Clinical Cancer Research The Future of Cancer Research: Science and Patient Impact
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, W.
Right arrow Articles by Noh, D.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, W.
Right arrow Articles by Noh, D.-Y.
Clinical Cancer Research Vol. 11, 4775-4778, July 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

A Haplotype Analysis of HER-2 Gene Polymorphisms: Association with Breast Cancer Risk, HER-2 Protein Expression in the Tumor, and Disease Recurrence in Korea

Wonshik Han1, Daehee Kang2, Jong Eun Lee5, In Ae Park3, Ji-Yeob Choi2, Kyung-Mu Lee2, Ji Yeon Bae4, Sook Kim5, Eun-Soon Shin5, Jeong Eon Lee1, Hyuk-Jae Shin1, Seok Won Kim1, Sung-Won Kim1 and Dong-Young Noh1,4

Authors' Affiliations: Departments of 1 Surgery, 2 Preventive Medicine, 3 Pathology, and 4 Cancer Research Institute, Seoul National University College of Medicine, and 5 DNA Link Inc., Seoul, Korea

Requests for reprints: Dong-Young Noh, Cancer Research Institute and Department of Surgery, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Phone: 82-2-760-2921; Fax: 82-2-766-3975; E-mail: dynoh{at}plaza.snu.ac.kr.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: A single-nucleotide polymorphism (SNP) in codon 655 of HER-2 has been extensively studied with inconclusive results. The purpose of this study was to investigate the association between common variants of HER-2 and breast cancer risk, HER-2 expression, and survival using a haplotype-based stepwise approach.

Experimental Design: Twenty-nine SNPs listed in the National Center for Biotechnology Information database were screened to identify novel polymorphisms of HER-2 gene in 90 healthy Korean women. Six of 29 SNPs were polymorphic and had greater than 10% of minor allele frequencies. Using these six SNPs, linkage disequilibrium and haplotype patterns were characterized. We tested association between the haplotypes and breast cancer in a large case–control study (n = 1,039 cases and 995 controls). Six-hundred two breast cancer patients with follow-up at least 24 months were analyzed for outcome in relation to haplotype. Expression of HER-2 protein was determined by immunohistochemistry in 1,094 cases of invasive breast cancer.

Results: All six SNPs showed a strong linkage disequilibrium pattern and were considered to belong to one haplotype block. Two haplotype-tagging SNPs (I655V and P1170A) for three common haplotypes (>5%) were genotyped in cases and controls. The haplotypes and individual SNPs were not associated with breast cancer risk. In patients with at least one copy of haplotype I (the most common haplotype), HER-2 expression was 1.5 times higher (P = 0.009) and the prognosis was worse (P = 0.032) compared with patients without having that haplotype.

Conclusions: Our results suggest that the currently identified genetic polymorphisms of HER-2 are not associated with an increased risk of breast cancer in Korean women, whereas one haplotype does affect protein expression of the tumor and disease outcome.

The HER-2 (also known as erbB-2 or neu) proto-oncogene, a member of the epidermal growth factor receptor family, is located at chromosome 17q21 and encodes a transmembrane glycoprotein (p185) with tyrosine kinase activity (1, 2). Amplification and/or overexpression is the main mechanism of HER-2 gene activation in human cancers (3–5). Alterations of this gene are found in more than 20% of breast tumors and have been shown to be associated with steroid hormone receptor–negative tumors, higher nuclear and histologic grades, tumor aneuploidy, higher rates of proliferation, a reduced response to chemotherapy and hormonal therapy, and poorer prognoses (5). More attention is being paid to HER-2 and its role in breast cancer because of the recent introduction of trastuzumab, a humanized murine monoclonal antibody directed against the extracellular domain of HER-2, for the treatment of patients with HER-2–overexpressing advanced breast cancer (6, 7). HER-2 is particularly significant in Korea as it is amplified/overexpressed more frequently in Korean breast tumors than in those of Caucasian women (8).

