MODIFIERS OF RISK OF HEREDITARY BREAST AND OVARIAN CANCER

© 2002 Macmillan Magazines Ltd

A small proportion of cancers in adults is attributable tothe effects of mutations in known cancer-susceptibilitygenes. The hereditary proportion varies according tothe type of cancer, and the relative importance ofgenetic and environmental factors varies between pop-ulations. The hereditary proportion of ovarian cancer isamong the highest of all common forms of cancer inadults1. Malignancies for which the hereditary propor-tion is similar or higher include pheochromocytoma2,medullary thyroid cancer3 and cancer of the fallopiantube4, but these are rare. There are familial componentsfor most other common cancers, and genetic testing isnow available for familial breast cancer, colon cancerand melanoma, but for each of these the hereditaryfraction is probably below 5%. The genes responsiblefor the hereditary forms of prostate, lung, pancreaticand testicular cancer are yet to be found.

The best studied of the adult cancer syndromes ishereditary breast and ovarian cancer. Multinationalstudies of gene carriers have been used to generate riskestimates and have led to the identification of severalrisk modifiers. Knowledge of risk, and of modifiers ofrisk, can help women to choose treatments; for exam-ple, depending on her level of risk, a woman might optfor intensive screening or for prophylactic surgery. Theelucidation of modifying factors might also help to clar-ify pathogenic mechanisms. So, what are the modifiersfor hereditary breast and ovarian cancer?

Approximately 10% of women who are diagnosedwith breast cancer report a family history of the condi-tion, but only a minority of these will be found to carry

a germ-line mutation in one of the two known suscep-tibility genes — BRCA1 and BRCA2 (BOX 1). A BRCA1or BRCA2 mutation will be found, using conventionalsequencing, for most families with an inherited suscep-tibility to breast–ovarian cancer, but for fewer than halfof the women from families that have many cases ofbreast cancer, in the absence of ovarian cancer5.However, the probability that a mutation will be foundin a family depends on the number and age-of-onset ofthe breast and ovarian cancers in the family, the pene-trance of the gene (see below) and the prevalence ofmutations in the underlying population. For example,it is estimated that one in 45 Jewish women carries a BRCA1 or BRCA2 mutation6, and families ofAshkenazi Jewish ancestry with an increased incidenceof cancer are particularly likely to harbour mutationsin these genes7. Countries with a high prevalence ofmutations owing to FOUNDER EFFECTS include Iceland8

and Poland9 (BOX 1).

PenetrancePenetrance refers to the probability of developing dis-ease in a carrier of a deleterious mutation and is usu-ally defined in terms of a given age (for example, toage 70). However, as a non-negligible risk of cancerexists among carriers of two non-mutant alleles (spo-radic cancer rate), and penetrance estimates are basedon the number of cancer cases observed in a cohort ofsusceptible individuals, the estimate is the sum of therisk attributable to the susceptibility allele and theunderlying sporadic rate.

MODIFIERS OF RISK OF HEREDITARYBREAST AND OVARIAN CANCERSteven A. Narod

Hereditary breast and ovarian cancer is among the most commonly encountered adult geneticdisease, and it is increasingly important that geneticists, oncologists, surgeons and gynaecologistsare aware of the issues regarding risk assessment, prevention and management of women withinherited susceptibility to cancer. Genetic risk can be modified by external factors, but what arethese factors, and how might our knowledge of them help us to better define the risks forindividual women and to develop strategies for cancer prevention?

FOUNDER EFFECT

The occurrence of manyindividuals in a population withthe same mutation of aparticular gene, owing to itsinheritance, over manygenerations, from a commonancestor (founder).

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The Centre for Research on Women’s Health,Women’s College Hospital,790 Bay Street, Room 750,University of Toronto,Toronto, Canada M5G [email protected]: 10.1038/nrc726

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period immediately following the disclosure of genetictest results. Increased efforts at early detection will havethe effect of increasing the penetrance estimate over theshort term.

Because of these difficulties, alternative methods havebeen used. These are based on family studies, or on thepedigrees of unselected cancer cases or members of thegeneral population. In general, the genotype is establishedfor the PROBAND (ascertained or sampled individual) andpenetrance is estimated by examining the phenotypes(and sometimes genotypes) of the first-degree relatives.For example, Struewing and colleagues sampled 5,318Ashkenazi Jews in the Washington area, performedBRCA1 or BRCA2 genotyping on them, and then esti-mated the cumulative incidence of breast cancer in thefirst-degree relatives of the carrier and non-carrier sub-groups6. Penetrance was estimated using the kin-cohortmethod devised by Wacholder10 (BOX 2). The kin-cohortmethod is appealing in its simplicity, but is only feasible inpopulations in which a founder effect is present andwhen the prevalence of the mutant allele is high (forexample, at least 1%). Otherwise, it would be necessary togenotype hundreds of individuals to identify a single car-rier. Penetrance estimates that are based on a single com-mon mutation of a gene in a founder population mightnot be applicable to all mutations and to all populations.Other penetrance estimates have been constructed usingunselected cases of breast and ovarian cancer in Jewishwomen11,12, and of ovarian cancer in Ontario1.

It is difficult to estimate penetrance by studyingfamilies with a high incidence of cancer who attend agenetics clinic, because it is likely that the number ofaffected individuals in the family influences the chancethat the family will attend a genetics clinic (‘ascertain-ment’) and that genetic testing will be offered. Familieswith BRCA1 or BRCA2 mutations vary substantially interms of the number of affected relatives — a familywith five cases of early-onset breast cancer is muchmore likely to attend a clinic and be offered testingthan a family with only two cases. If only the mostextremely affected families are used to construct therisk estimate, then penetrance might be overestimated.However, it is possible to adjust for ascertainment andto model penetrance using only unaffected women infamilies. For example, consider an unaffected 40-year-old daughter of a known BRCA1- or BRCA2-mutationcarrier. If the penetrance of the gene is 100% to age 40,then her likelihood of being a carrier is zero. By con-trast, if the penetrance is zero, then her likelihood is50%. By sampling and genotyping unaffected first-degree relatives of carriers, and then calculating theproportion of carriers by age, it is possible to generatean unbiased estimate of penetrance.

