HRAS Protooncogene Polymorphism and Breast Cancer1 · age and race. Our results, adjusted for race and age, showed a positive overall association between the presence of rare HRAS

Vol. 2, 131-138, March/April /993 Cancer Epidemiology, Biomarkers & Prevention 131

HRAS Protooncogene Polymorphism and Breast Cancer1

Peter A. Garrett,2 Barbara S. Hulka, Yoon L. Kim, andRosann A. Farber

Department of Epidemiology, University of North Carolina School ofPublic Health [P. A. G., B. S. H.], and Department of Pathology,

University of North Carolina School of Medicine, [Y. L. K., R. A. F.],Chapel Hill, North Carolina 27599

Abstract

The potential association of polymorphism in the HRASprotooncogene variable repeat region withsusceptibility to cancer has become a controversialtopic. A number of studies have produced results thatappear inconsistent. We report here a multidisciplinarystudy with a combined molecular and epidemiologicalapproach, addressing the specific question of theassociation of rare HRAS alleles and breast cancer.

Extensive questionnaire data and peripheral bloodfor DNA extraction were obtained from 160 cases ofincident breast cancer and from two control groupstotaling 405 unaffected women from five outpatientclinics in North Carolina between April 1990 and June1991. Controls were frequency matched to cases onage and race.

Our results, adjusted for race and age, showed apositive overall association between the presence ofrare HRAS alleles and breast cancer. This relationshipwas somewhat stronger in control group 2 (odds ratio= 3.0; P < 0.01) than in control group 1 (odds ratio =

2.0; P < 0.05). The relationship was 3-6 times strongerin blacks than in whites. In the case series, rare HRASalleles were associated with hormone receptor negativetumors. This association was stronger in blacks andyounger women. There was no confounding or effectmodification by any other breast cancer risk factors.

We conclude that rare HRAS alleles are associatedwith breast cancer and that this association may bestronger in black women than in white women. RareHRAS alleles may also be related to more aggressivetumors, particularly in blacks and younger women.HRAS alleles have the potential to become a valuablescreening biomarker for women at increased risk forbreast cancer.

Introduction

Despite many years of biomedical research, the patho-

genesis of breast cancer is still largely unknown. Epide-

miological and molecular studies strongly suggest thatthe disease is multifactorial, with both genetic and envi-ronmental components (1). In 1981 mutation in the HRASprotooncogene was shown to be associated with theproduction of bladder cancer (2, 3). This was the firstdirect evidence for the existence of a human oncogene.Further study of HRAS showed that it exhibited restrictionfragment length polymorphism (4). Later it was foundthat this polymorphism was produced by differences inthe copy number of a DNA sequence of VNTRs3 con-sisting of 28 bases, located approximately 1 kilobase

downstream from the coding region of the gene (5). Likeall nuclear DNA sequences, these VNTRs are inheritedin Mendelian fashion.

In 1985 Krontiris (6) first suggested that individualswith uncommon alleles of the HRAS VNTR might bepredisposed to a variety of cancers. Since then, a numberof published studies have attempted to confirm this work.All have utilized the case-control study design. Althougha wide variety of cancers have been investigated forassociation with rare HRAS alleles, the overall results havebeen inconclusive. Seven studies have been directed atthe relationship between HRAS alleles and breast cancer.Three have shown a statistically significant relationship(7-9), two were negative (10, 11), and two showed pos-itive but not statistically significant associations (12, 13).

This inconsistency may be due to a number of fac-tors. Study sample sizes have been uniformly small byepidemiological standards. No previous study has ex-amined the possible confounding or modifying effects oftraditional suspected risk factors for breast cancer. Mostinvestigators failed to differentiate between prevalentand incident cases of breast cancer, thus potentiallyselecting for prognostic rather than etiological factors. Informer work it is generally unclear whether the laboratoryanalysis was blind and whether the controls were se-lected from the populations which produced the cases.

Our case-control study considers 160 incident casesof breast cancer diagnosed at breast referral clinics inDurham and Chapel Hill, North Carolina, and two controlgroups obtained from local clinic populations that werefrequency matched on race and age. The research ques-tions to be addressed include: (a) the association of raregermline HRAS VNTR alleles and incident breast cancer;(b) confounding or modification of this relationship bysuspected risk factors for breast cancer; and (c) thepossible association of HRAS alleles and either tumorhormone receptors or local tumor metastasis.

Received 6/30/92.1 Supported by the National Cancer Institute (1R03CA52447-01( and theAmer/can Cancer Society (IN-15-31).2 To whom requests for reprints should be addressed, at t)epartment ofEpidemiology, CB# 7400 McGavran-Greenherg Hall, University of North

Carolina School of Public Health, Chapel Hill, North Carolina 27599.

