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© 2000 American Society for Clinical Oncology HER-2/neu Amplification in Benign Breast Disease and the Risk of Subsequent Breast CancerFrom the Department of Epidemiology, Department of Pathology and Laboratory Medicine, Department of Biostatistics, and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC; Department of Pathology and Laboratory Medicine, Northwestern University, Chicago, IL; Department of Laboratory Medicine and Pathology, and Department of Health Sciences Research, Mayo Clinic, Rochester, MN; and Division of Clinical Sciences, National Cancer Institute, Bethesda, MD. Address reprint requests to Kathleen Conway, PhD, University of North Carolina at Chapel Hill, Campus Box 7400, 2101 McGavran Greenberg Hall, Chapel Hill, NC 27599; email kconway{at}med unc.edu.
PURPOSE: The purpose of this study was to determine whether the presence of HER-2/neu gene amplification and/or overexpression in benign breast disease was associated with an increased risk of subsequent breast cancer. PATIENTS AND METHODS: We conducted a nested case-control study of a cohort of women who were diagnosed with benign breast disease at the Mayo Clinic and who were subsequently observed for the development of breast cancer. Patients who developed breast cancer formed the case group, and a matched sample from the remaining cohort served as controls. Benign tissue samples from 137 cases and 156 controls and malignant tissues from 99 cases provided DNA or tissue for evaluation of HER-2/neu amplification and protein overexpression. RESULTS: Among the controls, seven benign tissues (4.5%) demonstrated low-level HER-2/neu amplification, whereas 13 benign (9.5%) and 18 malignant (18%) tissue specimens from cases exhibited amplification. HER-2/neu amplification in benign breast biopsies was associated with an increased risk of breast cancer (odds ratio [OR] = 2.2; 95% confidence interval [CI], 0.9 to 5.8); this association approached statistical significance. The risks for breast cancer associated with benign breast histopathologic diagnoses were OR = 1.1 (95% CI, 0.6 to 1.9) for lesions exhibiting proliferation without atypia and OR = 1.5 (95% CI, 0.4 to 5.6) for the diagnosis of atypical ductal hyperplasia. For women having both HER-2/neu amplification and a proliferative histopathologic diagnosis (either typical or atypical), the risk of breast cancer was more than seven-fold (OR = 7.2; 95% CI, 0.9 to 60.8). Overexpression of the HER-2/neu protein product, defined as membrane staining in 10% or more of epithelial cells, was found in 30% of the breast tumors but was not detected in any of the benign breast tissues. Case patients who had HER-2/neu gene amplification in their malignant tumor were more likely to have had HER-2/neu amplification in their prior benign biopsy (P = .06, Fisher’s exact test). CONCLUSION: Women with benign breast biopsies demonstrating both HER-2/neu amplification and a proliferative histopathologic diagnosis may be at substantially increased risk for subsequent breast cancer.
HISTOPATHOLOGIC diagnosis is the prevailing method of assessing the risk of breast cancer among women diagnosed with benign breast disease (BBD).1 Although histopathologic risk classification can accurately identify a subset of women with BBD (ie, atypical hyperplasia) who are at considerably increased risk of breast cancer, less than 4% of women with biopsy-diagnosed BBD have atypical hyperplasia, and the majority of these women will not go on to develop carcinoma.2-4 Conversely, some of the 96% of women diagnosed with conditions other than atypical hyperplasia are at increased risk of subsequent invasive breast cancer, and for them, the histopathology is not considerably informative. Further, interobserver and/or intraobserver variability, differences in pathologic criteria, and limitations in the quantity of tissue available for examination have raised concerns about the robustness of histopathology in predicting the risk of breast cancer among women diagnosed with BBD.5-8 Because the present method of histopathologic risk classification identifies only a small portion of breast cancer cases that develop among women diagnosed with BBD, application of molecular techniques to identify possible genetic alterations in BBD may provide an avenue for improving risk prediction. Specifically, amplification of the HER-2/neu oncogene in BBD might serve as a sensitive biomarker of susceptibility for subsequent invasive breast cancer. HER-2/neu amplification is restricted to carcinomas of glandular epithelial origin and is at least three times more common in breast cancer than adenocarcinomas of other sites.9 HER-2/neu was initially identified as a marker of advanced-stage tumors and a prognostic marker.10 In 1988, van de Vijver et al11 described overexpression and amplification of HER-2/neu in carcinoma-in-situ, a finding that was later confirmed by two independent laboratories.12,13 The high frequency of HER-2/neu alterations in the early