The lifetime risk of breast cancer in women whocarry a deleterious BRCA1 or BRCA2 mutation isestimated to be as high as 80%13,14 — roughly tentimes greater than that of the general population —but several estimates are lower6,8. So far, methodsthat are based on family studies have generatedhigher risk figures than methods based on singlecases, unselected for family history. One estimate of

Penetrance estimates are commonly used by geneticcounsellors to communicate risk estimates, and are alsoused to model and to describe the effect of modifyingfactors. The magnitude of the effects of MAJOR GENES andof GENETIC MODIFIERS is described by relative risk. Formajor genes, such as BRCA1 and BRCA2, the relativerisk is usually defined as the incidence of the conditionamong carriers, divided by the incidence among non-carriers. The risk of cancer among women who carry amutation in BRCA1 or BRCA2 can be modified by asecond gene or by an environmental factor. For modify-ing factors, the relative risk is the penetrance of the disease among individuals with the modifying factor,compared with the penetrance of the disease amongthose without the modifying factor. Relative risks can beestimated directly from the HAZARD RATIO in a cohortstudy, or can be estimated from the ODDS RATIO in acase–control study, providing that the disease is rare(BOX 2). However, the rare disease assumption might nothold for a population of mutation carriers.

Measuring penetrancePenetrance can be estimated in several ways. Ideally, acohort of healthy carriers of the susceptibility gene inquestion is followed for a defined period of time andincident (new) cases of cancer are recorded. The cancerrate — calculated from the number of new cases and theperson-years of observation — is used to construct apenetrance estimate. This method does not depend onfamily members reporting cancers of relatives; instead,large cohorts of healthy carriers are used, and often tenor more years of follow-up are required. It is importantthat the relevant ‘at-risk’ group is properly defined. Forexample, when estimating the penetrance of ovariancancer among carriers of BRCA1 or BRCA2 mutations,the cohort should be restricted to women with intactovaries, and when the penetrance of breast cancer is esti-mated, women with prophylactic mastectomy should beexcluded. A further concern is related to the accelerationof diagnosis by screening or prophylactic surgery in the

MAJOR GENE

A gene for which there are oneor more alleles that confer a highlifetime risk of cancer (or otherdisease). Refers to the high riskof disease, not the frequency ofmutations.

GENETIC MODIFIER

A gene for which there are oneor more alleles that confer amodest, but significant, alteredrisk of cancer (or other disease).Usually refers to a gene thatmodifies the risk of the diseasein carriers of a mutation in amajor gene, but might alsomodify risk of disease in thegeneral public.

HAZARD RATIO

The risk of cancer (or otherdisease) in the exposed group,divided by the risk in theunexposed group. Derived froma cohort study.

ODDS RATIO

The odds of developing cancer(or other disease) in the exposedgroup divided by the odds ofdeveloping it in an unexposedgroup. Usually derived from acase–control study.

PROBAND

The family member who wasinitially ascertained (that is,came to the attention of theresearcher) in a study of familialaggregation of cancer (or otherdisease).

Summary

• Most, but not all, women who carry deleterious mutations in BRCA1 or BRCA2 willdevelop breast or ovarian cancer. The risk of cancer among mutation carriers variesbetween families and countries. Several study designs can be used to estimate diseasepenetrance and modifiers of penetrance.

• Reasons for variation include different mutations in the same gene (allelic variation)and the effect of modifying genes.

• Genetic modifiers of breast cancer risk include the androgen receptor, the steroid-hormone receptor coactivator NCOA3 and possibly the DNA-repair gene RAD51.

• Genetic modifiers of ovarian cancer risk include the HRAS1 oncogene and, possibly,the androgen receptor.

• Preventive oophorectomy, a non-genetic modifier, reduces the risk of ovarian cancerand of breast cancer in BRCA1-mutation carriers.

• Non-genetic modifiers of breast cancer risk include tamoxifen and reproductivehistory (such as parity and breastfeeding).

• Non-genetic modifiers of ovarian cancer risk include oral contraceptives and (inBRCA1-mutation carriers) tubal ligation.

© 2002 Macmillan Magazines LtdNATURE REVIEWS | CANCER VOLUME 2 | FEBRUARY 2002 | 115

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tion carriers continues to rise, which could implythat ovarian hormonal factors are relevant for main-taining risk in BRCA1-mutation carriers to a greaterdegree than in BRCA2-mutation carriers.

Modifiers of penetrance: allelic variationThe penetrance figure is an average for a population,but penetrance might vary between populations,within populations and between individuals. Thereare several potential sources of variation. Allelic varia-tion is due to different mutations of a single gene —for example, the effect of a truncating BRCA1 orBRCA2 mutation might differ from that of a missensemutation. Also, the position of the BRCA1 or BRCA2mutation within the coding region of the gene might

breast cancer penetrance among BRCA1-mutationcarriers has been made using prospective data. In acohort of 76 BRCA1-mutation carriers fromRotterdam, eight invasive cancers were detected dur-ing 318 person-years of follow-up15 — an annualincidence of 2.5%. Assuming a constant incident rateover 30 years of risk, this translates to a penetranceof approximately 75% for the Dutch population.This prospective study supports the validity of thehigher, family-based risk estimates. Examination ofthe differences in the penetrance curves of BRCA1-and BRCA2-mutation carriers might provide cluesabout potential modifiers. For example, after age 50,the risk of breast cancer in BRCA1-mutation carriersseems to stabilize, whereas the risk for BRCA2-muta-

Box 1 | BRCA1 and BRCA2

BRCA1 functions• Transcription factor: involved in the regulation of oestrogen receptor activity and related to the control of oestrogen-

induced proliferation of breast tissue.