No point mutations in the HER-2 gene have been identified to date (9). One common variant, a single-nucleotide polymorphism (SNP) encoding either isoleucine (I) or valine (V) at codon 655 in the transmembrane region of HER-2, has been extensively studied (10). Xie et al. (11) first reported that the I655V SNP is associated with a significantly increased risk of breast cancer development [odds ratio (OR), 1.4]. However, several following studies have cast doubt on this association, which remains controversial (12–16). Fleishmann et al. (17) first suggested a functional implication for this SNP by showing that the I655V polymorphism might enhance active dimeric conformations of HER-2 protein that result in increased autophosphorylation and tyrosine kinase activation.

In the present study, we employed a genetic haplotype approach to examine the contribution of common variations at the HER-2 locus to breast cancer risk among Korean women. Haplotype-based association studies have been proposed to be powerful comprehensive approaches for the identification of causal genetic variations that underlie complex diseases (18, 19). Recent studies have shown that the human genome is comprised of genomic segments (blocks) that display little evidence of historical recombination and low haplotype diversity (18–20). Due to the high degree of linkage disequilibrium observed between SNPs within these blocks, ancestral disease variants may be uncovered through evaluation of the underlying haplotypes. This methodology does not require the causal variant to be identified or directly tested, but rather has the potential to highlight physical regions that harbor putative disease-associated variants (21).

The effect of different haplotypes on HER-2 protein expression in the tumors of breast cancer patients as well as on their prognosis was also evaluated in this study because HER-2 has been thought to play a critical role in both breast cancer development and progression (22, 23). Any functional polymorphism can potentially affect breast cancer risk as well as cancer phenotype and outcome. In a previous study, we showed that polymorphisms of certain breast cancer susceptibility genes may affect various clinicopathologic phenotypes of breast cancer (24).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study subjects. To determine the novelty and frequency of SNPs of HER-2 gene in Korean populations, 90 Korean women were randomly accrued from the women who visited Health Promotion Center in Seoul National University Hospital between 2002 and 2003 and who had no previous history of malignant disease and no family history of breast cancer in their first degree relatives. Their age distribution was between 35 and 65. These 90 women were not enrolled in the case-control study described below. The case-control study subjects were selected from three teaching hospitals located in Seoul, Korea (Seoul National University Hospital, Borame Hospital, and Asian Medical Center) between January 1995 and March 2004. Women with histopathologically confirmed breast cancer were selected as cases, and controls with no present or previous history of cancer were recruited from the same hospitals. The majority of eligible controls were being treated for gastrointestinal problems such as acute appendicitis, cholecystitis, gall bladder or bile duct stones, hemorrhoids, and inguinal hernia. Women with amenorrhea, a previous history of hysterectomy, oophorectomy, hormone replacement therapy,hormone-related diseases such as thyroid disease, or systemic diseases such as diabetes mellitus or chronic liver disease were excluded from both groups. Approximately 21% of otherwise eligible breast cancer cases and 6% of otherwise eligible controls were excluded from the study because they refused to participate or failed to be interviewed or have blood drawn. In total, 1,039 cases and 995 controls were eligible for the case-control study that evaluated the association between common genetic variations and breast cancer risk. Evaluation of the influence of haplotype on HER-2 expression and prognosis was supplemented with 635 additional breast cancer patients diagnosed between August 1992 and March 2002. Of 1,674 total patients, those who had distant metastasis at the time of surgery (n = 37) and whose follow-up time was shorter than 24 months (n = 1,035) were excluded from survival analysis. Because more than 50% of the patients underwent operation after Jan. 2001 and the study for recurrence and survival status was done between 2002 and 2003, only 602 patients (37%) met the criteria and entered into the analysis.

Immunohistochemical assays for HER-2 protein expression were done for 1,094 invasive breast cancer cases from Seoul National University Hospital. Noninvasive cancers, such as ductal carcinoma in situ and lobular carcinoma in situ, were excluded from immunohistochemical study.

This study was conducted under the approval of the Institutional Review Board of Seoul National University Hospital. Informed consent was obtained at the time of blood sampling. Information on demographic characteristics, education, marital status, family history of breast cancer in first- and second-degree relatives, reproduction and menstruation history, lifestyle habits, and diet were collected by a trained interviewer using a questionnaire.