stages of breast cancer suggests that this gene may have a role in the inception of breast tumors. Low-level HER-2/neu (c-erbB-2) expression in benign breast lesions has been reported by several independent investigators.14,15 Lizard-Nacol et al,16 however, could not confirm HER-2/neu gene amplification in BBD, but these investigators used the Southern Blot technique, which requires approximately 50-fold more DNA than polymerase chain reaction (PCR) and thus is considerably less sensitive.17 Further, the intralaboratory reproducibility of Southern Blot analysis is inferior to that of PCR, potentially leading to misclassification error.18 In this study, we report that amplification of the HER-2/neu gene occurs in BBD and that its assessment can improve the accuracy of predicting the risk of subsequent breast cancer among women diagnosed with BBD. HER-2/neu amplification offers the promise of a valid surrogate end point for breast cancer susceptibility.19,20
Study Design and Population We conducted a case-control study nested within a unique cohort of 6,805 women who were diagnosed and treated for BBD in Rochester, MN, at Mayo Clinic hospitals (Saint Mary’s Hospital and Rochester Methodist Hospital) and the Olmsted Community Hospital, between January 1, 1967, to December 31, 1981. The cohort was observed for development of breast cancer through inpatient and outpatient medical record review, postal communication, and/or telephone contact. The median length of follow-up for the subsequent development of breast cancer was 10.2 years. As of January 1, 1987, 236 cases of breast cancer were diagnosed in the cohort. Diagnoses were confirmed through histopathologic review (n = 217) or from pathology reports and associated medical records (n = 19). Controls, women with no clinical manifestation of breast cancer as of January 1, 1987, were selected from the same cohort using a stratified random sampling approach. Strata were defined based on the year of the original benign breast biopsy (three strata: 1969 to 1971, 1972 to 1976, and 1977 to 1981), age at the time of biopsy (seven strata of 10 years each, from < 25 to > 74 years), and county of residency (two strata; Olmsted County or non–Olmsted County). Controls were selected to be frequency matched within strata to the cases. To limit the workload for molecular assays, only one control was randomly selected per case. We were able to access tissue blocks from 149 cases with paired benign and malignant tissues and 160 of their respective controls. Because of the nature of the sampling plan, cases and controls were reasonably well matched on age, time of biopsy, and residence. The same matching scheme was retained in the smaller tissue set as in the original case-control study.
Tissues and Pathology Review
HER-2/neu (c-erbB-2) Expression
Gene Amplification
Differential PCR reactions were carried out (in a 50-µL reaction mixture containing 100 ng genomic DNA or 2 to 5 µL DNA lysate extracted from paraffin-embedded tissues) using a standard protocol: 500 nmol/L of each HER-2/neu primer, 500 nmol/L of each reference gene primer, 1 x PCR buffer (10 mmol/L TrisHCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2), 200 µmol/L of each deoxyribonucleotide triphosphate, and 1.25 units AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA). PCR cycle conditions were as follows: one cycle of 94°C for 1 minute followed by a hot start at 80°C. The reaction was continued with 33 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute The reaction was completed with an additional cycle of 94°C for 1 minute and 60°C for 1 minute. Formalin-fixed, paraffin-embedded normal human spleen and the SKBR3 cell line, with four- to eight-fold amplification of the HER-2/neu gene, were used as negative and positive controls, respectively. The PCR product sizes obtained for each primer set were: 98 base pairs (bp) for HER-2/neu, 127 bp for PGR, and 85 bp for IFN- PCR products were resolved on 10% polyacrylamide gels and visualized using ethidium bromide staining and ultra violet irradiation. The ratios of intensities of the target to reference gene products, run on the same gels, were derived from densitometric analysis and were used as a measure of the relative copy number of HER-2/neu. Differential PCR is sensitive enough to detect gene amplification in the range of two- to four-fold.24 Assay results were interpreted independently by two of the investigators (A.T.S. and K.C.) who were blinded to the case or control status of the study participants. A sample was considered to be amplified if both investigators scored it as such, and this result was verified in a second differential PCR reaction. If there was disagreement between readers in the first or second assay, differential PCR was repeated a third time. If the readers disagreed on the results of two of the three assays, the sample was considered negative for amplification. Assay reproducibility was also assessed by repeating the differential PCR on a random selection of 20% of the samples.