3 The abbreviations used are: VNTR, variable number tandem repeat;OR, odds rat/i); Cl, confidence interval; BMI, body mass index; ER,estrogen re eptor; PR, progesterone receptor.

on April 8, 2021. © 1993 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

/32 Rare HRAS Alleles and Breast Cancer

Materials and Methods

Study Population. Data were collected prospectively be-tween April 1990 and June 1 991 . Newly diagnosed casesof breast cancer were ascertained from the Duke Uni-versity Multidisciplinary Breast Clinic, the University ofNorth Carolina Hospital Breast Clinic, and the DurhamClinic. There were two control groups. Controls wereselected from these three breast referral clinics plus twogeneral medical clinics at the University of North CarolinaHospitals. All study participants were patients attendingone of these five clinics. They submitted to an inter-viewer-administered questionnaire and donated 20 cc ofperipheral venous blood during the clinic visit. Interviewswere carried out before the patient was seen by a clini-cian and, therefore, prior to a definitive diagnosis ofbreast cancer.

Case Ascertainment. Incident cases consisted of newlydiagnosed patients (less than 6 months postdiagnosis;patients had not received chemotherapy at the time ofentry into the study) seen at one of the three breastreferral clinics. Cases had biopsies that were positive forprimary breast adenocarcinoma, as determined by one

of the university staff pathologists. The majority of caseswere entered into our study in the perioperative period,generally within 2 weeks of diagnosis.

Comparison Groups. Control group 1 consisted of pa-tients seen at the breast cancer referral clinics who hadno history of breast cancer. They had answered thequestionnaire, donated blood, and were diagnosed to befree of breast cancer. All members of control group 1had current mammograms and a clinical breast exami-nation on the day of entry into the study. Patients withcurrent or past breast diagnosis of epithelial hyperplasia,with or without atypia, were excluded from the study.Eligible controls formed a pool of 466 women, fromwhich 202 were jointly frequency matched to cases onage (within 3 years) and race. Selection into the studyfroni the control pool was based on the matching cate-gories and temporal proximity of the enrollment of thecontrol to the diagnosis of each matched incident case.

Control group 2 consisted of patients without ahistory of proliferative breast disease who were attendingtwo general medical clinics at the University of NorthCarolina for non-breast-related complaints. These partic-ipants were also frequency matched to the cases on raceand age. Since this group was selected late in the study(after the cases were known), all eligible patients whoconsented to participate from this group were enteredinto the study.

The Questionnaire. The questionnaire was administeredto all participants by a trained interviewer. It had beenpretested on another clinic population for ease of admin-istration and comprehensibility. Virtually all known orsuspected risk factors for breast cancer were evaluatedvia the questionnaire. These included race, age, bodybuild, educational attainment, ethnicity, alcohol and cig-arette use, obstetric and gynecological history, sex hor-mone exposure, personal and family history of cancer,

and past breast biopsy and mammography.

Patient Recruitment. Participation at the five study sitesis summarized in Table 1. Of 1055 identified, eligibleparticipants, 1009 were interviewed. The 46 others metour inclusion criteria but refused participation in thestudy. A total of 871 samples of peripheral blood were

Ti/i/c 1 PartiCipation iif siihje Is h� studs linic

LJN(1-1 UN(F-1 UN(IDurham Duke Total

Br: Ft Mcii.

Refused 0 .30 7 1 1/ 46Nii blood 0 1 09 6 3 2/) 1 .3/3lntervie�s 1)1(1 bloiid 39 544 /34 3 5 160 87/

Total 39 683 97 /9 197 1055

., LJNCF I Br., 1.)niversity iif North Carolin.i I liispital Bre’,ist Cliiie ; UNC) IF I’, Uni� c’rsity 1)1 North Carolina Hi)spit,il F,ui#{236}il�Pr,i( Ii(e (.lini : UNC)

Med., L)i#{236}iversitv ut Niirtb C,iriilina I 051)/Ill tsic’di il (lini

obtained (86.3% of those interviewed). Participation pro-

portion was approximately equal at each of the breastreferral clinics. Outright refusals were few, and theseoccurred at the referral clinics in proportions similar tothose at the two general medical clinics. Only 16 of the46 refusees were cases. The mean age and ethnic distri-

butions were similar for the participants and the refusees.

DNA Extraction. Peripheral blood was collected by phle-botoniy at the conclusion of the clinic visit. The wholeblood was stored at -80’C until DNA extraction fromleukocytes. The DNA was purified from nuclei and iso-Iated from whole blood, using the salting out method ofMiller and Bulbrook (14).

