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[Cancer Research 62, 1481-1488, March 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Molecular-Cytogenetic Analysis of HER-2/neu Gene in BRCA1-associated Breast Cancers1

Tatyana A. Grushko, M. Anne Blackwood, Phil L. Schumm, Fitsum G. Hagos, Moses O. Adeyanju, Michael D. Feldman, Melinda O. Sanders, Barbara L. Weber and Olufunmilayo I. Olopade2

Section of Hematology/Oncology, Department of Medicine, Committees on Genetics and Cancer Biology [T. A. G., F. G. H., O. I. O], and Department of Health Studies [P. L. S.], University of Chicago, Chicago, Illinois 60637-1463; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6021 [M. A. B.]; Laboratory Corporation of America, Elmhurst, Illinois 60126-1539 [M. O. A.]; Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia Pennsylvania 19104-6021 [M. D. F., M. O. S.]; and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 9104–6160 [B. L. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The BRCA1 tumor suppressor gene and the HER-2/neu oncogene are located in close proximity on the long arm of chromosome 17 (17q11–21). Absence of BRCA1 or functional overexpression of the HER-2/neu gene presumably contributes to the somatic phenotype of breast cancer in premenopausal women, characterized by unfavorable prognostic features such as high tumor grade, hormone receptor negativity, and high proliferation rate. To examine whether amplification of HER-2/neu contributes to the aggressive biology of BRCA1-associated tumors, we have performed fluorescence in situ hybridization on formalin-fixed paraffin-embedded breast tumor tissue sections from 53 BRCA1 mutation carriers and 41 randomly selected, age-matched sporadic breast cancer cases. Although BRCA1-associated and sporadic tumors were equally likely (19% versus 22%) to exhibit HER-2/neu amplification [defined as a ratio of HER-2/neu copies to chromosome 17 centromere (CEP17) copies >= 2], 6 (15%) of the sporadic tumors were highly amplified (defined as a ratio >= 5) versus none of the BRCA1-associated tumors (P = 0.048). HER-2 protein overexpression as measured by immunohistochemical analysis was not observed among the BRCA1-associated cases (P = 0.042). Four of 21 (19%) sporadic tumors exhibited strong membranous staining of HER-2 (intensity level of 3+) as compared with 0 of 39 BRCA1-associated tumors. Our data suggest that a germ-line mutation in the BRCA1 tumor suppressor gene is associated with a significantly lower level of HER-2/neu amplification. Thus, it is possible that BRCA1-associated and HER-2/neu-highly amplified tumors progress through distinct molecular pathways, and the aggressive pathological features of BRCA1-associated tumors appear unrelated to amplification of the adjacent HER-2/neu oncogene.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer results from the accumulation of multiple genetic alterations, including point mutations, chromosomal rearrangements, and genomic amplifications and deletions (1) . Gene amplification primarily involves oncogenes the overexpression of which leads to growth deregulation, whereas deletions commonly affect tumor suppressor genes that control cell cycle progression and DNA damage response pathways.

BRCA1 is a breast cancer susceptibility gene, the mutant form of which predisposes to both breast and ovarian cancers (2 , 3) . BRCA1 functions as a classical tumor suppressor gene on 17q12–21, and loss of the wild-type allele is required for tumorigenesis in mutation carriers. BRCA1 encodes a multifunctional protein which, together with other proteins, contributes to homologous recombination, DNA damage response, and transcriptional regulation (4) , processes also known to be involved in gene amplification (5) . BRCA1-associated tumors display aggressive pathological features (3) . However, the molecular mechanism(s) contributing to tumor progression in women with germ-line BRCA1 mutations is largely unknown. Specifically, the contribution of the adjacent HER-2/neu oncogene (localized on 17q11–12) to the aggressive behavior of BRCA1-associated tumors has not been clarified.

HER-2/neu (HER2, ERBB2) encodes a 185-kDa transmembrane cell surface receptor glycoprotein with tyrosine kinase activity that belongs to the epidermal growth factor receptor family (6) . The HER-2/neu proto-oncogene is involved in the regulation of normal cell growth and division and is expressed at low levels in many normal epithelial cells. The HER-2/neu oncogene is associated with tumor aggressiveness and enhanced chemoresistance of cancer cells through the mechanism of gene amplification, followed by increased transcription and higher levels of protein expression. HER-2/neu amplification/overexpression has been reported in 20–30% of human breast cancers (7) and in varying degrees in tumors of the ovary, endometrium, and other organs (6) . In multiple studies, HER-2/neu amplification/overexpression has been shown to be an independent prognostic and predictive marker of response to therapy with Herceptin (8, 9, 10) . However, the mechanism(s) by which HER-2/neu is selectively amplified/overexpressed in some cancers remains poorly understood. In addition, the chromosomal machinery through which the oncogene amplification is generated is largely unknown.

