, P D 0.003) compared to others. Most patients were

postmenopausal (76.0%), overweight or obese (62.9%), and

never smokers (51.7%) with no family history of breast cancer

in their first-degree relatives (80.8%). The majority of patients

had early stage (60.5% in-situ or Stage 1), ER positive

(74.9%), PR positive (59.3%), and HER2 negative (56.5%)

hormone status. There were no differences in FH, smoking his-

tory, body mass index, menopausal status between the two

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groups whereas TNBC cases had marginally significantly

more advanced cancer stage at diagnosis compared with others

(P D 0.053).

The 6 nsSNPs selected for the polygenic model based on

univariate analysis were ERCC4 R415Q, MSH3 R940Q,

MSH6 G39E, POLD1 R119H, XPC A499V, and XRCC1

R194W, as shown in Table 2. Among these 6 nsSNPs risk gen-

otypes, only MSH6 G39E (OR D 2.83, 95% CI D 1.30–6.15;

GE/EE as the risk genotypes) showed significant association

with TNBC risk whereas the other 5 nsSNPs showed sugges-

tive association: ERCC4 R415Q (OR D 4.98, 95% CI D 0.86–

28.85; QQ as the risk genotype); MSH3 940 R940Q (OR D2.86, 95% CI D 0.86–9.55; QQ as the risk genotype); POLD1

R119H (OR D 2.43, 95% CI D 0.92–6.38; RR as the risk geno-

type); XRCC1 R194W (OR D 4.44, 95% CI D 0.75–26.34;

WW as the risk genotype); and XPC A499V (OR D 2.07, 95%

CI D 0.93–4.61; AA as the risk genotype). The results for 12

other nsSNPs genotypes were summarized in Supplemental

Tables 1–3 by DNA repair pathway.

Supplemental Table 4 lists all the dietary factors and nutri-

ent intake levels in TNBC and others. None of the dietary fac-

tors except for zinc supplementation were significantly

different between TNBC and non-TNBC patients. Total

energy intake was 1681.2 § 820.3 Kcal, 14.1% from protein

and 37.3% from fat. In Table 3, we show that 3 micronutrients

were associated with TNBC: significant association with low

TABLE 1

Demographic and clinical characteristics

Others (n D 312) TNBC (n D 42)

Characteristic Categories Total n (%) n (%) P valuea

Age (yr) Mean § SD 57.7 § 12.7 54.3 § 15.2 0.12

�40 32 23 (72%) 9 (28%) 0.007

>40 322 289 (90%) 33 (10%)

Race African American 55 42 (76%) 13 (24%) 0.003

Caucasian 299 270 (90%) 29 (10%)

FHb No 286 250 (87%) 36 (13%) 0.39

Yes 68 62 (91%) 6 (9%)

Smoking historyc Never 183 165 (90%) 18 (10%) 0.53

Former 90 77 (86%) 13 (14%)

Current 43 39 (91%) 4 (9%)

Missing 38 31 (82%) 7 (18%)

BMI (kg/m2) Mean § SD 28.1 § 6.2 29.1 § 8.1 0.33

<25 128 115 (90%) 13 (10%) 0.92

25–29.99 115 102 (89%) 13 (11%)

�30 102 90 (88%) 12 (12%)

Missing 9 5 (56%) 4 (44%)

Menopausal status Pre 85 74 (87%) 11 (13%) 0.72

Post 269 238 (88%) 31 (12%)

Tumor stage In-situ 3 3 (100%) 0 (0%) 0.053

I 210 192 (91%) 18 (9%)

II 109 92 (84%) 17 (16%)

III 30 23 (77%) 7 (23%)

Missing 2 2 (100%) 0 (0%)

ER status Positive 265 265 (100%) 0 (0%) <0.001

Negative 89 47 (53%) 42 (47%)

PR status Positive 210 210 (100%) 0 (0%) <0.001

Negative 143 101 (71%) 42 (29%)

Missing 1 1 (100%) 0 (0%)

Her2 status Positive 154 154 (100%) 0 (0%) <0.001

Negative 200 158 (79%) 42 (21%)

TNBC D triple negative breast cancer; ER D estrogen receptor; PRD progesterone receptor; FH D family history.aP value for test of difference between phenotype groups; t-test for continuous variables and chi-square or Fisher’s exact test for categorical variables.bFirst-degree relatives with breast cancer (mother and/or sister).cLifetime smoking history of at least 100 cigarettes.