DNA Analysis. DNA was double-digested with restrictionenzymes Mspl and HpaIl. This digested genonhic DNAwas subjected to electrophoresis in O.8% agarose gels inTris-acetate buffer at 45 V for 22-24 h, denatured, andtransferred from the gel to MSI nylon membrane by theblotting method of Southern (15). The membrane wasthen washed and placed under a shortwave (254 nm) UVlight source for 30 s to bind the DNA fragments to thenienibrane (16). It then underwent prehybridization (atechnique to block the nonspecific attachment of theprobe to the nylon membrane) and hybridization with a1 .0-kilobase HRAS VNTR probe prepared by the randomprimer labeling method of Feinberg and Vogelstein (17)

using a Boehringer Mannheim labeling kit. After hybridi-zation the blots were washed five times under increas-ingly stringent conditions. Blots were exposed to X-rayfilm for autoradiography for 24 to 48 h.

Gels were run so that the smallest allele (1 .0 kilobase)migrated at least 18 cni. A 1-kilobase DNA ladder (BRL)and standards for each of the four comnioti alleles wererun on each gel. Autoradiograms were interpreted by anindividual who was blinded to disease status for all casesand referral clinic controls. Medical clinic controls wereenrolled near the conclusion of the study. Their DNAwas run separately and read independently by two of us,without disagreement.

DNA samples for which band sizes were equivocal

were mixer] with an internal standard of the nearestcommon allele and run again on another gel to moreprecisely differentiate uncommon from common allelesizes (see Fig. 1).

Two different hormone receptor assays were used

on each tumor specimen: a biochemical (dextran-coated

charcoal) (18) and a monoclonal antibody assay (19). Thesample was considered positive for hormone receptorsif either assay result was in the positive range.

Data Analysis. Descriptive statistics of breast cancer riskfactors were �valuated by a Wilcoxon rank-sum test for



Tab/c 2 ! !RAS allele distributions, by length in kilobases

Numbers represent the allele frequencies. Because each person contrib-

utes two alleles, the numbers on this table are twice the size of therespective sample sizes for individuals in our study population.

Allele category Cases (%) Control 1 (%( Control 2 (%(

obcdefghi

Conimon�1.0001.450

2.050

2.500

Total

Intermediate50.950

0.9751.0251.0501.0751.1001.150

1.175

2.450

2 (0.6)

4 (1.3)4 (1.3)2 (0.6)5 (1.2)

2 (0.6)4 (1.3)

0 (0.0)

2 (0.6)

4 (1.0)

2 (0.5)1 (0.3)4(1.0)5 (1.2)

5 (1.2)1 (0.3)

3 (0.7)4 (1.2)

#{149}-2.500

-2.050

I 450- I.

179 (55.9) 21 1 (52.2) 233 (57.4)

38(11.9) 53(13.1) 42 (10.3)

1.175- I 050

0.950 - . #{149}51� .� .� , � : �27 (8.4) 50 (12.4( 34 (8.4)24(7.5) 43(10.6) 49 (12.1)

268 (83.8) 357 (88.4) 358 (88.2)

6 (1.5)

2 (0.5)fig. 1. Autoracliogram of a representative Southern blot showing IIRAS 1 (0.3)alleles of various sizes. Lane a, homozygosily for the 0.950 allele; Lane h, 5 ( 1 .2)homozygosity for the 1 .000 allele. All others are heterozygous, except 1 (0.3)for Lane e, which demonstrates a mixed sample with a known standard. 1 1 (2.7)This allows differentiation between the similarly sized 1.000 and 1.050 2 (0.5)alleles. 4 ( 1 .0)

4 (1.0)

Total 25 (7.8) 29 (7.2) 36 (8.9)

continuous variables (20) and the Pearson x2 statistic (21)for nominal categorical variables. Estimates of relativerisk were computed using unadjusted and adjusted Man-

Rare’� 1(0.3) 1(03) 1(0.3)

11275 0 1 (03) 0tel-Haenszel ORs and 95% CIs to control individually for 1:300 1 (0.3) 0 0

breast cancer risk factors (22). Unconditional logisticregression via a backward elimination strategy was used

for joint control of these factors as well as for the evalu-

1.350 0 0 1 (0.3)

� � 1(03) 1 (03)11600 � 0.3) 1 (0.3) 0

ation of effect modification (23). Individual and global 1.650 2 (0.6) 0 1 (0.3)

tests for effect modification were evaluated on a multi-plicative scale with maximum likelihood ratio x2 esti-mates at a 0.10.