• DNA repair: homologous recombination and repair of transcription-coupled oxidation-induced DNA damage.

• Cell-cycle checkpoint control: interacts with RB, ESF1 and p53.

• Chromatin remodelling.

BRCA1 domains

• The RING-finger domain is a protein–protein-interaction domain, and binds BRCA1-associated RING domain 1(BARD1). The carboxy-terminal BRCT domain is necessary for BRCA1’s function in DNA repair and transcriptionalactivation.

• The highlighted mutations (denoted by *)are the two common founder mutations found in Ashkenazi Jews.

BRCA2 functions

• DNA repair: homologous recombination.

• Cell-cycle checkpoint control: interacts with p53.

• Chromatin remodelling.

BRCA2 domains

• Exon 11 contains both the ovarian cancer cluster region (OCCR) and eight BRC repeats, which are required forbinding to RAD51. Mutations in this region are associated with a greater risk of ovarian cancer and a lower risk ofbreast cancer than are mutations outside this region.

• The highlighted mutations (denoted by *) are the common founder mutations found in Ashkenazi Jews and in Iceland.

1 32 4 5 6 7 8 109 11

19 2021 22

23 25 26

17 18 23

12 13

14 15 16 17 18 24 27

Ovarian cancer cluster region (OCCR)

BRC repeat

Distal exon 27mutations notpathogenic

RING-fingerdomain

BRCT Domain

2 3 5 6 7 8 11 12 13 14 15 16 24

19 2021

229 10

BRCA1

1,863 amino acids

BRCA2

3,418 amino acids

* *

* *

* Jewish mutation * Icelandic mutation


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exon 9 (999 del 5) (BOX 1; REF. 8). In both countries, a sin-gle mutation accounts for most cancer cases that areattributable to BRCA2, and therefore penetrance esti-mates that are derived from studies in these countries aremutation specific. By contrast, for the United States orthe United Kingdom, the penetrance estimate is based ona wide range of deleterious mutations.

Modifiers of penetrance: modifying genesAnother potential source of variation is that of modi-fying genes. The penetrance of a major gene might beconditional on the presence of one or more specificalleles of a genetic modifier. There could be one ormore risk alleles (and one or more protective alleles)of the modifier. Modifier genes might or might nothave an effect on cancer risk in the absence of a muta-tion in the major gene. If the modifying gene is LINKED

to the major gene then a modifier allele might CO-SEGREGATE with the mutant allele and most carriersin the family will have at least one copy of the modi-fier allele in common. In different families, however,carriers might have different alleles of the modifier,and significant interfamilial penetrance differencesthat are attributable to the modifier might occur. Ithas been proposed that the gene for 17β-hydroxy-steroid dehydrogenase-2 is a linked modifier of ovar-ian cancer risk in carriers of BRCA1 mutations20, butthese data are preliminary. A more likely scenario isthat the modifier is unlinked to the major gene. Inthis case, different carriers within a given family arelikely to be discordant at the modifier locus.

It is possible that a particular allele of a modifier geneis frequent in one population and infrequent in another.In this case, intrapopulation variation in penetrance canresult. So far, such an occurrence has not been seen.However, the frequency of certain alleles of candidatecancer-susceptibility alleles — such as those of the

influence the risk of breast or ovarian cancer: BRCA2mutations that occur in the terminal portion of exon27, and which result in the production of a nearly full-length protein, are believed not to be deleterious16

(BOX 1). The reasons for the observed differences inpenetrance that are associated with the position of theBRCA1 or BRCA2 mutation are not yet known.Possible reasons include whether residual function isassociated with partial-length proteins; whether criti-cal binding regions of the protein are involved (partic-ularly for missense mutations); whether a dominant-negative effect is present (that is, the mutant proteinimpairs the activity of the normal gene product of theother allele); and whether the stability of the mutantRNA molecule is impaired (FIG. 1). Several studies haveshown that the risk of ovarian cancer among carriersvaries with the position of the mutation within thegene for both BRCA1 and BRCA2. Among BRCA2-mutation carriers, the risk of ovarian cancer is greatestfor women with mutations contained within the ovar-ian cancer cluster region (OCCR: defined by nucleo-tides 4,075–6,503) (BOX 1). The risk of ovarian canceris increased 1.9-fold for mutations within this region,and the risk of breast cancer seems to be decreased17.Among BRCA1-mutation carriers, the relative pro-portion of ovarian cancer compared with breast can-cer seems to be greater for mutations within the 5′ two-thirds of the gene18. It has not yet been estab-lished whether this gradient is due to a lower risk of ovarian cancer or a higher risk of breast cancer for3′ BRCA1 mutations, but a recent study by Risch andcolleagues1 supports the latter hypothesis.

Allelic variation can explain differences in penetrancebetween families and between countries. For example,the common BRCA2 mutation in Israel is a one-base-pair deletion in exon 11 (6,174 del T)19 and the commonBRCA2 mutation in Iceland is a five-base-pair deletion in

GENE LINKAGE

Two genes, or genetic sequencevariants, that are situated closelyon the same chromosome andthat tend to co-segregate inmeiotic cell divisions.

CO-SEGREGATE

The co-inheritance of linkedsequence variants (for example,a marker and a disease gene)through two or moregenerations in a family.

Box 2 | Types of epidemiological study

• Cohort study. A study that follows a group of people, who do not initially have the disease, over time and measures therate of disease that ensues. Cohort studies are usually defined in terms of two groups — one of which is subjected to aspecific exposure or intervention, such as oral contraceptives — the disease experiences of which are then compared.A historical cohort study traces the experience of the group from some time in the past until the present. A prospectivestudy follows the group in real time from study entry to a time in the future.