Both BRCA1-associated and HER-2/neu-amplified tumors occur in a subset of young women with histologically aggressive, hormone receptor-negative, and highly proliferative breast cancers. To date, few studies have evaluated HER-2/neu protein status in BRCA1-associated breast cancers, and the results have been inconclusive (11, 12, 13, 14, 15) . These studies have included relatively small numbers of samples, and the results are confounded by the false-positive rates associated with the different antibodies used to evaluate HER-2/neu protein expression (16 , 17) . In the present study, using breast cancers from BRCA1 mutation carriers, we tested the hypothesis that the presence of a germ-line BRCA1 mutation might be associated with amplification at the HER-2/neu locus. To our knowledge, this is the first comprehensive analysis of HER-2/neu gene amplification in BRCA1-associated tumors using molecular-cytogenetic techniques.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Materials.
The studies were conducted under research protocols approved by the University of Chicago and the University of Pennsylvania Institutional Review Board. Only tumors from women with protein truncating or splice site and missense mutations that affect protein function were included (18 , 19) . Informed consent was obtained from each BRCA1 mutation carrier from whom a tissue block was obtained. Sporadic breast tumors were identified as a random sample of breast cancer cases operated on at the participating hospitals. Sporadic cases were frequency matched to carriers on age (<50, >=50). Diagnosis was confirmed by review of medical records, and data were collected on clinicopathological features including age, tumor size, histological type, tumor grade, hormone receptor status, nodal status, and tumor stage.

Cell Lines.
Human breast cancer cell lines MCF-7, known to have no HER-2/neu amplification (20) , and HCC1937, known to have no HER-2/neu protein overexpression (21) , and HS578T and BT-474, known to have low and high levels of HER-2/neu gene amplification, respectively (20 , 22) , were used as negative and positive controls. Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to recommended conditions. Metaphase cell preparations and formalin-fixed paraffin-embedded sections from cell lines and from normal peripheral blood lymphocytes were performed according to routine protocols (23) .

DNA FISH3 Probes.
Probes and corresponding hybridization mixtures (Vysis, Inc., Downers Grove, IL) included HER-2/neu labeled with SpectrumOrange, and chromosome enumeration probes CEP17, labeled with SpectrumGreen and CEP9, labeled with SpectrumOrange. The HER-2/neu probe contains DNA sequences specific for the HER-2/neu human gene locus and hybridizes to 17q11.2–12 of human chromosome 17. The CEP17 probe contains {alpha}-satellite DNA that hybridizes to the centromeric region of chromosome 17 (17p11.1-q11.1). The CEP17 probe was used in dual hybridizations with the HER-2/neu probe to differentiate increases in HER-2/neu signal because of increases in the number of chromosome 17 homologues from true gene amplification, and as an internal control for chromosome 17 aneusomy. The CEP9 probe, labeled with SpectrumOrange, contains {alpha}-satellite DNA that hybridizes to the centromeric region of chromosome 9 (9p11-q13). The CEP9 probe was used as control to detect ploidy.

FISH Assay.
Fluorescence in situ hybridization for detection of HER-2/neu gene amplification in formalin-fixed, paraffin-embedded breast tumor tissue specimens was performed using the Vysis, Inc. Paraffin Pretreatment Kit, PathVysion HER-2 DNA Probe Kit and Post-Hybridization Rapid Wash "LSI Procedure" adjusted for use with archival material (24) .

FISH Results Interpretation.
Scoring of fluorescent signals was performed without knowledge of the results of prior clinical and IHC analysis by two investigators (in instances of equivocal slides). In each tumor sample an average of 114 (30–200) well-defined malignant nuclei, and in each normal sample an average of 50 (20–100) nonmalignant nuclei were scored (25) . Both the absolute number of HER-2 signals and the ratio of HER-2 signals to chromosome 17 centromere signals were recorded. On the basis of the published studies (24 , 25) , we adopted the following standardized criteria for determining HER-2/neu gene amplification status. Tumors with a HER-2:CEP17 signal ratio of <2 were considered to be nonamplified, whereas those with a ratio of 2 or greater were considered to have "low amplification" (2.0–3.0), "moderate amplification" (3.1–5.0), or "high amplification" (>5).