1.700 2 (0.6) 1 (0.3) 0

� 3(1.0) 1 (03) 1(0.3)1.775 1 (0.3) 0 0

1.800 1 (0.3) 0 01.825 1 (0.3) 0 0

Results 1.900 i (0.3) 0 0

The original case series consisted of 202 patients seen,interviewed, and phlebotomized at the three breast can-

3(0.7) 1(0.3)

11975 � (03) 0 0

cer clinics. The cases were frequency matched to con- 2.000 0 2 (0.5) 0

trols by age and race. Within each breast referral clinicthe number of cases and controls was virtually identical.After data collection was completed, 41 of the cases

2.125 0 0 1 (0.3)� � (0.6) � (0 5) 0 (0.3)

2.375 i (0.3) 0 1 (0.3)

were found to be prevalent and were removed from thecase series. One additional case of malignant phyllodestumor was also deleted. The remaining case series (n =

160) was restricted to adenocarcinomas: 34 patients with

2.400 1 (0.3) 0 1 (0.3)

� � � �

2900 � (0.3) 0 03.300 o 0 i (0.3)

intraductal carcinoma; 121 with invasive ductal carci-noma; and 5 with lobular carcinoma. Total 27 (8.4) 18 (4.4) 12 (2.9)

Table 2 shows the HRAS allele distributions by study

series using the established classification of HRAS alleles“ 0 “ ‘ ‘ ,‘

as common (frequency >7 /o), intermediate (fre-quency 0.5%-1 .5%), and “rare” (frequency <O.5%) (24).

Grand total 320 404 406

Population gene frequency >0.07., Population gene frequency 0 005-0 015.

‘ Population gene frequency <0.005.

Our population presented a total of 44 different allelesofthe HRAS VNTR. As has been demonstrated previously(24), the four common alleles represent approximately

90% of the control population. The stated sizes of the in Table 3. The table indicates the frequency-matchedcommon alleles are based upon our careful measurementof their migration under the electrophoretic conditions

age and race variables, as well as others, including edu-cation, BMI, family history of breast cancer (two or more

of our protocol and may differ slightly from sizes pub- first- or second-degree relatives aged 50 years or older,lished elsewhere. The case series contains a higher fre- or one of more first- or second-degree relative youngerquency of rare alleles (8.4%) than control group 1 (4.4%) than 50 years at diagnosis), alcohol use (at least once aor control group 2 (2.9%). There is no specific allele size month for 6 months), reproductive history, and oral con-that is overrepresented in either series. traceptive and hormone replacement therapy use. Infor-

A summary of selected breast cancer risk factors for mation on ethnic background of the study participantsthe case series and for each control group is presented was obtained for parents and grandparents. A total of 34

Cancer Epidemiology, Biomarkers & Prevention 133



1,ihle I S(’l(’(I(’(l l)reast C ,lii( er risk fa tons by study series

lndi .11(11 P V,)li.ies repr(’s(’nI diff(’rences betwe(’n a specifi Control group

,,I’ � 0.05.i, I’ � 0.01.

‘ B,ised upon a n)oditi(’d Ftollingshead scale.d �iI)t(’(t5 were asked for country of origin of I)arents and gr,1n(l�)an(’nts:50111)’ of this information svas unknown to the sul)/(’(I.

1.14 Rare HRAS Alleles and Breast Cancer

4 P. A. Carr(’It, subn#{236}itlecl for �illi ,ition.

and ti ie ( ,)S(’ series on i partiCular fa Ion.

- .Risk f,� Ion

Cases (%((ii = 160)

Control 1 (%)

(n = 202 )Control 2 (%)

ri = 203)

Age 54.2 54.1 54.0

Ra �‘

White 1 139 (86.9) 1 73 (851,) 1 74 (85.7)

Black 18 (1 1.2) 25 (12.4) 24 (11.8)

Other 3 1.9) 4 (2.0) 5 (2.5)

BMIMean 26.3 25.0” 27.7

Edu ,ifloo<High S( hool grantI- 6gb S( hool ,incl sonit’

C ollegeFour or 11)1)0’ sears of

( ollege

O � i.ipatiofl group’I (iSS

134 6

FI gb7 9

Aliihol drinkerPresentFonriier

Never

M(’iiar( he (nie,oi age)

Menopause ( man agi’(

No. of �regnani esMean .3. 1 2 .8 3.8”

Age first pregnantMean 22.1/ 23.1 21.0”

Age at first birthMean 2-1/) 2-1.)) 2t.7�

Parity (iiiean( 2.7 2. 3 3.0�

Ever or,il (. oot r,iceI)t iv).’

U5(’

‘(‘(‘S 8 3 15 1 .9) 1 (3 3 ) S 1 .0) 98 48. 3)

No 77 48. .3) 49 49.0) 1 05 ( S 1.7)

Hiirnl(ine repla( emenl1i5(’

Yes 56 ) 35.0) 80 1.39.0) 70 ( 34.6)No I 04 (65.0) 1 22 (6 1 .0) 1 32 (65.4)

Past ili,inlilii)gr,iii)s

Me,ul no. �3#{149}() 4.9 3.4

I 1/story of lienigii I)reasl

(lisease‘�(‘s 6) /4 3.4) 1 1 3 155.9)” 7.3 1 t(.0)

No 90 (56.6) 89 (44. 1 ( 1 I/o (64.0)

First-degree relatives(an( en 80 (50.0) 96 (47.5) 9 1 (44.8)Breast Cancer .31 (19.4) 47 (2.3.3) 25 12.3)

