Affiliations of authors: D. Xie, Z. Deng, W. Zheng, Department of Epidemiology and Biostatistics, University of South Carolina School of Public Health and South Carolina Cancer Center, Columbia; X.-O. Shu, W.-Q. Wen, Department of Epidemiology and Biostatistics, University of South Carolina School of Public Health and South Carolina Cancer Center and Department of Pediatrics, University of South Carolina School of Medicine and South Carolina Cancer Center; K. E. Creek, Departments of Pediatrics and Pathology, University of South Carolina School of Medicine and South Carolina Cancer Center; Q. Dai, Department of Epidemiology and Biostatistics, University of South Carolina School of Public Health and South Carolina Cancer Center, and Department of Epidemiology, Shanghai Cancer Institute, People's Republic of China; Y.-T. Gao, F. Jin, Department of Epidemiology, Shanghai Cancer Institute.
Correspondence to: Wei Zheng, M.D., Ph.D., University of South Carolina, 15 Richland Medical Park, Suite 301, Columbia, SC 29203 (e-mail: wei.zheng{at} rmh.edu).
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ABSTRACT |
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INTRODUCTION |
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An increasing body of evidence indicates that genes that encode growth factor receptors can be activated through mutations in their coding sequences (11-15). In 1986, Bargmann et al. (13) reported that a point mutation in the transmembrane region of the neu gene, a rat homologue of human HER2 gene, increased the transforming capacity of this gene. Transgenic mice that carry a mutationally activated HER2 gene or an overexpressed normal HER2 gene frequently develop mammary adenocarcinoma (16-19). Although mutations in the human HER2 gene have not been identified (20-22), sequence analysis of human HER2 complementary DNA clones identified a polymorphism in the transmembrane coding region at codon 655. This polymorphism encodes either isoleucine (Ile; ATC) or valine (Val; GTC) (23). The pivotal role that the HER2 protein p185 plays in cell proliferation and the critical role that the transmembrane domain plays in the function of p185 make this genetic polymorphism a strong candidate for a breast cancer susceptibility factor.
In this article, we report results from a population-based, case-control study that examined the association of HER2 polymorphisms with the risk of breast cancer.
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METHODS |
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Case patients and control subjects in this study were participants of the Shanghai Breast Cancer Study, a recently completed large-scale, population-based, case-control study (24,25). The Shanghai Breast Cancer Study was conducted among Chinese women in Shanghai, the largest city on the east coast of China with a population of more than 6 million residents. This study was designed to recruit all eligible patients aged 25-64 years who were diagnosed with breast cancer from August 1996 through March 1998, as well as a representative random sample of control subjects from the general population. All case patients and control subjects were permanent residents of urban Shanghai who had no histories of cancer and could be interviewed directly. Through a rapid case-ascertainment system, supplemented by the population-based Shanghai Tumor Registry, 1602 eligible case patients with breast cancer were identified during the study period, and in-person interviews were completed for 1459 (91%) of them. The major reasons for nonparticipation were refusal (109 case patients, 6.8%), death before the interview (17 case patients, 1.1%), and the inability to locate (17 case patients, 1.1%). Cancer diagnoses for all patients were confirmed by two senior study pathologists through a review of tumor slides.
Control subjects were randomly selected from the female general population and were frequency matched to case patients by age (5-year intervals). The number of control subjects in each age-specific stratum was determined in advance according to the age distributions of the case patients, with incident breast cancer reported to the Shanghai Tumor Registry from 1990 through 1993. The Shanghai Resident Registry, which keeps registry cards for all adult residents in urban Shanghai, was used to randomly select control subjects. For each age-determined control subject needed, a registry card identifying a potential control subject in the same 5-year age group was randomly selected. Only the women who lived at the address during the study period were considered to be eligible for the study. In-person interviews were completed for 1556 (90%) of the 1724 eligible control subjects identified. Excluded from the study were 168 potential control subjects because of refusal (n = 166; 9.6%) and death or a prior cancer diagnosis (n = 2; 0.1%).