Statistical Analysis
Archival tissue blocks from 160 control (benign only) and 149 case women (benign and tumor) were initially available for study. A number of these blocks (three control benign, two case benign, and 19 tumors) were eliminated from further analyses because they either had insufficient malignant tissue remaining for study or they contained tissue other than breast or primary tumor (usually lymph node or skin). Additional tissues were removed from further analysis because HER-2/neu could not be PCR-amplified despite our repeated efforts, presumably because of DNA degradation. These included 31 tumors, one control benign tissue, and 10 case benign tissues. Of the 11 benign tissues excluded from analysis, 10 were diagnosed as fibrocystic (one control and nine cases), and one case was a fibroadenoma. The histopathologic diagnoses and HER-2/neu amplification results presented are for 156 control benign tissues, 137 case benign tissues, and 99 case malignant tissues for which PCR amplification was successful. Of these, there were 87 paired benign and malignant specimens from case women. As listed in Table 1, benign breast parenchyma with fibrosis and fibroadenoma were two of the most commonly diagnosed benign conditions among the cases (38 [28%] of 137 cases and 15 [11%] of 137 cases, respectively) and controls (50 [32%] of 156 cases and 17 [11%] of 156 cases, respectively). Other commonly diagnosed conditions were adenosis (10% in cases, 16% in controls), cysts (10% in cases, 8% in controls), apocrine metaplasia (10% in cases, 8% in controls), and moderate to severe ductal hyperplasia without atypia (12% in cases, 7% in controls). Four percent of cases and 2.5% of controls had a diagnosis of ductal hyperplasia with atypia, which placed them at the greatest increased risk for subsequent breast cancer. There was no significant difference between cases and controls in major histologic diagnoses (nonproliferative, proliferative without atypia, and atypical hyperplasia) of benign tissues (P = .84; Fisher’s exact test).
HER-2/neu gene amplification in the benign and malignant tissue samples was evaluated by the differential PCR approach. Low-level (two- to four-fold) HER-2/neu oncogene amplification was detected in the benign breast biopsies of seven control women (4.5%) and 13 case women (9.5%) and in 18 malignant tumors from cases (18%). The differential PCR assay demonstrated significant reproducibility between repeated runs (  = 0.76; P < .001), and the concordance between the two reviewers was statistically significant ( = 0.66; P < .001). An example of differential PCR detection of HER-2/neu amplification is shown in Fig 1.
The risk estimates for the development of subsequent breast cancer based on proliferative status, HER-2/neu amplification, or both in benign tissues are listed in Table 2. The estimated risk of developing breast cancer for women with HER-2/neu amplification in their benign tissue was more than two-fold (OR = 2.2, 95% CI, 0.9 to 5.8). This was greater than the estimated risks of breast cancer conferred by a histopathologic diagnosis of proliferation without atypia (OR = 1.1, 95% CI, 0.6 to 1.9) or with atypia (OR = 1.5, 95% CI, 0.4 to 5.6). Proliferative histologic diagnoses were more common among the HER-2/neu–amplified case benign tissues than control benign tissues; six of 13 case benign breast lesions exhibiting HER-2/neu amplification had proliferative histopathologic diagnoses, whereas only one of seven control benign biopsies with HER-2/neu amplification demonstrated proliferative activity. Consequently, women with benign breast biopsies that exhibited both HER-2/neu amplification and a proliferative diagnosis (both with or without atypia) were estimated to have a substantially increased risk of subsequent breast cancer (OR = 7.2, 95% CI, 0.9 to 60.8) compared with women whose benign biopsies did not exhibit HER-2/neu amplification or epithelial proliferation.