Se i)n(I-degr(’e relal ives(an.er /3.3 (51.9) 144 71. 3)’ 108 /5.3.2)

Bre.isl ( ,Incer .35 (2 1 .9) 70 1.34.7)” 2 5 ( 1 2 . 3

Ethnic gR)i.i�)5 )%(

Britain ,ind Ireland �3C)#{149}5 4 1 .2 34.2

Niirlhern Europe 1 7.8 1 4.4 18.6

Southern Europe 2.5 4.7 1 .4Afri an American 8. 3 /3.9 10.3Native Anienican 1 .6 2.4 .3.8

Asian 1 .3 0.7 1.0South Aiiierican 0. 3 1 .0 0.5

Middle East 0.0 1). 5 0.2

Unknown” 28.8 26.2 25.0

27 (16.9)87 )54.4(

46 (28.7)

/7 (11.7)

78 /513.4)

St /34.9)

(is (40.6)12 (7.5)

/3.3 (51.4)

1 2.6

44.9

17 (8.4)”

111 (55.0)

74 ( 36.6)

13 (7.1)

95 (51.9)

75 (41(3)

10�I (54.0)’

15 (7.4)

78 (138.6)

1 2.8

413.7

64 (31.5)’81 /40.0)

6(/ (29.5)

55 .31.1)”64 (.3(,.t)

58 (.32.13)

(,4 (31.5)’

3)) (14./3)/�i3 /537)

1 2.6

41.6’

ethnic groups were represented in our study POI)ulatiOn.There was no significant difference in ethnic distributionbetween cases and either control group.

It can be seen that the control groups differ some-what both from each other and from the case series. Thebreast referral clinic control group (control group 1 ) isstatistically significantly less obese, has a higher educa-tional attainment, is more likely to drink alcohol, has astronger past history of benign breast disease, and hashigher proportions of relatives with cancer and breastcancer than the case series. Of these six differences, fiveare opposite in direction from concensus associations of

former studies (24). This inverse association is consistentwith the demographic make-up and referral patterns tothe I)reaSt clinics, i.e., patients may be referred basedpartially on the presence of known risk factors.

The medical clinic control group (control group 2)

contains subjects with lower educational and occupa-tional attainment, fewer alcohol drinkers, lower age atmenopause, higher gravidity and parity, lower age at firstpregnancy and live birth, and fewer relatives with breastcancer than the case series. All of these nine significantdifferences are in a direction imparting a lower risk ofbreast cancer to those women in control group 2 than inthe case series, which is consistent with former studies.

Potential confounders were evaluated for their as-sociation with HRAS allele status in our combined controlpopulation (Table 4). Race and oral contraceptive usewere associated with !IRAS allele category. Race wasstrongly associated with [IRAS allele status, with blacks

having approxiniately 6 tinies the population gene fre-quency of rare alleles (0.1 8) as whites (0.03).� Oral con-traceptive use was also positively associated with rare

IIRAS allele status. These variables were evaluated aspotential confounders in the analysis.

Crude odds ratios were calculated for the case seriesand each control group, separately, for the association ofindividuals bearing (a) an intermediate allele (Table 5,interniediate) verstis those with only comnion alleles or

(I)) a rare allele (Table 5, rare) versus only common alleles.The ORs were stratified on the frequency-matched fac-

tors, age and race, individually and jointly using uncon-ditional logistic regression. Univariate adjustment forother factors, including BMI, education, occupation, fam-ily history of breast cancer, alcohol intake, smoking his-tory, pregnancy, �)arity, age at first pregnancy, oral con-traceptive use (ever, first, duration), and hormone re-placertient therapy use (ever, first, duration, current) hadvirtually fl() effect on the odds ratio.

Joint confounding by any combination of these risk

factors was tested by simultaneously placing all risk fac-tors in the model with the HRAS allele category. Theresultant ii estimates and ORs were changed less than2% for either control group froni those models using only

race and age.Adjusting for race, the associations were generally

stronger (a) for individuals with a rare allele versus thosewith only common alleles compared to those with anintermediate versus only common alleles for both controlgroups and (1)) in the comparison of cases with controlgO)U�) 2 than in the coniparison of cases with control




Table 4 Associations of potential confounders with HRAS alleles in individuals in the combined control groups

Each risk factor is tested for association with ra re alleles versus all others combined.