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dietary zinc intake (OR D 4.27, 95% CI D 1.59–11.42) and

suggestive associations with low dietary b-carotene intake

(OR D 2.76, 95% CI D 0.99–7.67) or low folate supplementa-

tion (OR D 2.24, 95% CID 0.95–5.27; marginally significant),

adjusted for age, race, and total energy intake.

In Table 4, the analyses using a polygenic model show that

TNBC phenotype was associated with increasing numbers of

risk genotypes and dietary deficiencies, separately and com-

bined. In Table 4A, TNBC was associated with higher num-

bers of risk genotypes compared to women with other breast

cancer phenotypes [2 (OR D 3.57, 95% CI D 1.48–8.64) and

at least 3 (OR D 13.90, 95% CI D 4.50–42.93), respectively]

with a significant dosage-dependent manner (linear trend P <

0.001). In Table 4B, TNBC was associated with at least 2

nutritional deficiencies compared to others (OR D 3.53,

95% CI D 1.50–8.34, P D 0.004). In Table 4C, TNBC was

significantly associated with increasing numbers of combined

genetic and micronutrient risk factors: 2 (OR D 2.77, 95% CI

D 1.01–7.64) and at least 3 (OR D 10.89, 95% CI D 3.50–

33.89), respectively with a dosage-dependent manner (linear

trend P < 0.001).

DISCUSSION

Defective response to DNA damage and repair plays a key

role in carcinogenesis (23). To date, there is limited scientific

knowledge regarding the molecular mechanisms of TNBC.

Our data support the hypothesis that multiple genetic varia-

tions in DNA damage/repair and dietary factors may have

combined effects on TNBC phenotype. We observed an asso-

ciation between TNBC and six DNA repair related nsSNPs,

including BER genes, XRCC1 and POLD1, NER genes,

ERCC4 and XPC, and MMR genes, MSH3 and MSH6. Fur-

thermore, there was an association between TNBC and low

dietary zinc, dietary b–carotene, and folate supplementation.

The 2 BER genes associated with TNBC in our study were

XRCC1 and POLD1. The XRCC1 gene has a polymorphic var-

iant that has been associated with breast cancer risk in Asian

populations (24). In a meta-analysis of over 10 case-control

studies investigating the relationship between XRCC1 R194W

and breast cancer risk found no association (25). However,

none of these previous studies have evaluated subtypes of

breast cancer. The other polymorphic gene POLD1 has not

been associated with breast cancer. It has 3’-5’-exonuclease

activity that removes DNA lesions in close proximity to ioniz-

ing radiation-induced DNA single-strand breaks (26). Alter-

ation in function of this gene could, in theory, predispose

women to radiation-induced breast cancer.

This is the first study to report an association between

TNBC and 2 NER genes, ERCC4 and XPC. Both genes play

important roles in DNA repair. ERCC4/XPF functions as a 50-endonuclease and forms a tight complex with ERCC1 in NER

and it has been implicated in homologous recombination (HR)

and inter-strand cross-link repair (27), and XPC plays a critical

role in DNA damage recognition (28). The 2 MMR genes

associated with TNBC in our study have been associated with

breast cancer in other studies (29,30). High-frequency micro-

satellite instability is detected more frequently in bilateral but

not in unilateral breast cancers (29) and losses of heterozygos-

ity and/or microsatellite instability were detected in 83% of

the skin samples from breast cancer patients, which suggest a

potential role of MMR in breast cancer susceptibility (30).