Definition of the Chromosome 17 Copy Number Alterations.
The chromosome 17 copy number alteration was estimated by scoring the reduction of CEP17 signals to one copy (monosomy) and the gain of CEP17 signals to three or more copies (polysomy). The cutoff point for chromosome 17 copy number alterations in malignant cells was estimated from comparison with corresponding normal breast epithelium and separately for each group of specimens according to published standards (17 , 22 , 26 , 27) .

Tumor ploidy was identified by scoring and comparing the mean number of CEP9 and CEP17 signals per cell in a dual-color FISH experiment conducted on a set of six tumors from both BRCA1-associated and sporadic groups.

IHC Assay.
IHC of HER-2/neu protein was done in batches on tissue sections adjacent to those analyzed by FISH using a mouse monoclonal antibody to the intracellular domain of HER-2/neu protein (1:10, NCL-CB11; Novocastra Laboratories, Newcastle, United Kingdom) as described previously (17) .

Each slide was scored in a blinded fashion by a single pathologist. The immunostaining was read in a semiquantitative manner and graded as follows: 0 (no staining), 1+ (equivocal or weak staining in most tumor cells or stronger staining in less than 25% of cells), 2+ (definitive staining of moderate intensity in most tumor cells or stronger staining in 25–75% of cells), and 3+ (strong staining in more than 75% of tumor cells). Intensity scores of 0 or 1+ were designated as negative expression, whereas scores of 2+ and 3+ were designated as positive expression.

Statistical Analysis.
Data from the three cell lines that were analyzed both in suspension and in paraffin were used to compute the mean number of HER-2 and CEP17 signals per cell for each of two repetitions within each of the six cell lines by preparation combinations. A linear regression was then fitted to the logarithm of these means using both preparation and cell line as covariates (28) .

Demographic and disease characteristics were compared between BRCA1-associated and sporadic cases using either a two-sample t test (to compare two means) or Fisher’s exact test (to compare two percentage distributions). For each tissue sample (both normal and tumor), we computed the mean number of both HER-2 and CEP17 signals per cell, the percentage of cells with only one CEP17 signal, and the percentage with three or more CEP17 signals. The mean and SD of these values are reported separately for the BRCA1-associated and sporadic cases. A t test was used to compare BRCA1-associated tumors to sporadic tumors (two-sample test) and to compare tumor tissue to normal tissue (paired test).

The mean number of HER-2 signals in each tumor sample was plotted against the mean number of CEP17 signals and depicted separately for the BRCA1-associated and sporadic tumors, and the ratio of the number of HER-2 signals to the number of CEP17 signals was computed for each sample. A two-sample t test (assuming unequal variances) was used to compare the mean of the log-transformed ratios in the BRCA1 mutation-positive cases to that in the sporadic cases.

Finally, we compared the mean number of copies per cell of chromosome 9 to that of chromosome 17 in six tumors using a t test performed on the log-transformed values.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Probe Specificity and HER-2/neu Gene Detection in Normal Human Lymphocytes and in Breast Cancer Cell Lines.
Inherent probe hybridization efficiency was confirmed by performing FISH on normal lymphocyte metaphase chromosomes and interphase nuclei from two samples. HER-2 and CEP17 signals were located close to each other on chromosome 17 (Fig. 1A)Citation . The mean number (±SD) of HER-2 and CEP17 signals per cell was 2.0 ± 0.01 and 1.98 ± 0.01, respectively. The mean HER-2:CEP17 ratio was 1.0. Scoring the chromosome 17 ploidy gave the following results (mean ± SD): 95.1 ± 0.1% of disomic signals, 4.1 ± 0.1% of monosomic signals, and 0.9 ± 0.2% of polysomic signals.



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  		Fig. 1. Representative photomicrographs of reference cells from normal human lymphocytes (A) and breast cancer cell lines (B–E) after FISH. The HER-2/neu gene is localized by red fluorescent signals, and chromosome 17 centromere (CEP17) is localized by green fluorescent signals. The cells were counterstained with 4',6-diamidino-2-phenylindole (blue). A, partial metaphase cell of a normal mitogen-stimulated human lymphocyte. Two signals for each probe can be detected on chromosome 17 as a normal pattern. B, partial metaphase cell from low-amplified HS578T cell line showing an isochromosome 17 with a duplicated HER-2/neu pattern (ratio 2.5). C, metaphase spread from MCF-7 cells in suspension and an interphase nucleus in paraffin (inset in C) showing no HER-2/neu amplification. Two HER-2 signals and three CEP17 signals are visible per nucleus. Arrow, the chromosome 17 derivative with only the green signal visualized. D, BT474 cells in metaphase from cell suspension and in interphase from paraffin (inset in D), showing multiple bright spots representing highly amplified and nonamplified, and translocated and nontranslocated HER-2/neu gene copies on different chromosomes. Four to six CEP17 signals per nucleus are visible. E, HCC1937 BRCA1-deficient cells in metaphase from cell suspension and in interphase from paraffin (inset in E), showing no amplification. Cells were mostly tetrasomic (53%) and trisomic (32%) for chromosome 17 with concordant multiplication of HER-2/neu gene.