Single-nucleotide polymorphism selection and genotyping in the 90 single-nucleotide polymorphism screening set. Twenty-nine SNPs from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/dbSNP) were genotyped across 36.3 kb of the ERBB2 locus, from 5.1 kb upstream of exon 1 to 2.8 kb downstream of the transcribed region (exon 27), in 90 women with no previous history of any type of cancer. All known SNPs in the ERBB2 coding region were included, and we attempted to select additional SNPs that were as evenly spaced throughout the region as possible (Table 1). Of 29 SNPs, 14 that were either monomorphic or had a low allele frequency (<10%) in this population were removed from the haplotype analysis. Nine of the assays on the remaining SNPs yielded poor genotyping results, leaving six SNPs with allele frequencies ≥10% that were included in the haplotype analysis. The average spacing between these six SNPs was 6.8kb, and their allele frequencies did not deviate from Hardy-Weinberg equilibrium.


View this table:
[in this window]
[in a new window]
  		Table 1. Twenty successfully genotyped SNPs for screening in 90 Korean women

 
Blood samples were collected in 10-mL heparinized tubes and DNA was isolated from WBC fractions using the Qiagen Blood Kit (Qiagen, Chatsworth, CA). Genotyping was done by the SNP-IT assay using the SNPstream 25K System (Orchid Biosciences, Princeton, NJ). Briefly, the genomic DNA region spanning the polymorphic site was PCR amplified using one phosphothiolated primer and one regular PCR primer. The amplified PCR products were then digested with T7 exonuclease. The 5' phosphothiolates protected one strand of the PCR product from T7 exonuclease digestion, resulting in the generation of a single-stranded PCR template. The single-stranded PCR template was overlaid on a 384-well plate that contained a covalently attached SNP-IT primer extension primer designed to hybridize immediately adjacent to the polymorphic site. The SNP-IT primer was extended for a single base with DNA polymerase and a mixture of appropriate acycloterminators complementary to the polymorphic nucleotide that were labeled with either FITC or biotin. The identity of the incorporated nucleotide was determined by serial colorimetric reactions with anti-FITC-AP and streptavidin-horseradish peroxidase, respectively. Resultant yellow and/or blue color development was analyzed using an ELISA reader and the final genotype calls were made using a QCReview program (Orchid Biosciences).

Haplotype reconstruction, block determination, and haplotype-tagging single-nucleotide polymorphism selection. The D' statistic was used as a pairwise measure of linkage disequilibrium between the six SNPs used in the haplotype analysis. The linkage disequilibrium block structure was examined using the criteria of Gabriel et al. (19). A pair of SNPs is defined to have "strong linkage disequilibrium" if the one-sided upper 95% confidence interval (95% CI) boundary on D' is >0.98 and the lower boundary is >0.7. Conversely, SNP pairs are said to have "strong evidence of historical recombination" if the upper CI boundary on D' is <0.9. A haplotype block is defined as a region over which a very small proportion (<5%) of comparisons among informative SNP pairs shows strong evidence of historical recombination.

Haplotype structures and their frequencies were estimated from genotype data for the common six SNPs within the linkage disequilibrium block using the Haploview version 2.05 (http://www.broad.mit.edu/personal/jcbarret/haploview), which estimates haplotype by an accelerated EM algorithm similar to the partition/ligation method described by Qin et al. (25). The Haploview program was also used to calculate pairwise linkage disequilibrium (D' and r2) for each marker pair. Haplotype-tagging SNPs were easily identifiable without using a computer program. We included two coding SNPs as haplotype-tagging SNPs to predict the common haplotypes.

Genotyping in the case-control study and quality control. Haplotype-tagging SNPs for common haplotypes (>5%) were genotyped in cases and controls. The genotyping method used for case-control set was same as that used for screening set. Genotyping was done by the SNP-IT assay using the SNPstream 25 K System (Orchid Biosciences). Laboratory personnel were blinded to case-control status and the clinical/pathologic information of the patients. Replicate blinded quality control samples [368 of 2,034 (18%)] were included to assess reproducibility of the genotyping procedure; less than 0.3% (1 of 368) of the matched quality control pairs were discordant.