HER-2/neu amplification and c-erbB-2 overexpression were compared in the benign tissues as listed in Table 3. None of the benign tissues from cases or controls were positive for c-erbB-2 overexpression at the designated level of 10% or more of epithelial cells (one example shown in Fig 2). Six control benign and four case benign biopsies showed minimal c-erbB-2 membrane staining in 1% to 9% of cells, and none of these exhibited HER-2/neu amplification. None of the seven control benign or 13 case benign biopsies that demonstrated low-level HER-2/neu amplification overexpressed c-erbB-2.
Of the 99 malignant tumors, 18 (18%) exhibited HER-2/neu amplification, and 30 (30%) overexpressed the c-erbB-2 protein. Ten tumors (10%) exhibited both HER-2/neu amplification and c-erbB-2 overexpression, 61 (62%) were negative for both, and 28 (28%) were discordant, displaying either HER-2/neu amplification or c-erbB-2 overexpression (data not shown). The malignant tumors with HER-2/neu amplification had higher expression of c-erbB-2 than the nonamplified tumors (P = .003; Wilcoxon rank sum test) (Fig 3).
The relationship between HER-2/neu amplification in the benign biopsy and subsequent malignant tumor was evaluated for the 87 case women for whom matched tissues were available (Table 4). There was a marginally significant association between HER-2/neu amplification in the benign biopsies and subsequent malignant tumors from cases (P = .06; Fisher’s exact test), although the association was weak (Kendall’s tau-b = 0.23). HER-2/neu amplification occurred more often in the malignant tumors if the corresponding prior benign biopsies exhibited amplification; four (44%) of nine women who had HER-2/neu amplification in their benign biopsy showed amplification in their malignant tumor, whereas only 12 (15%) of 78 women with HER-2/neu–negative benign biopsies subsequently exhibited HER-2/neu–amplified tumors.
We compared the relative breast locations of the paired benign and malignant lesions from 85 case patients for whom data was available. In 54 cases (64%), malignancies arose in the same breast as the prior benign lesion. Of the nine cases with HER-2/neu–amplified benign biopsies, five developed a malignant tumor in the same breast.
The results of this study suggest that amplification of the HER-2/neu gene in benign breast biopsies and a proliferative histopathologic diagnosis are associated with a substantially increased risk of subsequent breast cancer (OR = 7.2, 95% CI, 0.9 to 60.8). HER-2/neu amplification or hyperplasia alone was each associated with only modest increases in breast cancer risk in this study. Although HER-2/neu gene amplification was found more frequently in biopsies with moderate to florid hyperplasia, many benign breast biopsies exhibited either HER-2/neu amplification or hyperplasia but not both, suggesting that these features can occur independently. That HER-2/neu amplification and hyperplasia may occur independently is consistent with many other studies that failed to find an association between HER-2/neu gene amplification or overexpression and a high proliferative capacity or growth fraction in breast carcinomas as measured by S phase markers such as Ki-67 staining.25-29 Overexpression of c-erbB-2, defined as staining in greater than 10% of epithelial cells, was not detected in any of the benign tissues with or without HER-2/neu amplification. Most previous studies have similarly found a lack of c-erbB-2 protein overexpression in benign breast biopsies, suggesting that overexpression of HER-2/neu generally occurs at the transition from hyperplasia to ductal carcinoma-in-situ.12,30-34 The recent study of Rohan et al,35 however, reported a low prevalence of c-erbB-2 overexpression (at the level of > 10% immunopositive cells) in the benign biopsies of cases and controls but no increased breast cancer risk associated with HER-2/neu overexpression. In contrast to our findings in benign breast tissue, we found a statistically significant correlation (P = .003) between HER-2/neu amplification and protein overexpression in the malignant tumors, although amplification and overexpression were not perfectly concordant. Approximately 20% of breast tumors exhibited overexpression of c-erbB-2 in the absence of HER-2/neu gene amplification. Previous reports by other investigators have identified similar percentages of malignant tissues overexpressing HER-2/neu while having diploid copies of the gene,36-39 indicating that mechanisms other than gene amplification can contribute to elevated expression of the c-erbB-2 protein. Several recent studies have shown that HER-2/neu overexpression may result from transcriptional deregulation involving cis-acting enhancer elements near the HER-2/neu promoter and from increased expression of transcription factors that bind to these sequences.40-43 Differences in the technical sensitivities of differential PCR and immunohistochemistry may also partially