 
The scoring efficiency was confirmed by performing FISH on breast cancer cell lines with known HER-2/neu amplification status. The breast cancer cell lines MCF-7 (no amplification), HS578T (low amplification), and BT-474 (high amplification) were used as standards. The results are presented in Fig. 1Citation . MCF-7 cells had no HER-2/neu amplification and were polysomic for chromosome 17 as reported previously (20) . In addition, we observed that the reported 2:3 ratio of HER-2:CEP17 in MCF-7 was because of the presence of a third abnormal chromosome 17 and deletion of one HER-2 signal (Fig. 1C)Citation . HS578T displayed a ratio of 2.5 ± 1.21 and was monosomic for chromosome 17 (the percentage of cells with one CEP17 signal was 58 ± 5%). HS578T was classified previously as a low HER-2/neu-amplified cell line (22) . However, further analysis of metaphase preparations revealed that the low-amplified ratio in HS578T cells was because of the formation of one isochromosome 17q, resulting in duplication of the HER-2/neu locus. Concomitant loss of the other chromosome 17 was observed in about 50% of cells analyzed (Fig. 1BCitation , partial metaphase cell). Furthermore, we observed that the absolute number of HER-2/neu copies per cell in HS578T cells was 4 ± 1. As expected, BT-474 cells had 11- to 12-fold amplification ({approx}50–60 signals/cell) and were highly polysomic for chromosome 17 (four to six copies per nucleus). HER-2 signals were grouped in clusters and were always integrated in chromosomes. Amplicons were accompanied by complex translocations of the amplified regions to other chromosomes as described previously (Fig. 1DCitation ; Ref. 20 ). The breast cancer cell line HCC1937, derived from a BRCA1 germ-line mutation carrier, was reported previously as having no HER-2/neu protein overexpression (21) . The results of FISH analysis presented here for the first time show that this cell line is HER-2/neu nonamplified and polysomic for chromosome 17 (Table 1)Citation . Increases in chromosome 17 copy number were accompanied by concordant increases in HER-2/neu copy number (Table 1Citation , Fig. 1ECitation ). We observed three (in 32% of cells) to four (in 53% of cells) fluorescent CEP17 and HER-2 signals per cell (ratio 1.0).


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  		Table 1 Concordance between FISH on nuclei in suspension and FISH on nuclei in paraffin from three cell lines

 
Next, we asked whether the results of FISH, performed on paraffin-embedded cells sectioned and mounted on a slide, were comparable with results from cytology imprint preparations. Because whole nuclei are not included in 4-µm-thick sections, the signal number may be lower in paraffin sections than in complete nuclei. Comparative HER-2:CEP17 FISH on MCF-7, BT-474, and HC1937 cell lines in cell suspension and in paraffin are presented in Table 1Citation and Fig. 1, C–ECitation (insets). A regression model fit to the data for all three cell lines revealed that the cells embedded in paraffin had approximately 15% fewer HER-2 copies per cell (P = 0.002) and 8% fewer CEP17 copies per cell (P = 0.038) than the cells in suspension (Table1)Citation . Thus, the type of cell preparation affects the number of fluorescent signals available for analysis. Nevertheless, results from paraffin studies did meet the criteria for HER-2/neu and chromosome 17 copy number status in all three cell lines described above.

Patient Characteristics.
The 94 breast tumors analyzed in the present study were composed of 53 specimens from BRCA1 germ-line mutation carriers and 41 randomly selected specimens from patients with breast carcinomas. Table 2Citation summarizes the clinical characteristics of these patients. Despite the predominance of infiltrating ductal carcinoma in both groups, medullary carcinoma was overrepresented in the BRCA1 mutation-positive cases, and ductal in situ carcinoma and lobular in situ carcinoma were more likely to be represented in the sporadic cohort (P = 0.019). The average disease stage was more variable among the sporadic tumors (P = 0.006). The overall tumor grade of BRCA1-associated cancers was significantly higher than that of controls (P = 0.038). Finally, estrogen receptor expression was more common among sporadic cases than those with BRCA1 mutation (43% versus 31%), but this difference was not statistically significant (P = 0.390).