Statistical analysis. Differences in variation of known risk factors between groups were evaluated by Student's t test or the {chi}2 test. The {chi}2 test was also used to determine associations between the frequencies of the haplotypes and the HER-2 protein expression in tumors, and to ensure the independence of alleles (Hardy-Weinberg equilibrium). The ORs and 95% CIs were calculated using an unconditional logistic regression model after adjustment for age, education level (at least a high school education versus less than a high school education), family history of breast cancer in first- and second-degree relatives, and alcohol usage (<1 versus ≥1 drink/mo). Follow-up duration was calculated from the date of diagnosis to the date of death or last contact. Disease-free survival was defined as the time between diagnosis and the first clinically recognized evidence of local or distance recurrence, death, or loss to follow up. Survival estimates were computed using the Kaplan-Meier method and differences between survival times were assessed using the log-rank test. Hazard ratios were calculated with Cox proportional hazard model adjusted for age (years), tumor size (≤2 versus >2 cm), and lymph node metastasis status (negative versus positive). Disease staging was done using the American Joint Committee of Cancer system, 6th edition (26). SPSS version 10.0 (Chicago, IL) was used for all statistical analyses.

Immunohistochemistry. Immunohistochemical analysis was done to determine the expression of HER-2 protein in paraffin-embedded tissues from 1,094 breast cancer patients. Sections were first deparaffinized in xylene. After hydration, sections were microwaved twice in 10 mmol/L citrate (pH 6.0) for 5 minutes and washed thrice in distilled water for 5 minutes. Sections were then placed in 3% H2O2 for 10 minutes to inhibit endogenous peroxide activity, washed thrice with PBS for 5 minutes, and placed in normal horse serum for blocking at room temperature for 30 minutes. Samples were incubated in primary antibody directed against the HER-2 protein, NCL-CB11 (Novo Castra, New Castle, United Kingdom), at a dilution of 1:40 at 4°C for 24 hours, and sections were then washed thrice with PBS for 5 minutes. Biotinylated anti-mouse immunoglobulin was used as the second antibody and 3,3-diaminobenzidine tetrahydrochloride was used as a chromogen. Sections were counterstained with hematoxylin. A single pathologist who had no information regarding genetic and clinical variables scored and interpreted all immunohistochemical staining results. The following scoring system was used to characterize HER-2 membrane staining intensity and pattern: 0, no staining; 1+, ≥10% cells showing weakly positive staining; 2+, moderate homogeneous staining; 3+, strong homogeneous staining.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Linkage disequilibrium and haplotype structure of the HER-2 locus. We assembled an SNP map across the HER-2 locus to determine linkage disequilibrium blocks and haplotype structure in 90 Korean women with no history of cancer (Fig. 1A). Six SNPs with allele frequencies of >10% were selected to make a haplotype structure. Nine of the fifteen SNP pairs showed a strong linkage disequilibrium pattern as defined by Gabriel et al. (19). None of the pairs had strong evidence of historical recombination, so we were able to conclude that these six SNPs are in the same haplotype block (Fig. 1B). This block covers 33.9 kb and spans the entire coding region of HER-2, including a 4.1 kb 5'-untranslated region and a 2.8-kb 3'-untranslated region. Within this block, we identified seven haplotypes, which we designated I to VII in order of increasing frequency (Fig. 1C). The most common three haplotypes (i.e., those with a ≥5% frequency) comprise 93.8% of the total predicted haplotype variation. We were able to distinguish these three common haplotypes with only two haplotype-tagging SNPs, I655V in exon 17 and P1170A in exon 27, which were both genotyped in the case-control study described below.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. A, the six SNPs used in the haplotype analysis and their location in the HER-2 gene [vertical arrows (numbered 1 to 6 from 5' to 3'): 1, rs2643194; 2, rs2934971; 3, rs1565923; 4, rs1801200; 5, rs1058808; 6, rs3809717]. Exons are depicted as small blocks. B, pairwise measure of linkage disequilibrium between the six SNPs in (A) with D'statistics (values in the bottom left area of the table). The pairs exhibiting a strong linkage disequilibrium pattern are shaded. r2 values are shown in the top right area of the table. C, haplotype pattern across the HER-2 gene and the estimated frequency of each haplotype. The haplotypes are designated from I to VII in order of increasing frequency in 90 Korean women.