.Risk factor

Rare alleles�(n = 29)

All other alleles”(n = 376)

P OR

Race

Black 10 39 <0.0002 4.4

White 19 328

Age

<50 15 146 0.17 1.7�50 14 230

Education<High school 3 76 0.42 NA’High school-some college 16 176Four or more years of college 10 124

BMIHigh 17 193 0.45 1.34

Low 12 183

Family history of breast cancer

Yes 10 144 0.68 0.85No 19 232

Alcohol useYes 14 204 0.53 0.79No 15 172

Times pregnant

<2 5 92 0.12 NA

2-3 18 160>3 6 124

Age first pregnant

<18 6 112 0.23 NA18-29 29 240

>20 4 24

Oral contraceptive use

Never 9 195 0.04 NA<5 years 9 107

�5 years 1 1 74

Hormone replacement therapy useNever 19 237 0.94 NA<5 years 6 79�5 years 4 60

Individuals either heterozygous or homozygous for a rare allele.

b Individuals without rare alleles.

C Odds ratio is not computable, as dl is greater than 1.

group 1. The strongest relationships were produced foreach control group when the common and intermediatealleles were combined into one category (Table 6). Allassociations on Tables 5 and 6 are stronger when controlgroup 2 is used as the comparison group.

Selected risk factors were modeled individually andwith their product terms with HRAS allele category, usingunconditional logistic regression. Maximum likelihoodratio x2 estimates and P values (df = 1) were calculatedto assess interaction on a multiplicative odds scale (Table7). Modeled variables included were race, age, familyhistory of breast cancer, BMI, education, alcohol use,

age at first pregnancy, gravity, parity, oral contraceptiveuse (ever, first, and duration), and hormone replacementtherapy (ever, first, duration, and current use). ORs werestronger for blacks than whites using either control group,but the effect for blacks compared to whites was greaterfor case comparisons to control group 2 (OR = 1 1.0versus 1.7) than control group 1 (OR = 4.0 versus 1.5).

No effect modification on a multiplicative scale by anyother variables was found when these were modeledindividually.

To assess global interaction in the exposure-diseaserelationship, the two control groups were modeled sep-arately. The above-listed breast cancer risk factors weretested simultaneously. Maximum likelihood ratio x2 forinteraction were not statistically significant at P = 0.10for any factors, other than race, for either control group.

Associations of rare HRAS alleles with estrogen andprogesterone receptors in tumors and with the presenceof lymph node metastasis were evaluated for the caseseries (Table 8). Of the 160 cases there were only 104for whom data were available on hormone receptoractivity. This was due to (a) the presence of small tumorswith too little tissue for hormone receptor determinationand (b) some patients having their excisional surgerycompleted later at sites other than the referral clinichospitals. Of these 104 tumors, a few assays showed



136 Rare HRAS Alleles and Breast Cancer

Table 5 Odds ratios for cases versus each control group, separately

HRAS allele categories are classified as common,a intermediate,b andrare.’

Cases versus control group 1

Intermediate (n = Rare (n = 40) versus

39) versus common common (n = 283)In = 283)

OR 95% Cl P OR 95% Cl P

Crude 1.2 0.6-2.2 0.67 1.9 1.0-3.6 0.07

Control forRace 1.4 0.7-2.9 0.29 2.3 1.1-4.9 0.02

Age 1.2 0.6-2.2 0.62 1.9 1.0-3.8 0.06Race and age 1.5 0.8-3.0 0.25 2.4 1.1-5.1 0.02

Cases versus control group 2

Intermediate (n =Rare (n = 35) versus

45) versus commoncommon (n = 283)

(n = 283(

OR 95%Cl P OR 95%Cl P

Crude 1.0 0.5-1.8 0.88 2.7 1.3-5.5 <0.01Control for

Race 1.2 0.6-2.4 0.60 3.2 1.4-7.1 <0.01Age 1.0 0.5-1.8 0.76 2.7 1.3-5.7 <0.01Race and age 1.2 0.6-2.4 0.58 3.2 1.4-7.2 <0.01

a An individual homozygous for common alleles.

b An individual with at least one intermediate frequency allele and no

rare alleles.S An individual either heterozygous or homozygous for a rare allele.

borderline activity by both techniques used. These cases(representing 1 1 ER assays and 6 PR assays) have beenexcluded from the hormone receptor analysis. Therewere 121 cases for whom lymph node metastasis infor-mation was available.

There was a suggestion of an association betweentumor hormone receptor negativity, both ER (OR = 2.1)and PR (OR = 3.2), and rare HRAS alleles, but theserelationships were not statistically significant in the totalcase series (Table 8). There appeared to be no associationbetween lymph node metastasis and rare HRAS alleles(OR = 0.6). When stratified on selected risk factors, thestrongest relationships found were for race and age, withboth black and younger women being at greater risk forhormone receptor negative tumors, in the presence of arare HRAS allele. In a separate analysis, the effects of ageand race in this relationship were found to beindependent.

Although some of the relationships shown in Table8 are strong, most are not statistically significant, perhapsdue to the small sample size resulting from limiting theanalysis to the case series and from stratification intosubgroups. Nevertheless, there is a suggestion in our datathat young women and black women with breast cancerand rare HRAS alleles may be predisposed to having amore aggressive form of the disease (i.e., hormone re-ceptor negative tumors).