• Case–control study. A study in which a group of individuals who are affected with a disease is compared with a groupof individuals without the disease, for a given exposure(s). For example, case–control studies can be used to comparethe use of oral contraceptives or tubal ligation in women with BRCA1 mutations. If tubal ligation protects against ovariancancer, then women with ovarian cancer (cases) are less likely to have had a tubal ligation than women without ovarian cancer (controls). The results are expressed in terms of an odds ratio. An odds ratio of less than one isindicative of a protective effect.

• Association study. A type of case–control study in which the exposure variable is a particular allele of a candidatesusceptibility gene. The frequency of the particular allele is measured in a group of patients and compared with thefrequency in an ethnically matched control group.

• Meta-analysis. A statistical evaluative technique in which the results of several case–control studies, or cohort studiesor both, are combined to generate a summary measure of the odds ratio. The individual odds ratios are combined, butare weighted by some function of the variance of each odds ratio.

• Kin-cohort study. A study design used to estimate penetrance. The sample of individuals is unselected for familyhistory. Genetic testing is performed on the subjects, and the family histories of the mutation carriers are comparedwith the family histories of the non-carriers.


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In the case of hereditary breast and ovarian cancer,the candidate modifier genes that have been studiedare related to the metabolism of sex hormones and toDNA repair. Four genes have been suggested as poten-tial modifiers of risk in BRCA1- or BRCA2-mutationcarriers (TABLE 1), but the evidence for these is tooweak to be used to guide clinical practice.

Androgen receptor. The extent to which genetic varia-tion influences the level of steroid-hormone activity inthe breast is not yet known, but candidate genes includethose involved in hormone synthesis and metabolism,as well as hormone receptors and their co-regulators.The androgen receptor contains a polymorphic poly-glutamine tract (encoded by variable numbers of thecodon CAG), which ranges in length in the general pop-ulation from 17 to 26 glutamines. In vitro, the degree oftransactivation of the receptor by androgen variesinversely with the length of the repeat23. In an associa-tion study (BOX 2) of breast cancer cases from Quebec(unselected for family history), Giguere and colleagues24

reported that women with a mean CAG length of 19.5units or fewer had half the risk of breast cancer ofwomen with a mean repeat length of 20 units or longer(p = 0.007). The study group was relatively large (255incident cases) and the subjects were genetically homo-geneous (French–Canadians). These results indicatethat increased androgenic activity is protective againstbreast cancer, but two earlier studies did not find suchan association25,26. BRCA1 has been shown to be a co-activator of the androgen receptor27. Rebbeck andcolleagues studied allelic variation of the androgenreceptor in 304 BRCA1-mutation carriers28. They foundthat women with at least one long androgen receptorallele (>27 CAG repeats) were diagnosed with breastcancer at a younger age than those with two shorter alle-les, in keeping with the theory that high androgenicactivity is protective. Dagan et al. did not confirm theeffect in Jewish carriers of BRCA1 or BRCA2 muta-tions29, whereas Levine and Boyd30 found that amongJewish BRCA1-mutation carriers with ovarian cancer,those with a short androgen receptor allele were diag-nosed at an earlier age than women without a shortallele (p = 0.004). This observation is in keeping withthe finding of Helzlsouer et al.31 that women with highlevels of circulating androgen were at increased risk ofdeveloping ovarian cancer. Together, these studies indi-cate that the influences of androgen activity on breastand ovarian cancer risk might be in opposite directions.However, at present, there are too few data on theseassociations to support definite conclusions.

NCOA3. Oestrogen is required for normal mammary-gland development and for reproductive function32.Oestrogen exerts its cellular effects by binding to theoestrogen receptor. Nuclear receptor coactivator 3(NCOA3; also known as amplified in breast cancer 1,AIB1) is a member of the family of co-activators thatinteract with steroid-hormone receptors, to enhanceligand-dependent transcription32. NCOA3 is amplifiedin 10%, and overexpressed in 64%, of breast tumours33.

CYP3A4 gene, which codes for a member of thecytochrome P450 family and is involved in metabolismof certain steroid hormones — varies dramaticallybetween populations. Genetic variation in CYP3A4 hasbeen associated with the risk of prostate cancer inmen21. Population differences in allele frequencies mightcontribute to differential cancer susceptibility in differ-ent ethnic populations21,22.

a RNA stability

b Critical binding region disrupted

c Truncated proteins

d Missense mutations

BRCA1

Functional protein is not produced

Protein is unable to interact with its binding partners, so cannot carry out its normal function

Short proteins are oftennon-functional

Missense mutations have different effects, depending on whether they produce less/non-functional proteins or dominant-negative proteins

Figure 1 | Possible reasons for penetrance differenceswith different mutations in a given gene. a | Mutations thataffect RNA stability result in a reduction in the amount ofprotein that is produced by the mutant allele. This effectivelyalters the ‘dose’ of the active gene product in a cell. b | Mutations that affect critical binding regions, or otherimportant functional domains, can be deleterious. c | Truncation mutations — caused by frameshift or nonsensemutations — affect penetrance by producing proteins ofreduced length. These proteins are usually inactive, or showminimal residual function. For tumour-suppressor genes, this isa common mechanism of inactivation of the first allele (that is,the first hit). d | Missense mutations might affect penetranceaccording to whether they produce a protein with impairedfunction, or a dominant-negative protein that also interfereswith the activity of the normal gene product, which is producedfrom the other allele. Most missense mutations in BRCA1 arebelieved not to be deleterious, but those in critical domains,such as the RING-finger domain of BRCA1, are associatedwith an increased cancer risk. There are no known deleteriousmissense mutations in BRCA2.

Table 1 | Effects of modifier genes on breast and ovarian cancer risk

Breast cancer Ovarian cancer

BRCA1 BRCA2 BRCA1 BRCA2

Androgen receptor ↓↑ ? ↓↑ ?

NCOA3 ↑ ? ? ?

RAD51 – ↑ ? ?

HRAS1 ? ? ↑ ?