 
Case-control analysis. The mean age of the cases and controls was 48.4 and 49.3 years, respectively, which did not represent a statistically significant difference (Table 2). The distribution of established breast cancer risk factors was generally consistent with our expectations and was similar to what we have observed in previous studies (27, 28). Compared with controls, cases were more likely to be more educated, be older at menopause, be older at first full-term pregnancy, have breast-fed for a shorter duration, and have a greater family history (first- and second-degree relatives) of breast cancer. In addition, more cancer patients used alcohol (≥1 drink/mo) compared with controls.


View this table:
[in this window]
[in a new window]
  		Table 2. Selected characteristics of the study subjects

 
Two haplotype-tagging SNPs (I655V and P1170A) for three common haplotypes (>5%) were genotyped in cases and controls. To evaluate the effect of haplotype on breast cancer risk, we regarded each haplotype as an allele and analyzed them separately (Table 3). No significant association was observed between each haplotype and breast cancer risk. Haplotype III showed a nonsignificant protective effect when it is homozygous compared with when it is absent [i.e., homozygous null (OR, 0.5; 95% CI, 0.29-1.05)].


View this table:
[in this window]
[in a new window]
  		Table 3. Association between HER-2 haplotypes and breast cancer risk

 
Next, the two coding SNPs, I655V and P1170A, were analyzed separately for any possible association with breast cancer risk (table not shown). No significant correlations were found, although rare I655V homozygotes showed a nonsignificant protective effect on breast cancer (OR, 0.5; 95% CI, 0.28-1.01). In addition, no significant associations were found in subgroup analysis [e.g., within younger subjects (<45 years old), those who have a family history, or those with localized or in situ cancer; data not shown]. Previous studies (11, 14, 15, 29) have shown that the association between I655V status and breast cancer risk became more significant in these subgroups.

Effect of haplotypes on HER-2 protein expression and breast cancer patient survival. HER-2 protein expression as evaluated by immunohistochemical assay differed significantly according to the patients' HER-2 haplotypes (Table 4). In breast cancer patients with haplotype I (both homozygotes and heterozygotes), HER-2 protein overexpression in tumors was 1.5 times higher than in tumors from patients without haplotype I (OR, 1.5; 95% CI, 1.11-2.16; P = 0.009). Haplotype I also showed gene-dosage relationship with HER-2 expression (OR, 1.4 for heterozygote and 1.6 for homozygote; P for trend = 0.007). When we excluded HER-2 2+ cases by immunohistochemistry, the OR increased to 1.8 and the P value became more significant (0.004; data not shown). It has been reported that the 2+ scoring by immunohistochemistry is not definitive because it does not coincide with fluorescent in situ hybridization results as a test for HER-2 amplification (30). In patients homozygous for haplotype II and heterozygous for haplotype III, the HER-2 expressions in the tumor were significantly lower than those of patients without each haplotype (P = 0.014 and 0.038, respectively).


View this table:
[in this window]
[in a new window]
  		Table 4. Association between HER-2 haplotype and HER-2 protein expression in invasive cancer determined by immunohistochemistry

 
The association of each haplotype and postoperative disease-free survival was evaluated in 602 patients with a median follow-up of 41 months among 1,637 breast cancer patients without distant metastasis. The 602 patients were not different from the other 1,035 patients excluded from the survival analysis in terms of the prognostic factors, such as percentage of large (>2 cm) tumor size (47.0% versus 49.1%; P = 0.40) and lymph node positivity (34.5% versus 36.9%; P = 0.32). In a univariate Kaplan-Meier analysis (Fig. 2), patients with haplotype I had significantly shorter disease-free survival compared with patients who lacked that haplotype (P = 0.032). Lymph node status as well as the distribution of disease stage, classified as noninvasive, early stage (stage I or II), or advanced stage (stage III), was equivalent between the two groups (Table 5). The hazard ratio for disease-free survival associated with haplotype I was 4.1 (95% CI, 1.00-17.34) when adjusted for age, tumor size, and lymph node status. On the other hand, the homozygote for haplotype II and heterozygote for haplotype III, which showed association with HER-2 expression, did not have a significant effect on patients' postoperative disease-free survival (P = 0.352 and 0.328, respectively).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Disease-free survival curves for cases with haplotype I [(C)-(G)-(A)-A-C-(C)] versus those without it. Patients with haplotype I had significantly worse outcomes than patients lacking that haplotype (P < 0.032).