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  		Table 2 Clinicopathological features of BRCA1-associated and sporadic breast cancers

 
HER-2/neu Gene Copy Number in Nonmalignant Breast Samples.
To establish control values for tumor cells, we evaluated the adjacent nontumor breast epithelium from 41 BRCA1 mutation carriers and from 24 patients with sporadic tumors (Table 3)Citation . The mean number of HER-2 copies per cell was 1.84 ± 0.10 for the BRCA1 mutation-positive cases and 1.87 ± 0.10 for the sporadic cases, with no case from either group having more than four HER-2 signals per nucleus (29) . Similarly, the mean number of CEP17 copies per cell was 1.87 ± 0.10 for the BRCA1 mutation-positive cases and 1.81 ± 0.10 for the sporadic cases. More CEP17 copies per cell combined with fewer HER-2 copies per cell resulted in a slightly lower mean ratio (HER-2:CEP17) among the BRCA1 mutation-positive cases (0.99 versus 1.03; P = 0.002; Table 3Citation ). The mean proportion of cells having only one copy of CEP17 was 16 ± 10% in BRCA1-associated cases and 21 ± 10% in sporadic cases (P = 0.068). The mean proportion of cells with two CEP17 signals per nucleus was 83% (±9) and 75% (±18) in BRCA1 mutation carriers and sporadic controls, respectively. The mean proportion of cells with more than two CEP17 signals per nucleus was similar in the two groups (1.3 ± 2.2% and 1.1 ± 1.6% respectively).


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  		Table 3 Mean number of HER-2 and CEP17 copies per cell and mean ratio in normal and tumor tissues from BRCA1-associated and sporadic cases

 
HER-2/neu Gene Copy Number in BRCA1-associated and Sporadic Breast Cancers.
Among both BRCA1-associated and sporadic cases, tumor cells had on average more HER-2 copies per cell than did the adjacent normal cells (Table 3)Citation . Fig. 2Citation shows the mean number of HER-2 copies per cell plotted against the mean number of CEP17 copies per cell, depicted separately for tumors from BRCA1-associated (Fig. 2A)Citation and sporadic (Fig. 2B)Citation groups. Ten (19%) of the BRCA1-associated tumors and 9 (22%) of the sporadic tumors had a ratio of 2 or higher, both comparable with the 20–30% of breast tumors that have been reported to be amplified in the literature (7) . However, detailed analysis of the distribution of the mean number of HER-2 copies relative to CEP17 revealed significant differences between amplified BRCA1-associated and sporadic tumors. The mean HER-2:CEP17 ratio across all 10 amplified BRCA1-associated tumors was 2.4 ± 0.4 and was never higher than a ratio of 3.1. The mean absolute HER-2 signal per cell was 4 ± 1. These tumors (19%) were categorized as having low levels of amplification (Fig. 2ACitation and Fig. 3, C and DCitation ). Low HER-2/neu amplification was also observed in the sporadic tumors. Three of nine amplified sporadic tumors (or 3 of 41, 7%) had ratios of 2.0–3.0 (Fig. 2B)Citation with a mean HER-2:CEP17 signal ratio of 2.4 ± 0.2. The mean absolute HER-2 signals per cell was 4 ± 2, which was comparable with the low-amplified tumors from BRCA1 mutation carriers. In contrast, 6 of 9 amplified sporadic samples (or 6 of 41, 15%) had a ratio of >5.0 (high amplification), whereas none of the BRCA1 cases did (Fig. 2)Citation . Across the six highly amplified tumors, the mean HER-2:CEP17 signal ratio was between 5.1 and 16.7 (11.3 ± 4.6); the mean absolute number of HER-2 signals per cell was >10 (21 ± 8). A t test comparing the mean log ratio between BRCA1-associated and sporadic tumors yielded a P of 0.048, providing evidence that the distribution of the ratios differs between the two groups. However, except for the six highly amplified sporadic cases, the distribution of HER-2 and CEP17 in BRCA1-associated tumors was similar to that in sporadic tumors. Thus, the differences observed between the two populations of tumors were because of the presence of six highly amplified sporadic samples, which may be indicative of a distinct molecular pathogenesis for this subgroup of sporadic tumors. In HER-2/neu highly amplified tumor cells, the HER-2 signals were grouped in clusters (Fig. 3E)Citation , similar to those in interphase nuclei of the BT-474 cell line described above (Fig. 1D)Citation , and likely represent intrachromosomal amplification as defined previously (20 , 24) .



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  		Fig. 2. Distribution of the mean number of HER-2/neu copies relative to chromosome 17 centromere signals in BRCA1-associated (A) and sporadic (B) breast cancer tissues. , ratio >= 2, the cutoff point for HER-2/neu amplification. Specimens with a ratio of 2.0–3.0 were classified as having low amplification. Specimens displaying a ratio > 5.0 (····) were classified as highly amplified.