Previous studies have reported associations between poly-

morphisms in DNA repair genes and breast cancer (4,11,31–

33). One study evaluated genotypic variants of DNA repair

genes in the BER and NER pathways and found synergistic

effects of multiple genes on increased risk of breast cancer,

but only among women with a longer time of estrogen

TABLE 2

Association between DNA repair single nucleotide polymor-

phisms (SNPs) and triple negative breast cancer

(TNBC) phenotype

DNA-repair SNP Genotype Others TNBC OR (95% CI)a

ERCC4 R415Q RR 268 40 Referent

RQ 38 0 NA

QQ 4 2 4.32 (0.75–25.05)

RR/RQ 306 40 Referent

QQ 4 2 4.98 (0.86–28.85)

MSH3 R940Q RR 219 31 Referent

RQ 80 7 0.61 (0.25–1.46)

QQ 12 4 2.56 (0.76–8.66)

RR/RQ 299 38 Referent

QQ 12 4 2.86 (0.86–9.55)

MSH6 G39E GG 266 30 Referent

GE 31 11 3.25 (1.44–7.34)

EE 7 1 1.19 (0.14–10.18)

GG 266 30 Referent

GE/EE 38 12 2.83 (1.30–6.15)

POLD1 R119H RR 236 34 Referent

RH 60 5 0.35 (0.12–1.01)

HH 6 2 0.96 (0.16–5.73)

RH/HH 66 7 Referent

RR 236 34 2.43 (0.92–6.38)

XRCC1 R194W RR 268 37 Referent

RW 37 3 0.54 (0.15–1.86)

WW 4 2 4.23 (0.71–25.13)

RR/RW 305 40 Referent

WW 4 2 4.44 (0.75–26.34)

XPC A499V AA 180 33 Referent

AV 102 9 0.59 (0.26–1.31)

VV 21 0 NA

AV/VV 123 9 Referent

AA 180 33 2.07 (0.93–4.61)

OR D odds ratio; CI D confidence interval.aAdjusted for age (continuous) and race.

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exposure (32). Although this study did not focus on TNBC, it

supports our findings that multiple genetic variants in DNA

repair may have synergistic/additive effects on the risk of

TNBC.

The fact that MARS selected 2.7 mg/day zinc (from animal

dietary source), which is about one third of recommended die-

tary allowances (RDA, 8.0 mg/day for women 19C), as a cut-

off value implicates the importance of bio-active zinc intake

in optimizing zinc function in the body because zinc in plant

foods is less bio-available due to phytate (34). Our finding

associating low dietary zinc from animal source and TNBC

may be supported by previous studies. Farquharson and

colleagues (35) showed that ER- breast cancer tumor tissue

had approximately 80% lower zinc levels than that in ERCtumors. Other studies showed that women with breast cancer

exhibited lower zinc levels in whole blood and scalp (36) and

erythrocytes (37) when compared to controls. Zinc is an essen-

tial component required by many proteins and transcription fac-

tors that regulate important cellular functions, including

response to oxidative stress, DNA transcription, DNA damage

repair, DNA-binding proteins with zinc fingers such as XPA,

DNA damage recognition protein, and the single-strand break

repair protein PARP in the BER and NER pathways (17,38).

Zinc deficiencies may contribute to DNA damage and

TABLE 3

Association between micronutrient intakes and triple negative breast cancer (TNBC) phenotype

Micronutrientsa Others TNBC Crude OR (95% CI) P value Adjusted ORb (95% CI) P value

Zinc (mg/day)

�2.7 162 13 Referent Referent

<2.7 47 13 3.45 (1.50–7.94) 0.004 4.27 (1.59–11.42) 0.004

Folate (mcg/day)

�114.3 112 9 Referent Referent

<114.3 97 17 2.18 (0.93–5.12) 0.073 2.24 (0.95–5.27) 0.065

b-carotene (mcg/day)

�1131.8 185 19 Referent Referent

<1131.8 24 7 2.84 (1.08–7.46) 0.034 2.76 (0.99–7.67) 0.051

OR D odds ratio; CI D confidence interval.aZinc from dietary animal source, b-carotene from diet, and folate from supplementation.bAdjusted for age (continuous), race, and total energy intake.