 

View this table:
[in this window]
[in a new window]
  		Table 5. Comparison of disease stage and lymph node status between the cases with and without haplotype I that were involved in survival analysis

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, all previous studies of HER-2 polymorphisms with respect to breast cancer have focused on I655V, although the functional relevance of this variation has not yet been established, and the results of these SNP association studies have been inconclusive. In this study, we applied a stepwise haplotype-based association approach similar to the method of Haiman et al. (21). Rebbeck et al. (31) noted that causal variants need not be studied directly but that gene discovery studies can be accomplished using a strategy that relies on linkage disequilibrium between genetic variants. In the SNP screening step, we first selected SNPs from public databases and genotyped them to look for polymorphic loci in healthy Korean women. We then defined linkage disequilibrium blocks and constructed genetic haplotypes across the HER-2 locus. A reduced set of haplotype-tagging SNPs was selected and genotyped in a large case-control analysis to evaluate the relationship between these haplotypes and breast cancer risk. This approach was very efficient even when considering the possibility that some errors may have occurred in haplotype estimation and the fact that rare haplotypes (i.e., those with a frequency <5%) were ignored. We were able to evaluate virtually the entire HER-2 gene, including the promoter region, by genotyping only two SNPs.

This is the first study that describes the linkage disequilibrium and haplotype structure of the HER-2 gene. We identified only one linkage disequilibrium block in Korean women. The size of linkage disequilibrium blocks is known to depend on ethnicity (19, 32) and to vary widely between genes (33). Because the linkage disequilibrium structure of this gene is relatively simple and the number of common haplotypes in this ethnically homogeneous population was small, we were able to considerably reduce the number of haplotype-tagging SNPs needed for genotyping and, as a result, reduce the costs associated with this process.

One weakness of using a haplotype-based approach in this study is that the average spacing between the five SNPs used to construct the haplotype blocks was relatively wide (6.8 kb) and uneven because we were not able to find additional polymorphic loci between the third and fourth SNP during SNP screening. Gabriel et al. (19) have suggested a survey approach for candidate genes with 1 SNP/5 kb. The lower the density of SNPs in this haplotype approach, the higher the possibility that we could miss linkage disequilibrium blocks.

In this study, the individual coding SNPs and haplotypes were not associated with breast cancer risk. This finding supports recent studies that found no association between I655V status and breast cancer risk (12, 13, 15, 16). In contrast to the study of Xie et al. (11), which reported a significant correlation between the Val allele of this SNP and breast cancer, we found that Val/Val genotype has a borderline protective effect (OR, 0.5). This result agrees with the study in Japanese patients published by Hishida et al. (OR, 0.31 for Val/Val genotype; ref. 16). It still remains unclear whether the findings of Xie et al. (11) represent a true ethnic variation in the penetrance of the Val allele.

We found that in patients lacking haplotype I [(C)-(G)-(A)-A-C-(C)], the most common haplotype in the HER-2 gene, HER-2 protein expression was lower and prognosis was better compared with patients who were either homozygous or heterozygous for this haplotype. The relationship between the I655V genotype and HER-2 amplification has been suggested in previous studies (29, 34), which showed that I655V is associated with HER-2 amplification and/or protein overexpression in breast cancer, although the number of cases was small and the results were not significant. If our result is not an incidental finding, it suggests that polymorphisms other than I655V may be associated with HER-2 protein expression and breast cancer progression. Among the six common SNPs analyzed in this study, the coding SNP, P1170A, which was directly genotyped, and the two promoter region SNPs, rs2643194 and rs2934971, in the 5'-untranslated region of the HER-2 gene are possible candidates for a functional polymorphism that affects gene expression and/or cancer aggressiveness. Other promoter SNPs and coding region SNPs not detected in this study may also be associated with increased HER-2 expression and cancer progression. Because the mechanism of HER-2 gene amplification has not yet been identified, it is unknown how genetic polymorphisms may affect amplification of this gene. Further study is clearly warranted to elucidate the relationship between genetic polymorphisms and HER-2 gene amplification.