 


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  		Fig. 3. Representative photomicrographs of breast tumor tissue sections from BRCA1 germ-line mutation carriers (A-D) and a sporadic cancer patient (E) after FISH. The HER-2/neu gene is identified by red fluorescent signals, and the chromosome 17 centromere (CEP17) is identified by green fluorescent signals. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue). A, typical example of nonamplified BRCA1-associated cancer (ratio = 1.1), in which 83% of the nuclei displayed monosomy for chromosome 17 with a corresponding loss of one copy of the HER-2/neu gene. B, tumor showing 74% of cells with three to eight HER-2/neu signals per nucleus because of an increase in chromosome 17 copy number (ratio = 1.4). The tumor was classified as nonamplified polysomic. C and D, highly heterogeneous tumors with low levels of HER-2/neu amplification (ratio = 2.0–3.0). C, the mean copy number of HER-2/neu per cell was 3.1 (ratio = 2.9) and 91% of the nuclei displayed one CEP17 copy signal per cell. D, low amplification and polysomy for chromosome 17 in the same tumor cells as in C. HER-2:CEP17 ratio was 2.1, and 48% of the cells revealed three centromeric signals and three to eight HER-2 signals. E, a sporadic tumor with high levels of amplification (ratio = 16.7). HER-2/neu displayed a significantly increased gene copy number (20–40/nucleus), grouped in clusters presumed to be intrachromosomal amplicons; 62% of the cells revealed one CEP17 signal per nucleus. By IHC the tumors in A and B showed equivocal or weak staining (1+); the tumors in C and D showed no staining (0), whereas the tumor in E was positive for HER-2/neu protein overexpression (+3).

 
Chromosome 17 Copy Number Alterations and Relationship with HER-2/neu Amplification.
To differentiate the increases in HER-2 signals caused by increases in chromosome 17 copy number from true gene amplification, the assessment of CEP17 signals was also used to determine aneusomy for chromosome 17 in tumor cells.

Computation of the mean number of CEP17 signals per cell revealed that the majority of tumors from both BRCA1-associated (32 of 53, 61%) and sporadic (21 of 41, 51%) groups were definitely monosomic for chromosome 17. (A representative photomicrograph of a monosomic tumor is shown in Fig. 3ACitation .) The proportion of polysomic BRCA1-associated tumors was comparable with that of sporadic tumors and amounted to 19% (10 of 53) and 17% (7 of 41), respectively (Fig. 2)Citation . (Representative photomicrographs of BRCA1-associated tumors with three, four, and more than five copies of CEP17 are depicted in Fig. 3, B and DCitation .) The mean number of CEP17 copies per cell was similar across all BRCA1-associated and all sporadic tumors (1.75 versus 1.71, Table 3Citation ). Computation of the percentage of nondisomic nuclei revealed significant intratumor heterogeneity in each group. Nine (18%) BRCA1-associated tumors and seven (17%) sporadic tumors fall into the category of monosomic/polysomic group because of heterogeneous subpopulations of cells present in the same tumor.

To verify that the reduction in chromosome 17 copy number observed in the majority of tumors was a real genetic feature of the malignant cells, we performed dual-color FISH with a centromere 17 probe (CEP17) and a centromere 9 probe (CEP9) on a subset of six tumors monosomic for chromosome 17 (two BRCA1-associated and four sporadic). Across all six tumor samples the mean number of CEP17 copies per cell was 1.2, as expected, and was lower than the mean number of CEP9 copies per cell of 2.1 (P = 0.001). Thus, the significantly high proportion of tumors with monosomy for chromosome 17 found in our study likely represents a feature of the malignant process in breast cancer, rather than an artifact of tissue preparation.

The six highly HER-2/neu-amplified tumors found in the present study were distributed evenly over the full range of the number of CEP17 copies, implying no correlation between high oncogene amplification and chromosome 17 aneusomy. In contrast, all but one (Fig. 2Citation and Fig. 3DCitation ) low-amplified tumors were monosomic, suggesting that the mechanism(s) for low HER-2/neu amplification may involve reduction of chromosome 17 copy number, accompanied by gene translocation or isochromosome formation events.

