The HER2 I655V Polymorphism and Risk of Breast Cancer in Women < Age 40 Years¹

HER2³ is a transmembrane glycoprotein with tyrosine kinase activity that has several functions, including the control of cellular proliferation. Amplification and/or overexpression of HER2, present in 30% of breast cancers, is associated with poor prognosis (1, 2). The use of a recombinant humanized monoclonal antibody that specifically targets HER2 may increase the clinical benefit of first-line chemotherapy in women with breast cancers that overexpress HER2 (3).

An isoleucine to valine polymorphism at codon 655 (I655V) was reported to be associated with an increased risk of breast cancer in a Chinese population (4). Of the six subsequent studies that investigated the risk of breast cancer associated with this HER2 I655V polymorphism, two reported a positive association (5, 6), whereas four found no association (7, 8, 9, 10). It is difficult to interpret these studies given that the characteristics of the samples were different, and some lacked statistical power. Interestingly, in the two that reported a positive association, the only effect evident was in women diagnosed before age 45 years (4) or in women with a first-degree family history of breast cancer (5), groups in which inherited genetic effects on susceptibility are expected to be more pronounced. In the present study, we evaluated the association between the HER2 I655V polymorphism and breast cancer risk before the age of 40 years using Australian women.

Subjects were drawn from the Australian Breast Cancer Family Study, a population-based, case-control-family study of early onset breast cancer carried out in Melbourne and Sydney during the period 1992 through 1995 that has been described in detail previously (11, 12). Briefly, women under the age of 40 at diagnosis of a first primary invasive breast cancer were identified through the Victorian and New South Wales cancer registries. Controls were selected from the electoral roll (registration for voting is compulsory in Australia) by use of stratified random sampling and were frequency matched for age. Response rates were 73% in both cases and controls (11). Case subjects, control subjects, and their relatives were administered the same questionnaire on risk factors (see Ref. 11 for characteristics of study participants). For each case and control, a detailed family history was recorded for all first- and second-degree relatives, and verification of all family cancers was sought through cancer registries, pathology reports, clinical records, and death certificates. Blood samples were collected from subjects at the time of interview. Written informed consent was obtained from all subjects, and approval of the study protocol was obtained from the relevant ethics committees.

A total of 409 cases and 299 controls were genotyped for the HER2 I655V polymorphism using a dual color allele-specific PCR assay described previously (7). Briefly, PCR amplifications were performed using HEX- and FAM-labeled forward primers (5-CAGCCCTCTGACGTCCATCA-3 and 5-CAGCCCTCTGACGTCCATCG-3) and a common reverse primer (5′-TCCTCAGCTCCGTCTCTTTC-3′). Ten percent of the genotyping was confirmed with a PCR and restriction fragment length polymorphism assay described previously (13).

The Hardy-Weinberg equilibrium assumption was assessed by the standard method of comparing the observed numbers of individuals in the different genotype categories with those expected under Hardy-Weinberg equilibrium for the estimated allele frequency and assessing the Pearson goodness-of-fit statistic against the χ² distribution with one degree of freedom. Genotype distributions were compared with the use of contingency table analysis.

We conducted a case-control analysis using unconditional multiple logistic regression to fit models assuming: (a) dominant inheritance (i.e., women with one or two valine alleles had the same increased risk); (b) codominant inheritance (i.e., risk could differ across all three genotypes); or (c) recessive inheritance (i.e., only women with two valine alleles were at the increased risk). For all analyses, adjustment was made for demographic variables (study site, country of birth, education, and marital status) and putative breast cancer risk factors (age, parity, oral contraceptive use, height, and family history; Ref. 11).

All analyses were performed by the use of Stata 7.0 statistical software (StataCorp., College Station, Texas). All statistical tests and Ps were two tailed, and, after convention, statistical significance was taken as a nominal P of <0.05.

There was no evidence of a deviation from Hardy-Weinberg equilibrium among the cases (P = 0.5) or controls (P = 0.9). The frequency of the valine allele in controls (0.187; SE 0.016) was similar to that found in other studies of Caucasian populations (10, 11) and less than in cases (0.245; SE 0.015, P = 0.01).

Table 1 shows odds ratios and 95% CIs for the effects on breast cancer risk of HER2 I655V genotypes according to different models of inheritance. Odds ratios adjusted for demographic, reproductive, and lifestyle variables did not differ greatly from the crude estimates. There was evidence of an increased risk under dominant inheritance (P = 0.04), codominant inheritance (P = 0.01), and recessive inheritance (P = 0.005). The recessive model gave an increased risk of 2.8-fold (95% CI 1.3–6.2; P = 0.005) and provided the best fit under parsimony; the codominant model did not give an improved fit (P = 0.2), and the dominant model gave a worse fit for the same number of parameters (difference in log likelihood = 1.5). Stratification by body mass index, family history of cancer, alcohol consumption, or oral contraceptive use revealed no evidence for a modification of the recessive effect on breast cancer risk by these factors, albeit with limited power (data not shown).

Our study, of women diagnosed before the age of 40 years, has demonstrated that homozygosity for the valine allele was associated with an increased risk of early onset breast cancer. This finding is consistent with the increased breast cancer risk observed by Xie et al. (4) among Chinese women diagnosed before age 45 years. Overexpression of HER2 is detected in a large proportion of breast cancers, indicating that activation of this gene is an important step in breast carcinogenesis. Consequently, it is plausible that functional polymorphisms in this gene that enhance HER2 activity may represent breast cancer predisposing alleles.

There is currently no direct information regarding any biological role of the I655V polymorphism, located in the transmembrane region of HER2. However, oncogeneic mutations have been reported in the transmembrane region of the related mouse gene neu (14) and an amino substitution at position 659 in HER2 has also been shown to increase transforming ability in NIH3T3 cells (15). It is possible, therefore, that the I655V polymorphism in HER2 may also have functional consequences. In light of our finding of a recessively inherited risk of early onset breast cancer associated with this polymorphism, investigations into the functional implications of the isoleucine to valine substitution, and attempts to replicate this association in independent and sufficiently large studies, are warranted.