account for discordance in HER-2/neu gene amplification and protein overexpression in breast tumors. Higher levels of HER-2/neu amplification have been shown to be more closely associated with protein overexpression,12,37,44 suggesting that the HER-2/neu–amplified breast tissues in our study that failed to exhibit c-erbB-2 overexpression may have harbored only low levels of gene amplification. The correlation between HER-2/neu gene amplification and protein overexpression is also influenced by the particular c-erbB-2 antibody used.45 Furthermore, the sensitivity of differential PCR to detect gene amplification, which is estimated to be in the range of two- to four-fold,24 may be reduced if the lesion of interest contains a large proportion of normal diploid cells. Under this condition, lesions with low-level HER-2/neu amplification could appear negative for gene amplification by differential PCR, yet may demonstrate c-erbB-2 membrane staining in more than 10% of cells by immunohistochemistry. Application of more refined tissue dissection techniques, such as laser capture microdissection, should further enhance the sensitivity of molecular assays for detecting gene amplification.46 Finally, it is possible that imbalances of chromosome 17 on which the HER-2/neu gene resides, in addition to gene amplification, may be detected by the differential PCR approach.44,47 Concordance in HER-2/neu amplification status was found in 70 of 87 paired benign and malignant breast tissues from the case women (P = .06, Fisher’s exact test), although the association was weak (Kendall’s tau-b = 0.23). Only 17 cases showed discordance in HER-2/neu amplification status between their benign and malignant tissues, with either benign or malignant tissues, but not both, exhibiting amplification. HER-2/neu amplification in malignant but not prior benign tissues was not an unexpected finding, because amplification of this gene has been associated with advanced histologic grades and other poor prognostic features of breast tumors.48,49 Amplification detected in benign but not malignant lesions could be explained by a polyclonal origin of the benign lesions. Although the present data do not permit us to assess this possibility, others have shown polyclonality in benign conditions of the breast.50 The most plausible explanation for discordance in HER-2/neu amplification status, however, is that the benign and subsequent malignant lesions were probably derived from different precursor cells; this idea is supported by the fact that the original benign lesions were surgically removed. Several benign and malignant tissue pairs each exhibited HER-2/neu amplification, yet the malignant tumor arose in the opposite breast relative to the benign lesion. This pattern of multiple discrete foci of HER-2/neu–amplified cells supports the idea that molecular alterations can occur as a field effect in the breasts of some women. The existence of field effects in breast tissue are consistent with models of breast carcinogenesis that propose that certain benign lesions, such as hyperplasias, may be nonobligatory precursors of breast cancer, having a natural history that is parallel to, rather than serial with, breast carcinoma.2,3,12,51 Recent studies have suggested that genetic alterations, including p53 mutations, loss of heterozygosity, and microsatellite instability, can be detected in premalignant lesions of the breast.50,52-55 Our findings provide additional evidence that genetic alterations, including HER-2/neu amplification, may occur as relatively early events in the development of breast cancer. Amplification of HER-2/neu, in combination with a proliferative histopathologic diagnosis in benign breast lesions, was found to be associated with an increased risk of subsequent breast cancer. To our knowledge, this is the first study that has assessed HER-2/neu amplification in benign breast disease and estimated the risk of subsequent carcinoma based on this genetic perturbation. This work should stimulate further research into the prevalence and the role of genetic alterations in nonmalignant conditions of the breast and their application in screening and early intervention.
This work was supported by the Specialized Program of Research Excellence in Breast Cancer grant no. CA 58223, the National Institute of Environmental Health Sciences grant no. T32-ES07018, the National Cancer Institute grants no. CA 46332 and CA44768), and the American Cancer Society grant no. RPG-97-110-01-CCE. We thank Daniel Fried for his technical advice.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
). The following primer sequences were used: Her-2/neu: 5' - CCTCTGACGTCTTCGACCTC-3' and 5' - ATCTTCTGCTGCCGTCGTCGCTT-3' IFN-
10% of epithelial cells were positive for overexpression. 
Tumors without HER-2/neu amplification (n = 81). 
Tumors with HER-2/neu amplification (n = 18) had higher expression of c-erbB-2 (P = .003, Wilcoxon rank sum).