HER-2/neu Protein Expression by IHC and Correlation with FISH.
Because of limited patient material, only 39 BRCA1-associated and 21 sporadic breast cancers were evaluated for HER-2/neu protein overexpression. HER-2/neu positivity was significantly less frequent (5 of 39, 13%) in BRCA1 mutation carriers compared with sporadic cancers (33%, 7 of 21, P = 0.0421; Fig. 4, A and BCitation ). In addition, none of the tumors in BRCA1 mutation carriers showed strong (3+) membranous expression of HER-2/neu. We observed no increases in protein expression in the cases with low amplification (ratios of 2.0–3.1) or in the cases where increased number of HER-2 signals was because of gains of chromosome 17 copy number. However, seven tumors with HER-2:CEP17 ratios of 1.0–1.8 were scored as showing 2+ and 3+ protein expression (Fig. 4, A and BCitation ). Except for these cases, there was a strong positive correlation between staining intensity and HER-2:CEP17 ratio across all tumors (Fig. 4B)Citation .



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  		Fig. 4. Relationship between HER-2/neu amplification (HER-2:CEP17 FISH ratio) and HER-2/neu protein expression (IHC staining intensity) in BRCA1-associated (A) and sporadic (B) breast tissues. Observations labeled Not analyzed on the X axis are those for which the protein expression analysis was not performed. ····, ratios >= 2 and >= 5, the cutoff points for low and high amplification, respectively. , the mean HER-2:CEP17 ratio in a group of tumors with a given grade of staining intensity.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results from the present study indicate that high levels of HER-2/neu oncogene amplification do not occur or are rare in breast tumors from BRCA1 germ-line mutation carriers. In contrast, HER-2/neu was highly amplified in 15% of sporadic breast tumors and was consistent with the findings of other investigators (17 , 30) . Similarly to previous reports (20 , 24 , 30) , we also found discordance between IHC and FISH (seven tumors) in which HER-2 staining was scored as 2+ or 3+ positive by IHC, but the tumors demonstrated no gene amplification by FISH. Lastly, we observed significant differences in the pattern of chromosomal alterations observed in low-amplified tumors compared with tumors with high amplification, suggesting a distinct molecular pathogenesis for highly amplified tumors. To our knowledge, this is the first study in which the molecular-cytogenetic analysis of HER-2/neu gene in BRCA1-associated breast cancers was performed.

To date, five studies have reported HER2/neu status using IHC in BRCA1-associated tumors (Table 4)Citation . Among them, the study by Johannsson et al. (11) appeared to be the most representative, and the authors concluded that tumors from BRCA1 mutation carriers are generally HER-2/neu expression-negative. However, Johannsson et al. (11) and two other groups (13 , 14) reported rather high proportions of HER-2/neu-positive tumors (42–90%) among the sporadic breast cancer controls compared with the 20–30% found in other studies (7 , 17) . The remaining two studies were inconclusive (Table 4)Citation . In the present study, using the largest sample of BRCA1-associated tumors evaluated to date, we found significant differences between BRCA1 mutation carriers versus sporadic groups with regard to both HER-2/neu amplification and HER-2 protein expression. Across all tumors, we did not observe any relationship between IHC and FISH when the HER-2:CEP17 ratio was more then 2.0 but less than 5.0. However, both staining intensity and percentage of cells stained positive were notably good predictors of having a ratio of >5.0. Our findings are in agreement with published reports, which suggest that HER-2/neu low-amplified breast tissues are usually HER-2/neu-negative by IHC (17 , 31 , 32) .


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  		Table 4 HER-2/neu IHC studies in BRCA1-associated tumors reported in the literature and in the current study

 
In the present study, we observed low levels of HER-2/neu amplification in 19% of BRCA1-associated and 7% of sporadic tumors. We also observed that whereas tumors with high levels of amplification were usually disomic and polysomic for chromosome 17, tumors with low levels of amplification displayed reduction of chromosome 17 to one copy. Therefore, low amplification may be the result of loss of one homologous chromosome with concomitant oncogene duplication or an unbalanced translocation to nonhomologous chromosomes (33) , or the result of isochromosome formation, as we observed in the HS578 cell line (Fig. 1D)Citation . Thus, it is likely that tumors with low levels of amplification do not have true oncogene amplification and would not be expected to respond to Herceptin therapy. We also suspect that the five BRCA1-associated tumors and two sporadic tumors which demonstrated increased levels of protein expression without gene amplification likely represent false positives and would also not be expected to respond to Herceptin. Recent results of clinical trials using Herceptin as a single agent or in combination with chemotherapy support this premise because only the small fraction of patients whose tumors demonstrated high levels of HER-2/neu amplification/overexpression appear to benefit from the drug (10 , 34) .