↓↑, The androgen receptor polymorphism might decrease or increase the risk of breast or ovariancancer in BRCA1-mutation carriers, depending on the specific genotype; ↑, increased cancer risk; –, no modifying effect seen; ?, not studied.


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cancer types39. In the first study of a genetic modifier ofcancer risk in BRCA1-mutation carriers, Phelan andcolleagues40 reported that the presence of a rare allelewas associated with an elevated risk of ovarian cancer,but not of breast cancer (odds ratio 2.8; p = 0.002). It isnot yet known whether the VNTR polymorphism influ-ences HRAS activity or expression, or is a marker forgenetic instability. So far, only one study has been con-ducted on the association between HRAS and hereditarybreast–ovarian cancer, so it cannot yet be concluded thatthis is a modifying gene.

Modifiers of penetrance: hormonal factorsEnvironmental and lifestyle factors might also modifythe risk of breast and ovarian cancer among carriers ofBRCA1 or BRCA2 mutations (TABLE 2). Based on theinspection of multiple-generation families, it has beenproposed that the risk of breast cancer has increasedamong BRCA1-mutation carriers — that is, the pene-trance of the gene for women born after 1930 wasgreater than for those born earlier41. This trend indicatesthat environmental factors (of increasing prevalence)could modify the risk.

It should be noted that cohort effects, such as thosedescribed above, are different from the phenomenon ofgenetic anticipation. In genetic anticipation, the pene-trance of a gene increases with subsequent generationsafter the de novo occurrence of the mutation.Anticipation is often due to unstable (dynamic) muta-tions, but for some conditions — such as hereditaryretinoblastoma — the basis for anticipation is unex-plained42. However, in contrast to a cohort effect, there isalso no general relationship between penetrance andcalendar year with genetic anticipation. Furthermore,the common BRCA1 or BRCA2 mutations are believedto have occurred at least 50 or more generations ago43,and it is improbable that the penetrance could increasesignificantly over just a few generations because ofmutation instability.

Oophorectomy. In an early study, we found the incidenceof breast cancer in BRCA1-mutation carriers to be max-imal in the age group 40–55 and to decline slightlythereafter44. This observation indicates that ovarian hor-mones might be involved in promoting breast carcino-genesis. Oophorectomy — removal of the ovaries — hasbeen found to be protective against breast cancer inBRCA1-mutation carriers in two studies. Rebbeck et al.compared the breast cancer risk in a historical cohort ofBRCA1-mutation carriers, some of whom had under-gone an oophorectomy and some of whom had bothovaries intact45. The estimated relative risk of breast can-cer that was associated with oophorectomy was 0.53(95% CI 0.33–0.84), which corresponds to a 47%decline in breast cancer risk. Eisen and colleagues car-ried out a case–control study on BRCA1- or BRCA2-mutation carriers with breast cancer, and matched con-trols without breast cancer46. Among BRCA1-mutationcarriers, the risk of breast cancer in women who had hadan oophorectomy was decreased by 61% (odds ratio0.39; 95% CI 0.20–0.75). A protective effect was also

A CAG-repeat polymorphism in the NCOA3 genedetermines the length of a polyglutamine tract — sevenalleles have been identified that range in size from 20 to37 repeat units — and is believed to have a functionaleffect on sex-steroid-hormone signalling34. Rebbeck etal. found that BRCA1- or BRCA2-mutation carrierswho also carried an NCOA3 allele with 29 or morerepeats were at an approximately threefold greater riskof developing breast cancer than carriers with twosmaller alleles34. The biological basis of this observationis not known, but the study supports the hypothesisthat the oestrogen pathway also influences the risk ofbreast cancer in BRCA1- or BRCA2-mutation carriers.The NCOA3 gene polymorphism has also been associ-ated with bone mineral density35, but a relationshipbetween bone mineral density and breast cancer risk inBRCA1- or BRCA2-mutation carriers has not beenestablished so far.

RAD51. RAD51 is involved in the repair of double-strand DNA breaks, and interacts with both BRCA1 andBRCA2 (REF 36). In two studies, a single-nucleotide poly-morphism in the 5′ untranslated region of RAD51 wasreported to be associated with an increase in breast cancer risk in BRCA2-mutation carriers37,38.Wang et al.37

reported a positive association between the presence ofthis allele and breast cancer in BRCA2-mutation carriers(odds ratio 3.2; 95% confidence interval (CI) 1.4–4.0),and, in a study of Ashkenazi Israeli women, Levy-Lehadet al.38 confirmed this elevated risk (odds ratio 4.1; p =0.07). These results indicate a modifying effect ofRAD51, but both study populations were small and fre-quencies of the risk allele in the various control groupsvaried widely. Neither group saw a significant modifyingeffect for BRCA1-mutation carriers.

HRAS. A non-coding polymorphism of the VNTR(variable number of tandem repeats) type is situatedone kilobase downstream of the HRAS proto-oncogeneon chromosome 11. The frequency of the individualVNTR alleles varies within the population from verycommon (> 50% of alleles) to very rare (< 1%)39,40. Ithas been proposed that the presence of one or more ofthe rare alleles was a risk factor for a wide range of

Table 2 | Effect of modifying factors on breast and ovarian cancer risk

Breast cancer Ovarian cancer

BRCA1 BRCA2 BRCA1 BRCA2

Oophorectomy ↓ ↓? ↓ ↓

Pregnancy* ↑ ↑? ? ?

Breastfeeding ↓ ↑? ? ?

Oral contraceptives ↑? ↑? ↓ ↓

Tubal ligation – – ↓ ↓?

Tamoxifen ↓ ↓ – –

Hormone-replacement ? ? ? ?therapy

↑?, suggested increase in cancer risk, but uncertain; ↓?, suggested decrease in cancer risk, butuncertain; ↑, significant increase in cancer risk; ↓, significant decrease in cancer risk; ?, not studied; –, no modifying effect seen. * The pregnancy effect was seen for early-onset (40 years) breast cancer only.