A structured questionnaire was used to elicit detailed information on demographic factors, menstrual and reproductive histories, hormone use, dietary habits, prior disease history, physical activity, tobacco and alcohol use, weight, and family history of cancer. All participants were also measured for their current weight, circumferences of the waist and hip, and heights while sitting and standing. Blood samples (10 mL from each woman, collected in vacutainer tubes containing EDTA or heparin) were obtained from 1193 (82%) case patients and 1310 (84%) control subjects who completed the in-person interviews. Written informed consent was obtained from all tested subjects, and the study protocol was approved by relevant committees for the use of human subjects in research. Blood samples were processed on the same day at the Shanghai Cancer Institute. Buffy coats (white blood cells) for each participant were distributed into two 2-mL vials and stored at -70 °C. As soon as possible after a case patient was diagnosed with cancer, we conducted in-person interviews and collected biologic samples. For about 46% of the case patients, samples were collected before any cancer treatment. The average intervals from cancer diagnosis to the in-person interview and blood collection were 78 days and 79 days, respectively.
Because this project was initiated before the completion of the Shanghai Breast Cancer Study, at first, we included only the case patients and control subjects who were recruited in the study in 1996 and whose blood samples were shipped to our laboratory (25). Subsequently, we added to the study the subjects who were included in the ancillary project of the Shanghai Breast Cancer Study (24). The ancillary study included case patients whose samples were collected before cancer treatment. Case patients and control subjects (1 : 1 match) were matched on age (±3 years), menopausal status (yes or no), and the date of sample collection (±30 days). The final sample size in this study was 700 subjects (339 case patients and 361 control subjects). Genomic DNA from study participants was extracted from buffy coat fractions. Frozen white blood cells were thawed at room temperature and then digested overnight at 65 °C in 500 µL of lysis buffer (50 mM Tris-HCl [pH 8.5], 1 mM EDTA, 0.2% sodium dodecyl sulfate, and proteinase K [200 µg/mL]). The digest was precipitated directly with isopropanol, and the pellets were washed with 70% ethanol. Genomic DNA pellets (50-100 µg) were dissolved in 300-800 µL of Tris-EDTA buffer. About 10-100 ng of genomic DNA was used for each polymerase chain reaction (PCR).
PCR-Restriction Fragment-Length Polymorphism-Based Assay
HER2 genotypes were determined with a PCR-restriction fragment-length
polymorphism-based assay. The primers, based on the published sequence of human
complementary DNA of the HER2 gene (3,26), were as follows: F
= 5'-AGAGCGCCAGCCCTCTGACGTCCAT-3'; R =
5'-TCCGTTTCCTGCAGCAGTCTCCGCA-3'. DNA was amplified in a
Perkin-Elmer GeneAmp PCR System 9700 (The Perkin-Elmer Corp., Norwalk City, CT)
according to the manufacturer's protocol. These reactions were carried out in 50 µL
containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, all four deoxynucleoside triphosphates (each at 0.2 mM), and 1 U of Taq polymerase. The reaction mixtures were heated to 94 °C for 30 seconds followed
by 35 cycles at 94 °C for 30 seconds, 62 °C for 30 seconds, and 72 °C for 30
seconds. At the end, the reactions were extended 7 minutes at 72 °C. The PCR products
(148 base pairs [bp]) were digested with BsmAI and separated by gel
electrophoresis (3% Nusiev/SeaKem agarose; 2 : 1 [wt/wt]). Digestion of each
PCR product with BsmAI gives 116- and 32-bp fragments for the Val allele and a single
148-bp fragment for the Ile allele (Fig. 1). The HER2 genotypes were
identified for all control subjects but two, and so we analyzed data from 339 case patients and
359 control subjects.
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Statistics
Chi-squared statistics were used to evaluate the distribution of HER2 allele types and
genotypes among case patients and control subjects. Odds ratios (ORs) and 95%
confidence intervals (CIs), obtained from unconditional logistic regression, were used to measure
the strength of the association between a HER2 polymorphism and the risk of breast cancer. In
addition to age, educational level was adjusted to control for potential bias caused by
socioeconomic disparity. Subject-recruiting period (in 1996 or in the ancillary study) was also
adjusted. All risk factors for breast cancer that were identified in this study population were
evaluated to examine their potential influence on the association between HER2 genotypes and
the risk of breast cancer. Final fully adjusted models included study variable (HER2 genotypes),
age, educational level, study period, and all risk factors for breast cancer that affected the risk
estimate for the association between HER2 genotypes and breast cancer risk. Stratified analyses
by age (45 and >45 years) were performed to examine whether the association was
stronger among younger women. Age 45 years or younger was used as the cutoff point because it
has been traditionally used to define early-onset breast cancer. Because cumulative endogenous
estrogen exposure is believed to play a major role in breast carcinogenesis (27), a potential joint effect between HER2 polymorphism and factors related to
endogenous estrogen exposure was evaluated. Trend tests for dose-response relationships
between breast cancer risk and the number of the Val allele were performed by treating an
ordinal-score variable (with a value of 0, 1, or 2 for number of Val alleles) as a continuous
variable in logistic regression. All statistical tests are two-sided.