Although the finding of the difference in disease-free survival rate according to haplotype I is also interesting, the results should be cautiously interpreted. First of all, the follow-up durations for the majority of the cancer patients were too short. We excluded the patients with short follow-up time from the survival analysis to reduce the bias, and as a result only 36% of the cases were involved. Although we showed that the known prognostic variables are not different between these selected patients and all the other patients, we still cannot warrant the generalizability of our findings. Longer follow-up time is required to confirm the result on survival association.

In summary, we have identified one haplotype block in the HER-2 locus in an ethnically homogeneous Korean population. Our results suggest that the individual SNPs, including I655V, and the HER-2 haplotypes investigated in this study are not associated with an increased risk of breast cancer development in Korean women. However, one HER-2 haplotype was found to affect tumor protein expression and patient prognosis. Future studies should focus on the identification of possible functional genetic variations, possibly SNPs, in the HER-2 gene or its promoter region that are linked to this haplotype.


   Acknowledgments
 
We thank Dr. Kuk-Jin Choe, Keun-Young Yoo, and Tae-You Kim in Seoul National University College of Medicine for valuable discussions.


   Footnotes
 
Grant support: Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN07-0004), and Genomic Research Center for Lung and Breast/Ovarian Cancer, Korea (Dr. Yeul Hong Kim, chairman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/29/04; revised 3/10/05; accepted 4/ 8/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 1986;319:226–30.[CrossRef][Medline]
  2. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T. The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 1986;232:1644–6.[Abstract/Free Full Text]
  3. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[Abstract/Free Full Text]
  4. Jardines L, Weiss M, Fowble B, Greene M. neu/c-erbB-2/HER-2 and the epidermal growth factor receptor (EGFR) in breast cancer. Pathobiology 1993;61:268–82.[Medline]
  5. Revillion F, Bonneterre J, Peyrat JP. ERBB2 oncogene in human breast cancer and its clinical significance. Eur J Cancer 1998;34:791–808.
  6. Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 1998;58:2825–31.[Abstract/Free Full Text]
  7. Nabholtz JM, Reese DM, Lindsay MA, Riva A. HER2-positive breast cancer: update on Breast Cancer International Research Group trials. Clin Breast Cancer 2002;3 Suppl 2:S75–9.
  8. Choi DH, Shin DB, Lee MH, et al. A comparison of five immunohistochemical biomarkers and HER-2/neu gene amplification by fluorescence in situ hybridization in white and Korean patients with early-onset breast carcinoma. Cancer 2003;98:1587–95.[CrossRef][Medline]
  9. Lemoine NR, Staddon S, Dickson C, Barnes DM, Gullick WJ. Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene 1990;5:237–9.[Medline]
  10. Papewalis J, Nikitin AYu, Rajewsky MF. G to A polymorphism at amino acid codon 655 of the human erbB-2/HER2 gene. Nucleic Acids Res 1991;19:5452.[Free Full Text]
  11. Xie D, Shu XO, Deng Z, et al. Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 2000;92:412–7.[Abstract/Free Full Text]
  12. Baxter SW, Campbell IG. Re: Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 2001;93:557–9.[Free Full Text]
  13. Keshava C, McCanlies EC, Keshava N, Wolff MS, Weston A. Distribution of HER2(V655) genotypes in breast cancer cases and controls in the United States. Cancer Lett 2001;173:37–41.[CrossRef][Medline]
  14. McKean-Cowdin R, Kolonel LN, Press MF, Pike MC, Henderson BE. Germ-line HER-2 variant and breast cancer risk by stage of disease. Cancer Res 2001;61:8393–4.[Abstract/Free Full Text]