Discussion

Our findings have confirmed a positive relationship be-tween rare alleles of the HRAS protooncogene VNTR andbreast cancer. Previous molecular studies of this questionhave been inconsistent, perhaps due to factors outlinedabove. Among these is the problem of securing an ade-

Table 6 Odds ratios for cases versus each control group, separately

HRAS allele categoni es are classified as rare v ersus nonrare.

Cases (n = 160) ver-

sus control 1 )n =

202)

Cases )n = 160) versuscontrol 2 (ii = 203)

OR 95%Cl P OR 95%Cl P

Crude 1.8 0.9-3.6 0.08 2.7 1.3-5.6 <0.01Control for

Race 2.1 1.0-4.4 0.04 3.0 1.4-6.5 <0.01Age 1.9 0.9-3.6 0.07 2.7 1.3-5.6 <0.01Race and age 2.0 1.0-4.5 0.04 3.0 1.4-6.1 <0.01

quate comparison group. Our decision to utilize twocontrol groups was an attempt to (a) select a valid controlgroup from the source population which produced thecases and (b) detect and avoid a selection bias producedby overmatching controls to the cases.

Breast referral clinic comparison patients originatedfrom the same study base as the cases. These womenare at high risk for breast cancer and tend as a group tobe similar to the cases on most potential confoundingfactors, known and unknown. Descriptive data presentedin this study demonstrate that control group 1 is com-parable to the case series on most traditional breastcancer risk factors. This partial matching on potentialconfounders is an advantage of the use of members ofthe breast clinic populations without diagnosed breastcancer as a source of controls. However, there is theintrinsic possibility that HRAS VNTR allele status, ourfactor of interest, will become overmatched by the ex-clusive use of this control group. Therefore, our medicalclinic group was selected as a second set of controls toprovide a validation of the results produced using ourbreast clinic control group. As shown in Table 3, thissecond control group is less comparable to the cases onthe basis of known breast cancer risk factors than iscontrol group 1 . These differences are predictable, basedupon the known demographics of our clinic populations.There are no known disparities between this secondcontrol group and the case series which might producea spurious positive association with the factor of primaryinterest, HRAS alleles.

Both the crude and adjusted outcomes were positivewhen we used each control group, although they differin magnitude, being generally stronger when comparingcases with control group 2. This is supportive of the viewthat selection bias may be operating in the comparisonof control group 1 to the case series due to overmatch ingof the exposure variable.

We would expect any effect modification present tobe similarly affected by this process. The only strongeffect modifier in this study is race. Consistent with thisargument, the effect modification of rare HRAS allelesand breast cancer, by race, is stronger for control group2 than for control group 1 . This may be indicative of theselective referral of high-risk patients to the breast clinicsbased upon known risk factors, independent of race.Alternatively, an unidentified positive bias may exist incontrol group 2, accounting for the stronger odds ratioswhen comparing it to the case series. From the results ofstratified analysis and the separate and joint modeling ofbreast cancer risk factors we can conclude that there wasno confounding by any nonmatched risk factor in our




Table 7 Effect modification on selected variables for cases versus eachcontrol group

Stratum-specific odds ratios compare rare versus all other alleles with95% confidence intervals and P values for interaction.a

C ases v

gersus controup 1

rol C ases versus contrgroup 2

ol

n OR CI P” n OR Cl P

Crude 362 1.8 0.9-3.6 363 2.7 1.3-5.6

RaceBlackWhite

43312

4.01.5

1.1-14.70.6-3.7

0.22 42313

11.0 2.4-51.11.7 0.7-4.2

0.03

Age (yearsl

<50

�50

144

218

2.5

1.9

0.9-6.4

0.7-5.2

0.68 141

222

3.7 1.3-10.7

2.5 0.8-7.3

0.61

Family historyBreast cancer

Yes

No

130

232

2.3

2.10.6-8.90.9-4.7

0.91 141223

1.6 0.5-5.54.3 1.6-11.9

0.21

BMIHighLow

166196

1.43.6

0.5-3.51.2-10.8

0.17 192171

4.7 1.4-15.21.9 0.7-5.0

0.23

Education>High

school�High

school

218

144

1.9

2.6

0.8-4.6

0.8-8.8

0.66 179

184

4.0 1.2-13.2

2.3 0.8-6.4

0.47

a Interaction is on a multiplicative scale, calculated using maximum like-

lihood ratio x2.

b p the probability of no interaction by the specified subgroupings.

study population. Additionally, since individual HRAS

allele status is unknown prior to specialized laboratoryanalysis it is difficult to envision a selective force produc-ing a distortedly low prevalence of rare HRAS alleles incontrol group 2. This could, of course, occur by chance.