TABLE 4

Association between combined genetic and nutritional factors and triple negative breast cancer (TNBC) phenotype

Others TNBC

n % n % OR (95% CI)a P valueb

A. Total number of genetic risk factorsc (n D 354)

0 or 1d 145 95% 7 5% Referent

2 148 86% 25 14% 3.57 (1.48–8.64)

3C 19 66% 10 34% 13.90 (4.50–42.93) <0.001

B. Total number of nutritional risk factorse (n D 235)

0 or 1d 173 92% 15 8% Referent

2 C 36 77% 11 23% 3.53 (1.50–8.34) 0.004

C. Total number of combined genetic and nutritional risk factorsf (n D 235)

0 or 1d 127 95% 7 5% Referent

2 66 87% 10 13% 2.77 (1.01–7.64)

3C 16 64% 9 36% 10.89 (3.50-33.89) <0.001

aOdds ratio (OR) and 95% confidence interval (CI) adjusted for age (continuous) and race.bP values; test of linear trend for (A) and (C), or two group difference for (B).cNumber of risk genotypes from 6 nsSNPs selected by univariate analysis; ERCC4 R415Q (QQ as risk genotype);MSH3 R940 (QQ as risk genotype);MSH6G39E (GE/EE as risk genotypes), POLD1 R119H (RR as risk genotype), XPC A499V (AA as risk genotype), and XRCC1 R194W (WW as risk genotype).dThe risk levels of 0 and 1 were combined because they have similar distributions, also to increase sample size of the referent group.eNumber of low intake of three micronutrients: zinc < 2.7 mg/day, folate <114.3 mcg/day, and b-carotene<1131.8 mcg/day.fCombined numbers of genetic and nutritional risk factors.

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epigenetic alterations that could lead to cancer (38) such as

prostate, esophageal, and colon cancers (39,40). The hypothesis

that zinc may interact with DNA repair and contributes to

malignant transformation is supported by our current findings.

Another variable selected by MARS was folate supplemen-

tation and the cut-off value defined by MARS was at 114.3

mcg/day, which was the lowest amount among those who took

folate supplementation. Therefore, our data suggest that any

folate supplementation at least 114.3 mcg/day would be bene-

ficial in reducing the risk of TNBC. The association between

folate supplementation and breast cancer hormone receptor

status has been examined in several epidemiological studies.

First, the VITamins And Lifestyle (VITAL) cohort study

examined the association of the very high intakes of folate,

predominantly from supplements (�1272 dietary folate equiv-

alent) and breast cancer and they showed that women who

consumed higher levels of total folate had a 22% and 62%

decreased overall and ER- breast cancer risk, respectively,

compared to those who consumed low levels of folate (41).

Second, the Nurses’ Health Study also reported that women

who consumed high level of total folate intake, but not dietary

folate alone, had a 54% decrease in ER- breast cancer risk

compared to those consumed the lowest level of total folate

(42). The finding of present study is consistent with these stud-

ies. Third, a prospective cohort study of Chinese women have

found a significant inverse association between dietary folate

intake and overall breast cancer risk (OR D 0.58, 95% CI D0.34–0.99) and nonsignificant inverse relationship with ER-/

PR- breast cancer risk (OR D 0.84, 95% CI D 0.50–1.43) (43).