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Minnesota53. Mutation analysis was not performed. Theyfound that having ever used an oral contraceptive wasassociated with a significantly increased risk of breastcancer among sisters and daughters of the probands(relative risk = 3.3; 95% CI 1.6–6.7). These risks aremuch higher than those observed for the general popu-lation. In the Oxford overview analysis of oral contracep-tives and breast cancer, current use of birth-control pillswas associated with a relative risk of 1.2 (REF. 54). A large-scale, matched, case–control study of oral contraceptivesand breast cancer in BRCA1- or BRCA2-mutation carri-ers is now underway.

It is important to establish whether oral contracep-tives are hazardous to the breast, because their use hasbeen proposed as a preventive measure against ovariancancer. We have reported a protective effect of oral con-traceptives in three case–control studies of BRCA1- orBRCA2-mutation carriers55–57. In a recent study of 232ovarian cancer cases and 232 controls, oral-contracep-tive use was associated with a 56% reduction in the riskof ovarian cancer (p = 0.002). Cases and controls werematched for year of birth, mutation (BRCA1 orBRCA2) and country of residence. However, in acase–control study from Israel, Modan and colleaguesdid not find oral contraceptives to be protective againstovarian cancer in the subset of women with BRCA1 orBRCA2 mutations58. The reason why the Modan studydid not show an effect is unclear, but it might be due tothe fact that the controls were not closely matched tothe cases in terms of age (patterns of use of oral contra-ceptives are strongly related to the year of birth). Amongthe Israeli patients in our study, a strong protective effectwas seen (odds ratio 0.15; 95% CI 0.03–0.75)57.

Tubal ligation. Ligation of the fallopian tubes (tubal lig-ation) has been found to be protective against ovariancancer in the general population59 and among BRCA1-mutation carriers56. We have reported an adjusted rela-tive risk of 0.39 (95% CI 0.22–0.70) for tubal ligationand subsequent ovarian cancer (a risk reduction of61%). The combination of tubal ligation and oral con-traceptives provided 72% protection. The mechanismof risk reduction is unclear. Surprisingly, tubal ligationdoes not seem to affect the risk for BRCA2-mutationcarriers. It has been suggested that tubal ligation inter-rupts ovarian blood flow, but this mechanism is vague.It is also possible that tubal ligation blocks the passage ofcarcinogens from the external environment to theovary, but no offending carcinogens have been identi-fied. Piek et al. suggest that the cells of origin of ovariancancer in BRCA1-mutation carriers arise in the fallop-ian epithelium and are transported to the ovary, andthat tubal ligation interrupts this passage60.

Tamoxifen. TAMOXIFEN is an anti-oestrogenic drug that isroutinely used in the treatment of oestrogen-receptor-positive breast cancer (see the review article by Ali and Coombes on page 101 in this issue). Tamoxifenhas also been shown to be of value in reducing the risk of primary invasive and pre-malignant breast cancer in high-risk women in North America61, and of

observed among BRCA2-mutation carriers, but this didnot reach statistical significance (odds ratio 0.56; 95% CI0.16–1.95). Together, these studies indicate thatoophorectomy might be used as a strategy to decrease therisk of breast cancer among BRCA1-mutation carriers,although it must be recognized that in young women theprocedure is associated with acute and long-term sideeffects. Further work is required before this operation canbe recommended to BRCA2-mutation carriers.

Pregnancy. In the general population, pregnancy offersprotection against breast cancer after the age of 40, butseems to increase the risk for very early-onset breastcancer47. This is consistent with the hypothesis that theovarian hormones produced during pregnancy aremitogenic, and accelerate the growth of existingtumours. During pregnancy, breast differentiationoccurs, and afterwards the population of susceptiblecells is reduced48. This might explain why pregnancyprevents breast cancers at a later age. In the general pop-ulation, only a small proportion of breast cancers occurbefore age 40, and pregnancy confers an overall advan-tage. Early-onset breast cancers are typical amongBRCA1-mutation carriers, however, and a high propor-tion of cancers occur before age 40 in this group. Twogroups have found pregnancy to be a risk factor forearly-onset breast cancer in BRCA1- or BRCA2-muta-tion carriers. Johannsson and colleagues reported tenpregnancy-related breast cancers in 37 BRCA1- orBRCA2-mutation carriers, compared with the expected3.7 (REF. 49). Jernstrom et al. reported that the risk ofbreast cancer increased with each pregnancy in BRCA1-or BRCA2-mutation carriers before the age of 40 (REF. 50).This was true for BRCA1- and BRCA2-mutation carriers,but was only significant for the former group.

Breastfeeding. A preliminary case–control study ofbreastfeeding and breast cancer in BRCA1- or BRCA2-mutation carriers reported a protective effect in womenwith BRCA1 mutations, but not with BRCA2mutations51. The study group was matched for parityand for year of birth. BRCA1-mutation carriers whobreastfed for more than one year were 40% less likely tohave breast cancer than those who breastfed for ashorter period (p = 0.01). The risk of breast cancerdeclined as the lifetime duration of breastfeedingincreased (adjusted p for trend = 0.02).

Oral contraceptives. There is much concern amongwomen who are BRCA1- or BRCA2-mutation carriers,and their doctors, that oral contraceptives might increasetheir risk of breast cancer. This concern has been height-ened by two reports that showed a positive relationshipbetween taking oral contraceptives and breast cancer.Ursin and colleagues found that among Jewish breastcancer patients, long-term use of oral contraceptives waseight times more common in BRCA1-mutation carriersthan in non-carriers52, but there were only nine BRCA1-mutation carriers and two BRCA2-mutation carriers inthe study. In the second study, Grabick et al. studied agroup of familial breast cancer patients from

TAMOXIFEN

One of a group of anti-oestrogenic drugs. Acts byblocking oestrogen signalling bybinding to the oestrogenreceptor.

CONTRALATERAL BREAST

CANCER

After the initial diagnosis ofbreast cancer, the occurrence of asecond, independent primarybreast cancer in the other breast.