The odds ratios, adjusted for the frequency-matchedvariables, show a 2- to 3-fold increase in risk in breastcancer for those exposed to a rare HRAS allele. Thisadjusted relationship appears to be partially due to theeffect modification of race. In the United States, whiteshave a slightly higher incidence of breast cancer overall

than do blacks (25). However, given the presence of arare HRAS allele, blacks in our study population appearto be at a much greater risk (3-1 1 times) than whites(1.5-2 times) for breast cancer. This difference betweenracial groups reaches statistical significance at P = 0.05for control group 2 only.

Although the racial disparity in the frequency distri-bution of HRAS allele groups has been reported previ-ously (26, 27), the question of effect modification by racefor rare HRAS alleles and breast cancer has not beenaddressed, since earlier reports of the association of rareHRAS alleles and breast cancer have been restricted towhites. Although our results are novel, they should beviewed as exploratory and in need of confirmation inother, larger populations. Nevertheless, this is the firststudy showing an association between racial groups anda molecular marker in breast cancer.

Breast tumors in blacks and in younger women aremore likely to be ER and PR negative than are tumors ofwhites and older women (28, 29). ER negativity confersa worse prognosis and is found in more advanced tumors(30). We have found an association between rare allelesof HRAS and hormone receptor negative tumors both forblack and for younger women in our breast cancer series.Our results do not appear to be confounded by tumorstage, since there is no association between HRAS allelesand stage of disease in our study, as manifested by theabsence of an association of rare HRAS alleles with lymphnode metastasis. If the relationship is independent oftumor stage, we may infer that rare alleles of HRAS areindependently associated with more aggressive tumorsin both black and younger patients. This conclusion mustalso be regarded as a newly generated hypothesis, re-quiring verification. If supported, it could account, inpart, for the higher breast cancer mortality and lowersurvival rates known to exist in blacks (31).

The role of HRAS alleles in the pathogenesis of breastcancer is unclear. It has been suggested that they mayact as enhancers for gene transcription. This has beenshown to occur in vitro (32, 33). Alternatively, the HRAS

VNTR might coexist in linkage disequilibrium with an-

other unidentified gene on chromosome 1 1 that is itselfcausal of breast cancer (34). Finally, rare HRAS allelesmay represent a characteristic of inherited instability of

Table 8 Odds ratios for HRAS allele status by estrogen receptor, progesterone receptor, and axillary lymph node metastasis status within strata of race,age, and family history among breast cancer cases

HRAS allele classifications are rare versus all other alleles.

Estrogen receptor Progest erone receptor Lymph n ode metastasis

n OR 95% CI P” n OR 95% Cl P n OR 95% CI P

Crude 93 2.1 0.6-7.0 98 3.2 0.9-11 121 0.6 0.2-1.7

Race

Black 13 12.0 0.8-181.3 0.06 14 14.0 0.6-675.1 0.17 17 0.7 0.1-11.4 0.39White 78 0.5 0.1-4.7 81 1.4 0.1-15.0 104 2.6 0.3-59.4

Age<50 31 6.4 0.6-167.2 0.08 34 14.3 1.3-371.0 0.05 43 1.9 0.3-15.9 0.10�50 62 0.8 0.1-5.6 64 1.1 0.2-8.0 78 0.3 0.1-1.3

Family history

Positive 34 2.6 0.0-107.3 0.76 36 13.4 0.6-305.4 0.12 41 0.6 0.1-5.9 0.94Negative 59 1.6 0.3-7.7 62 1.7 0.4-7.9 80 0.6 0.2-2.6

a Probability of no inte raction by the specified subgroupings.



138 Rare HRAS Alleles and Breast Cancer

genomic DNA and be a marker for cancer susceptibility,rather than a causal agent.

At present there is no molecular marker for thoseeither at high risk for breast cancer or for those at risk fora more aggressive form of breast cancer. If the presentwork is verified, the development of HRAS alleles as ascreening marker, facilitating the identification of individ-uals at high risk, would be a positive public healthoutcome.

Molecular aspects of the etiology of breast cancerare just beginning to be appreciated. Although epidemi-ological risk factors for this disease have been studied foryears, their predictive value is limited. Perhaps multidis-ciplinary approaches to this problem, combining epide-miological design with the emerging molecular laboratorytechnology, will provide us with additional insight into

the pathogenesis of this major human cancer.

AcknowledgmentsWe are indebted to Drs. I. Dirk Iglehart, George S. Leight, lames F. Huth,

James S. Wilson, and Arthur T. Evans for providing access to their patientsin our recruitment of study participants. We are appreciative of Dr.

Kenneth McCarty’s providing us with hormone receptor assay results.We are especially grateful to Dr. loanne M. Garrett for her assistance incomputer and data analysis.

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1993;2:131-138. Cancer Epidemiol Biomarkers Prev P A Garrett, B S Hulka, Y L Kim, et al. HRAS protooncogene polymorphism and breast cancer.

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