Lastly, a case-control study conducted in China, also reported

protective effects of high dietary folate intake on overall breast

cancer risk (OR D 0.32, 95% CI D 0.21–0.49) with a sugges-

tive but not significant ER-/PR- breast cancer risk (OR D 0.68,

95% CI D 0.24–1.95) (44). Considering folate’s roles in DNA

methylation and DNA synthesis via folate-mediated one-

carbon metabolism (45), folate deficiency causes incorporation

of uracil into human DNA and chromosome breaks. However,

some studies did not report protective effects of folate on

breast cancer risk (46–49). Furthermore, there was potential

combined effect of low dietary folate intake and polymor-

phisms in the 5,10-methylenetetrahydrofolate reductase 1298

genotype on susceptibility of breast cancer in a cohort of

Chinese women (50).

b-carotene is an antioxidant with potential anticarcino-

genic effects. We observe an association between low lev-

els of dietary b-carotene and TNBC risk. b-carotene cut-

off value was 1131.8 mcg from diet and this is about 94

retinol activity equivalents and corresponds to 13.5% of

RDA of vitamin A. Considering data from NHANES III

(1988–1994) that reported women in the United States

obtain 34% of vitamin A as provitamin A carotenoids, cut-

off value for b-carotene suggests that women who have

lower intake of b-carotene rich foods such as leafy green

vegetables, orange and yellow vegetables, tomato products,

and fruits would have increased risk of TNBC. Several pre-

vious studies support its role in breast cancer prevention

(51–54) and one study reported an inverse association

between the consumption of carotenoid-rich fruits and veg-

etables and premenopausal but not postmenopausal breast

cancer risk (51). Another study showed that women with

higher plasma levels of carotenoids are 43% less likely to

develop new breast cancer (52) and plasma level of carote-

noids were inversely related to oxidative stress (53). Inter-

action between b-carotene and DNA repair genes is also

possible as b-carotene induces apoptosis, reactive oxygen

species, and peroxisome proliferator-activated-g expression.

These activities, in concert with DNA repair, might

account for its antitumor activity (54). However, it is ques-

tionable whether dietary carotenoids have biological func-

tions or are simply a proxy for fruit and vegetable intake.

To the best of our knowledge, this is the first study to sug-

gest multiple genetic and nutritional factors are associated

with TNBC. The strengths of this study include 1) hypothesis-

driven DNA repair SNP selection and testing and 2) incorpo-

rating an innovative data-mining tool, MARS modeling, to

select and define cut-off values of dietary predictors. The

major limitation of this pilot study is the relatively small sam-

ple size and associated statistical power. Our current ongoing

study with a targeted sample size of 2000 breast cancer cases

and 400 TNBC will focus on a more comprehensive evaluation

of DNA repair genotypes and micronutrient deficiencies. In

addition, we will also test several risk loci that were associated

with ER-negative breast cancer but not ER-positive breast can-

cer (55).

In summary, although our preliminary data require valida-

tion in larger independent populations, this pilot study pro-

vides new evidence that multiple nsSNPs from different DNA

repair pathways have additive/multiplicative effects on TNBC

risk, particularly when combined with low micronutrient

intakes. Our findings, if confirmed, will have important clini-

cal implication that newer therapeutic strategies target DNA

repair pathways, such as PARP and other DNA repair inhibi-

tors. Another potential implication is that dietary modulations

and micronutrient supplementations may prevent or reverse

TNBC phenotype for more effective and less toxic therapeutic

strategies, particularly in genetically susceptible women. In

principal, micronutrients may be used to restore ER expression

(9) and reverse TNBC phenotype; then tumors can be treated

with less toxic antiestrogen agents, such as tamoxifen or aro-

matase inhibitors (56).

ACKNOWLEDGMENTS

We acknowledge the contributions of Shirley Cothren,

Judy Lovelace, Nadine Shelton, Joel Anderson, Jianfeng

Xu, S. Lilly Zheng, Jorge L. Rodriguez Gil, Joseph Su,

and the Breast Care Center at the Wake Forest University

Health Sciences. We are grateful to study participants.

NUTRIGENETIC RISK MODELS OF TRIPLE NEGATIVE BREAST CANCER 7

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FUNDING

This work was supported by National Cancer Institute R01

CA73629 and the Florida Biomedical Bankhead-Coley Cancer

Research Program 1BG04 to Jennifer J. Hu.

SUPPLEMENTALMATERIAL

Supplemental data for this article can be accessed on the

publisher’s website.

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