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mutation carriers the odds ratio was 0.63 (95% CI0.20–1.50). The protective effect of tamoxifen was pre-sent in the subgroups of women with oophorectomy(odds ratio 0.36; 95% CI 0.07–1.81) and withoutoophorectomy (odds ratio 0.49; 95% CI 0.28–0.83).The odds ratio for tamoxifen combined with oophorec-tomy, compared with neither treatment, was 0.16 (95%CI 0.04–0.69), which is equivalent to a risk reduction of84%. This result indicates that the combination oftamoxifen and oophorectomy is more effective thaneither treatment alone, and that the two therapies couldbe complementary. The biological basis for this syner-gism is not known. Our study was of contralateralbreast cancers, and most individuals who were giventamoxifen had oestrogen-receptor-positive primarycancers. If the results of the study of contralateral breastcancer can be extrapolated to prevention of primarybreast cancers, then tamoxifen CHEMOPREVENTION inBRCA1- or BRCA2-mutation carriers is indicated.There is no consensus yet in this regard, and it will behelpful to generate data on contralateral breast cancerrisks among women with oestrogen-receptor-negativebreast cancers.

Hormone-replacement therapy. There are little data sofar to support the hypothesis that hormone-replacementtherapy (HRT) increases the risk of breast cancer inwomen at high genetic risk. In a meta-analysis (BOX 2),Steinberg and colleagues reported a positive interactionbetween HRT and breast cancer risk65 — that is, womenwith a family history of breast cancer were at greater riskof breast cancer if they took HRT. But it is also of interestthat in both of the European trials that assessed the effec-tiveness of tamoxifen in preventing breast cancer, the useof HRT was associated with a reduced risk of breast can-cer. In the Royal Marsden tamoxifen prevention trial66,women who were on HRT at the start of the trial had anincreased risk of breast cancer compared with non-users(relative risk = 1.9; p = 0.04), but those who began HRTafter the trial had begun had a significant reduction inbreast cancer incidence (relative risk = 0.4; p = 0.01).Women were also permitted to use HRT in the Italiantamoxifen prevention trial67. Among women who usedHRT, tamoxifen was associated with a large reduction inrisk, although the absolute numbers were small (relativerisk = 0.13, p = 0.02). In neither trial, however, was thesubgroup of BRCA1- or BRCA2-mutation carriers iden-tified. No studies have been conducted yet to investigatewhether HRT increases the risk of breast cancer inBRCA1- or BRCA2-mutation carriers.

Biological rationaleWe are beginning to see the emergence of different risk-factor profiles for BRCA1- and BRCA2-mutation carri-ers. The proposed modifiers of breast cancer risk inBRCA1-mutation carriers relate to oestrogen exposureand deprivation (FIG. 2). The risk of breast cancerdeclines after the menopause, the cancers are largelylimited to the breast and ovary, and breast cancer is pre-ventable by tamoxifen and oophorectomy. The proposed candidate genetic modifiers of risk (NCOA3

CONTRALATERAL BREAST CANCER in unselected women62. Thelarge National Surgical Adjuvant Breast and BowelProject (NSABP) breast cancer prevention trial reporteda highly significant reduction in the incidence of invasivebreast cancer in women who were randomized totamoxifen compared with those who received placebo61.In a subset analysis of the NSABP database, genetic-mutation status was determined by DNA sequencing on288 women with incident breast cancer63. Nineteen car-riers were identified; five of eight women with BRCA1mutations had taken tamoxifen, versus three of 11women with BRCA2 mutations. These data indicate thattamoxifen might be effective in the primary preventionof BRCA2-mutation-associated breast cancer, but not ofBRCA1-mutation-associated breast cancer. However, thedata set is very small and neither result is statistically sig-nificant. The power to detect a moderate protective effectwas low in this study.

Because of the difficulties encountered in conduct-ing historical cohort studies on BRCA1- or BRCA2-mutation carriers, a case–control study is preferable. Wehave studied tamoxifen and contralateral breast cancerin a case–control study of BRCA1- and BRCA2-muta-tion carriers64. The results of this study contrastmarkedly with the results of the NSABP breast cancerprevention trial, but the sample size was much greater.Tamoxifen use was reported by 10.5% of the 209 bilat-eral cases and by 21.1% of the 384 unilateral controls.This is equivalent to a relative risk of 0.45, or a 55%reduction in risk (p = 0.003). For the BRCA1-mutationcarriers and matched controls, the univariate odds ratiowas 0.38 (95% CI 0.19–0.74), and for the BRCA2-

CHEMOPREVENTION

The strategy of reducing the riskof cancer by using drugs(tamoxifen or oralcontraceptives), vitamins orother agents.

Androgen receptor RAD51

Tamoxifen

Oophorectomy

Oral contraceptives

BRCA1 BRCA2

NCOA3

Oophorectomy

Pregnancy

Tamoxifen

Breastfeeding

Androgen receptor

HRAS1

Oophorectomy

Oral contraceptives

Tubal ligation

a

b

Figure 2 | Factors that modify risk of breast or ovarian cancer. These differ betweenBRCA1- and BRCA2-mutation carriers in both breast (a) and ovarian (b) cancer. Most of theseproposed factors are based on suggestive results of a single study and require confirmation.