There are several possible mechanisms to explain why HER-2/neu is never highly amplified in the background of a BRCA1 germ-line mutation. First, BRCA1 and HER-2/neu are located in close proximity on chromosome 17 (17q11–21), and the absence of amplification may be because of a simple physical codeletion of one allele of HER-2/neu and nearby sequences during loss of heterozygosity (LOH) at the BRCA1 locus (11) . A number of studies of genes that become amplified while developing resistance to inductive circumstances, such as dihydrofolate reductase, have demonstrated that the presence of both alleles of the gene is essential for gene amplification (35, 36, 37) . These studies also demonstrated that gene amplification occurs only in one of the two chromosome homologues, whereas the other homologue retains a normal single copy of the gene. It appears that sequences flanking the gene to be amplified are crucial for amplicon formation, and the disruption of those sequences by LOH may abrogate the process of amplification (37) . Analysis of HCC1937 cells suggests that the inactivation of the BRCA1 tumor suppressor gene because of mutation and/or LOH may precede and hence restrain HER-2/neu oncogene amplification (21 , 38 , 39) .

Second, changes in chromatin conformation can also affect processes required for amplicon formation by causing inefficient double-strand break repair, alternative replication, alternative homologous recombination, inefficient sister chromatid exchange, and, consequently, abrogation of amplification (5) . It was shown previously that the deletion of DNA sequences might result in changes in chromatin structure (40) . Thus, mutation and/or LOH at the BRCA1 locus may suppress HER-2/neu amplification through an indirect structural mechanism, such as abnormal chromatin conformation on 17q (41) .

Lastly, it is conceivable that BRCA1 may functionally suppress amplification. BRCA1 contains several functional domains that interact directly or indirectly with a variety of molecules (42) , and is likely to serve as an important central component in multiple biological pathways (4) , including processes involved in gene amplification. Some gene amplification processes require functional nonhomologous end-joining (5) . Loss of this repair pathway in BRCA1 mutant tumors may limit gene amplification, as we have observed in this study. BRCA1-deficient cells are also incapable of repairing oxidative DNA breaks (43) . It is possible that in such cells, the oxidative stress-derived amplification structures that are formed initially (44) are not sustained, and amplification is abrogated. Further dissection of these various pathways may allow elucidation of the conditions required for HER2/neu amplification in human breast cancer cells.

Cancers arising in BRCA1 mutation carriers are commonly high grade, aneuploid, highly proliferative, and ER-negative. These same features also characterize HER-2/neu-amplified tumors. In fact, the six HER-2/neu highly amplified sporadic tumors in our study were clinically more similar to the BRCA1-associated cancers than to the other sporadic tumors (data not shown). Perou et al. (45) have recently suggested that estrogen receptor-negative breast cancers encompass at least two biologically distinct subtypes of tumors, basal-like and HER-2/neu-amplified. In a study comparing BRCA1- and BRCA2-associated tumors to sporadic tumors, Hedenfalk et al. (46) reported unique gene expression profiles for BRCA1-associated tumors. Their data also demonstrated that in addition to several genes that are distinctly expressed or down-regulated, HER-2/neu overexpression was not observed in BRCA1-associated tumors. Therefore, it is reasonable to conclude that HER-2/neu amplification/overexpression is not a feature of BRCA1-associated tumors, probably because the BRCA1 locus on chromosome 17 could potentially play a role in controlling amplification at the HER-2/neu locus. Thus, a better understanding of the molecular mechanisms underlying the functions of BRCA1 and HER-2/neu will lead to the rational design of effective therapeutic targets against the biologically aggressive, estrogen receptor-negative breast cancers, which disproportionately affect young women.


   ACKNOWLEDGMENTS
 
We thank Rafael Espinosa III for help in image preparations and Lise Sveen for culturing breast cancer cell lines. We are grateful to Dr. Michelle LeBeau and Dr. Katrin Carlson for useful comments and fruitful discussions.


   FOOTNOTES
 
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.

1 B. L. W. is supported by grants from the NIH (CA57601) and the Breast Cancer Research Foundation. B. L. W. is an investigator in the Abramson Family Cancer Research Institute at the University of Pennsylvania. O. I. O. is supported by an Academic Award from the United States Army Department of Defense Grant DAMD17–99-1–9123, the Falk Medical Research Trust, and Grant CA 14955 to the University of Chicago Cancer Research Center. O. I. O. is a Doris Duke Distinguished Clinical Scientist. Back

2 To whom requests for reprints should be addressed, at: Section of Hematology/Oncology, Department of Medicine, Committees on Genetics and Cancer Biology, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637-1463. Phone: (773) 702-1632, Fax: (773) 702-0963, E-mail: folopade{at}medicine.bsd.uchicago.edu Back

3 The abbreviations used are: FISH, fluorescence in situ hybridization; IHC, immunohistochemical analysis. Back

Received 7/18/01. Accepted 1/ 3/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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