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RESULTS |
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DISCUSSION |
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The HER2 gene (c-erbB2) is the human homologue of the rat proto-oncogene neu and is closely related to the gene that encodes the epidermal growth factor receptor (erbB1), a receptor tyrosine kinase. p185c-HER2/c-erbB2, the HER2 gene product, is a 1255-amino acid glycoprotein (185 kd) that is quite similar to other members of the epidermal growth factor receptor family including p160c-erbB3 and p180c-erbB4. All members of this family share common structural features, including a cysteine-rich extracellular ligand-binding domain containing several potential sites for glycosylation, a single hydrophobic transmembrane domain, a short juxta-membrane segment followed by the tyrosine kinase domain, and the carboxyl-terminal tail (30). Overexpression of p185c-HER2/c-erbB2, with or without HER2 gene amplification, has been clearly linked to the transformation of NIH 3T3 cells (31) and the neoplastic process in a variety of human cancers, especially in breast carcinoma (8,9). Alterations in the transmembrane segment of this protein may also have profound effects on its biologic activity and transforming ability. The only difference between the oncogenic and proto-oncogenic forms of neu is a single-point mutation (GTG to GAG) that results in the valine to glutamic acid amino acid substitution at position 664 in the transmembrane domain of the receptor. This point mutation in the neu oncogene apparently mimics ligand induction of receptor activation and results in the activation of the receptor tyrosine kinase (15). Of interest, in c-erbB2, a valine to glutamic acid (GTT to GAA or GAG) or valine to aspartic acid (GTT to GAT) substitution in codon 659 (analogous to position 664 in p185neu) resulted in increased (10- to 100-fold) transforming activity in NIH 3T3 cells and enhanced in vitro tyrosine kinase activity (32). Although a corresponding point mutation found in the neu oncogene has not been found in the HER2 gene in human tumors, the Val655Ile polymorphism (GTC or ATC) in the transmembrane coding region of the HER2 gene has been identified (23). Whether the polymorphic HER2 proteins differ in their ability to transform cells and/or have different tyrosine kinase activities has not been determined. However, our findings of an association between breast cancer and the HER2 Val allele suggest that this polymorphism may be functionally important. The prevalences of the HER2 Val/Ile and Val/Val genotypes in our control population were 21.7% and 0.3%, substantially lower than those (40% and 12%) observed in the Caucasian population (23). These data correspond well with the traditionally lower risk of breast cancer among Chinese women compared with Caucasian women and provide additional support for a potential role of the HER2 polymorphism in breast carcinogenesis.
The methodologic limitations of this study were few. The participation rate in this study was high, minimizing potential selection bias, which is common in many case-control studies. Chinese women living in Shanghai have a relatively homogeneous ethnic background; more than 98% of these women belong to a single ethnic group (Han Chinese). Therefore, a potential confounding effect by ethnicity may not be a major concern in our study. It has been suggested recently through simulation analyses that, in most situations, ethnicity is unlikely to be a major confounder in the study of the association between genetic factors and cancer (33). The frequency of control subjects homozygous for the Val allele was lower than expected under the Hardy-Weinberg equilibrium, although the difference was not statistically significant (P = .392). The distribution of the HER2 polymorphisms in the control group and in the general population should not differ substantially because control subjects were randomly selected from the general population and the response rate was very high. The frequency of the Val allele was high and should have been estimated reasonably well, given the large sample size of the study. This allele was more common in case patients (15.8%) than in control subjects (11.1%) (P = .01), and this difference was more pronounced among younger women. The association observed in this study could be a result of linkage disequilibrium of the Val655Ile polymorphism with the functional allele(s) at other site(s), given the uncertainty of the functional importance of the polymorphism under study. Nevertheless, information about a closely linked nonfunctional allele may also be helpful because it may be used to identify individuals at high risk of breast cancer who may benefit from cancer screening and chemoprevention.
In summary, from this population-based, case-control study, we found that genetic polymorphism of the HER2 proto-oncogene was associated with an increased risk of breast cancer, particularly for early-onset breast cancer. Women homozygous for the HER2 Val allele in codon 655 had a statistically significantly increased risk, suggesting that the Val allele may be an indicator of genetic susceptibility to breast cancer.
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NOTES |
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Manuscript received July 30, 1999; revised December 13, 1999; accepted December 20, 1999.
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