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including receptor competition, inhibition ofaromatase — an enzyme involved in oestrogen synthe-sis — and ovarian ablation (surgically or medically)(see the review article by Ali and Coombes on page 101in this issue). It is generally believed that oestrogenblockade is ineffective in the prevention of oestrogen-receptor-positive breast tumours, but this might notbe the case for BRCA1-mutation carriers. There is noconsensus yet as to whether tamoxifen should beoffered to unaffected women — if it were to be estab-lished that tamoxifen prevented oestrogen-receptor-negative contralateral cancers, then support forchemoprevention with tamoxifen would strengthen.Recent epidemiological studies indicate that the pro-tective effects of tamoxifen and oophorectomy mightbe independent and additive64. It is important to estab-lish the biological basis for the effectiveness of thiscombination. Studies on clinical outcome after treat-ment for both oestrogen-receptor-positive and oestro-gen-receptor-negative breast cancers in BRCA1-muta-tion carriers are underway, and it will be of interest tosee whether tamoxifen improves the survival ofwomen with oestrogen-receptor-negative tumours. Sofar, there are few data on the effectiveness of eitheroophorectomy or tamoxifen in BRCA2-mutation carriers, and further studies are needed.

In North America, concurrent use of HRT andtamoxifen is rare. In principle, the two drugs would beexpected to be antagonistic, but in the European trials,tamoxifen seemed to be effective in women on HRT.However, this was not a prior hypothesis of either trial,and the exposed subgroups were small. There has beensome success in studying the preventive effect of tamox-ifen on contralateral breast cancer using case–controlmethods; this is because the drug has been in wide-spread use for more than 20 years. By contrast, prospec-tive studies will be required to evaluate new drugs; can-didates for chemoprevention include raloxifene (andother selective oestrogen-receptor modulators(SERMS)), gonadotrophin-releasing hormone (GnRH)agonists and aromatase inhibitors. It is possible that oneor more drugs in these categories will be effective andwill have an acceptable toxicity profile, but it will be agreat challenge to collect the requisite clinical data tovalidate the efficacy.

Both BRCA1 and BRCA2 participate in DNA repair.Drugs or dietary supplements that reduce oxidativedamage, such as selenium74 or green tea75,76, or thatmodify oestrogen metabolism, such as indole-3-carbinol77, might therefore be of value. In the absence oflarge, randomized trials, it will be difficult to obtain thetype of data required to recommend routine dietarysupplementation.

A realistic goal for the next few years is to developrisk-assessment tools that consider all established riskfactors simultaneously, including age, mutation typeand position, reproductive history, exogenous hormoneuse, and the genotype of the relevant modifying genes.This could then be used to generate a specific risk esti-mate for an individual woman, which could be used as abasis for recommending a tailored intervention.

and androgen receptor) are involved in sex-hormonesignalling. By contrast, the evidence for the importanceof hormones in BRCA2-related carcinogenesis is muchless compelling (FIG. 2). The range of cancers seen amongBRCA2-mutation carriers is wide and includes typesthat are classically associated with exogenous carcino-gens, including melanoma and pancreatic cancer68.Breast cancer affects males as well as females, and thereis no apparent post-menopausal decline in breast cancerrisk. In one recent study, the risks of all cancers com-bined was similar for male and female relatives ofBRCA2-mutation carriers1. The first proposed geneticmodifier of risk for BRCA2-mutation carriers is RAD51,which is active in the DNA-repair pathway.

It is not yet possible to link the specific modifiers tothe biological functions of the gene products. A possiblereason for the sensitivity of BRCA1-mutation carriers toexogenous oestrogen relates to the role of BRCA1 inoestrogen-receptor signalling. BRCA1 has been foundto inhibit the signalling of the ligand-activated oestro-gen receptor69. It is believed that this function is relatedto minimizing DNA damage in the breast during therapid bursts of proliferation that occur during pubertyand pregnancy. In the presence of BRCA1, oestrogen isrequired for the transcriptional activation of oestrogenreceptor-α, but in cells with mutant BRCA1, oestrogenreceptor-α has transcriptional activity that is indepen-dent of oestrogen70. In a study on oestrogen-dependentovarian cancer cells, the introduction of antisenseBRCA1 enhanced the ability of the cells to grow in theabsence of oestrogen71.

Both BRCA1 and BRCA2 participate in the repair ofdouble-stranded DNA breaks through homologousrecombination72. The repair complex includes other pro-teins such as RAD51 (which directly binds to BRCA2)73.Cells with mutant BRCA2 are genetically unstable and areprone to chromosome rearrangements74. Mutagenicagents, such as smoking, ionizing radiation and genotoxicchemicals, can induce double-strand breaks, but there isno empirical evidence that exposures of this type increasecancer risk in BRCA1- or BRCA2-mutation carriers. Interms of ovarian cancer, the risk factors seem to be simi-lar in some respects, but not in all. The effect of oralcontraceptives is strong in both BRCA1- and BRCA2-mutation carriers, but tubal ligation seems to be effec-tive for BRCA1-mutation carriers only. The reason forthis discrepancy is not known.

Clinical implications and future directionsIt is hoped that emerging knowledge of gene–environ-ment and gene–gene interactions will lead to new pre-vention strategies for BRCA1- or BRCA2-mutationcarriers. At present, chemoprevention offers hope for adramatic reduction in breast cancer risk (and for theavoidance of prophylactic surgery), but it will be achallenge to identify the optimal agent and the properindications. There are several unanswered questions— foremost is the relationship between oestrogen-receptor status and hormone responsiveness. Mostproposed chemoprevention strategies are based oninterruption of the oestrogen signalling pathway,


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Online links

DATABASESThe following terms in this article are linked online to:CancerNet: http://cancernet.nci.nih.gov/breast cancer | colon cancer | thyroid cancer | melanoma | ovariancancer | pancreatic cancer | pheochromocytoma | prostatecancer | retinoblastoma | testicular cancerLocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/androgen receptor | BARD1 | BRCA1 | BRCA2 | CYP3A4 | HRAS |NCOA3 | oestrogen receptor-α | p53 | RAD51 | RBMedscape DrugInfo:http://promini.medscape.com/drugdb/search.aspraloxifene | tamoxifen

FURTHER INFORMATIONBIC (BRCA Information Core):nhgri.nih.gov/Intramural_research/Lab_transfer/BIC/Access to this interactive links box is free online.

Documents

MODIFIERS OF RISK OF HEREDITARY BREAST AND OVARIAN CANCER