  15. Wang-Gohrke S, Chang-Claude J. Re: Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 2001;93:1657–9.[Free Full Text]
  16. Hishida A, Hamajima N, Iwata H, et al. Re: Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 2002;94:1807–8.[Free Full Text]
  17. Fleishman SJ, Schlessinger J, Ben-Tal N. A putative molecular-activation switch in the transmembrane domain of erbB2. Proc Natl Acad Sci U S A 2002;99:15937–40.[Abstract/Free Full Text]
  18. Daly MJ, Rioux JD, Schaffner SF, Hudson TJ, Lander ES. High-resolution haplotype structure in the human genome. Nat Genet 2001;29:229–32.[CrossRef][Medline]
  19. Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science 2002;296:2225–9.[Abstract/Free Full Text]
  20. Patil N, Berno AJ, Hinds DA, et al. Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21. Science 2001;294:1719–23.[Abstract/Free Full Text]
  21. Haiman CA, Stram DO, Pike MC, et al. A comprehensive haplotype analysis of CYP19 and breast cancer risk: the Multiethnic Cohort. Hum Mol Genet 2003;12:2679–92.[Abstract/Free Full Text]
  22. Hynes NE. Amplification and overexpression of the erbB-2 gene in human tumors: its involvement in tumor development, significance as a prognostic factor, and potential as a target for cancer therapy. Semin Cancer Biol 1993;4:19–26.[Medline]
  23. Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology 2001;61Suppl 2:1–13.
  24. Han W, Kang D, Park IA, et al. Associations between breast cancer susceptibility gene polymorphisms and clinicopathological features. Clin Cancer Res 2004;10:124–30.[Abstract/Free Full Text]
  25. Qin ZS, Niu T, Liu JS. Partition-ligation-expectation-maximization algorithm for haplotype inference with single-nucleotide polymorphisms. Am J Hum Genet 2002;71:1242–7.[CrossRef][Medline]
  26. Greene FL, Page DL, Fleming ID, et al., editors. AJCC cancer staging manual. 6th ed. New York: Springer; 2002.
  27. Choi JY, Abel J, Neuhaus T, et al. Role of alcohol and genetic polymorphisms of CYP2E1 and ALDH2 in breast cancer development. Pharmacogenetics 2003;13:67–72.[CrossRef][Medline]
  28. Shin A, Kang D, Nishio H, et al. Estrogen receptor {alpha} gene polymorphisms and breast cancer risk. Breast Cancer Res Treat 2003;80:127–31.[CrossRef][Medline]
  29. Millikan R, Eaton A, Worley K, et al. HER2 codon 655 polymorphism and risk of breast cancer in African Americans and whites. Breast Cancer Res Treat 2003;79:355–64.[CrossRef][Medline]
  30. Dowsett M, Bartlett J, Ellis IO, et al. Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres. J Pathol 2003;199:418–23.[CrossRef][Medline]
  31. Rebbeck TR, Ambrosone CB, Bell DA, et al. SNPs, haplotypes, and cancer: applications in molecular epidemiology. Cancer Epidemiol Biomarkers Prev 2004;13:681–7.[Free Full Text]
  32. Reich DE, Cargill M, Bolk S, et al. Linkage disequilibrium in the human genome. Nature 2001;411:199–204.[CrossRef][Medline]
  33. Bonnen PE, Wang PJ, Kimmel M, Chakraborty R, Nelson DL. Haplotype and linkage disequilibrium architecture for human cancer-associated genes. Genome Res 2002;12:1846–53.[Abstract/Free Full Text]
  34. Zheng W, Kataoka N, Xie D, Young SR. Re: Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 2001;93:558–9.[Free Full Text]



This article has been cited by other articles:


Home page
 Ann OncolHome page
S Beauclair, P Formento, J. Fischel, W Lescaut, R Largillier, E Chamorey, P Hofman, J. Ferrero, G Pages, and G Milano
Role of the HER2 [Ile655Val] genetic polymorphism in tumorogenesis and in the risk of trastuzumab-related cardiotoxicity
Ann. Onc., August 1, 2007; 18(8): 1335 - 1341.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, W.
Right arrow Articles by Noh, D.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, W.
Right arrow Articles by Noh, D.-Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online