This study evaluated the relative frequencies of HER-2/neu gene amplificationin ductal carcinoma in situ (DCIS)-associated invasive breast cancer and DCIS alone. We examined archival tissue samples of 100 DCIS lesions with an invasive component (cases) and 100 without an invasive component (controls), with cases and controls matched by pathologic nuclear grade. HER-2/neu gene amplification was determined by fluorescence in situ hybridization. We compared HER-2/neu gene amplification in DCIS lesions only, irrespective of the presence or absence of an invasive component. HER-2/neu gene amplification occurred significantly less frequently in the cases (26%) than in the controls (40%), with an odds ratio of 0.35 (95% confidence interval, 0.17–0.72; P < 0.004) after adjustment for pathologic and quantitative-image-analyzed morphometric nuclear grade. The HER-2/neu gene also was amplified more frequently in higher- than in lower-grade DCIS alone (56% versus 19%, respectively; P < 0.0001) or in higher- than in lower-grade DCIS with invasive cancer (44% versus 2%, respectively; P < 0.00001). Future studies should examine the potential roles of HER-2/neu and other biomarkers (e.g., p21 and Rb) as markers of the risk of DCIS patients for invasive breast cancer and as molecular targets of chemoprevention in breast intraepithelial neoplasia.

DCIS3 accounts for 12–14% of newly diagnosed breast cancer cases in the United States, and ∼50% of untreated DCIS developed an ipsilateral invasive breast cancer within 24 years after the original biopsy (1, 2, 3, 4). The biological mechanisms of breast carcinogenesis are not fully understood. The growth factor receptor HER-2/neu generally is not overexpressed in normal or benign breast lesions (5, 6, 7) and is overexpressed in DCIS lesions. However, Allred et al.(7) and others have reported significantly lower HER-2/neu expression in invasive carcinoma than in DCIS (5, 6, 8). Despite findings that HER-2/neu overexpression in invasive breast cancer is associated with poor prognosis (9, 10), few studies have compared HER-2/neu gene amplification in DCIS alone versus in DCIS associated with invasive cancer. In this case-control study, we evaluated the relative frequencies of HER-2/neu gene amplification in DCIS-associated invasive breast cancer and DCIS alone. The evaluation of HER-2/neu gene amplification in DCIS may help identify the highest-risk women and provide a potential molecular target for chemoprevention in this setting.

Identification of Patient Material.

Paraffin-embedded archival breast tissue specimens and surgical pathology reports for 100 consecutively examined controls (DCIS alone) and 100 consecutively examined cases (DCIS with an invasive component) were identified from the pathology database of The University of Texas M. D. Anderson Cancer Center during the period 1986–1999. H&E-stained slides of each DCIS case and control were reviewed and graded by one pathologist (A. A. S.) using standard grading criteria, as described previously (11). We defined necrosis as the presence of karyorrhectic, pyknotic nuclei associated with eosinophilic debris. Only inspissated secretory material without nuclear debris was not considered to be necrosis. Necrotic material occupying less than or more than one-third of the ductal lumen was classified as focal or extensive necrosis, respectively. Cases and controls were frequency-matched according to nuclear grade. Sociodemographic characteristics and clinical variables were abstracted from patient medical records.

Analysis of HER-2/neu Amplification.

The analysis of HER-2/neu gene amplification was performed by the FISH method using the Vysis Path Vysion HER-2/DNA probe kit (Downers Grove, IL). Before using HER-2/DNA probe, one pathologist (N. S.) examined the H&E-stained specimen to locate the relevant DCIS region of each of the cases and controls, and then marked the DCIS region of interest for gene amplification. Briefly, tissue sections were baked overnight at 56°C. Slides were immersed in xylene to remove paraffin, and tissue sections were dehydrated in 100% alcohol for 5 min and air-dried. Next, slides were immersed in 0.2 n HCl for 20 min to solubilize nuclear proteins. The slides were then immersed in Vysis pretreatment solution for 30 min at 80°C followed by protein digestion, in which tissues were treated with pepsin at 37°C for 10 min. After 10 μl of probe was applied to the target area of the slide, a glass coverslip was placed over the probe and sealed with rubber cement. Denaturation of slides was performed on a hot plate at 73°C for 5 min. Slides were then allowed to hybridize overnight at 37°C in a humidified chamber. After the coverslips were removed, the slides were washed in 2× SSC/0.3% NP40 at 73°C to remove excess probes, then counterstained in 4′,6-diamidino-2-phenylindole.

The Path Vysion kit uses two directly labeled fluorescent DNA probes: locus specific identifier (LSI) HER-2/neu, which is specific for the HER-2/neu gene locus, and chromosome enumerator probe (CEP) 17, which is specific for the α satellite DNA sequence at the centromeric region of chromosome 17. The expected ratio of LSI-2/neu to CEP 17 is 2.0 for normal breast tissue. A ratio >2.0 was considered indicative of HER-2/neu amplification. Signals were counted for 60 tumor nuclei. Both positive and negative controls were included. We compared HER-2/neu amplification in DCIS lesions only, irrespective of presence or absence of invasive component.

Quantitative Morphometric Nuclear Grading (Z Score).

The nuclear images were captured from Feulgen-stained tissue sections and z scores were computed according to Bacus et al.(12). Z score is a common scale used for objective quantitative morphometric nuclear grading system. Z scores of normal epithelia are always distributed about a mean value of zero. The individual feature z scores are weighted by a coefficient and summed to compute the quantitative morphometric nuclear grade. Four nuclear features and their weighting coefficients used include nuclear area, DNA, entropy, and valley.

Statistical Analysis.

HER-2/neu gene amplification status was categorized as amplified or not amplified. Odds ratios and 95% confidence intervals were computed using the logistic regression model. The χ2 test was used to compare proportions of HER-2/neu gene amplification status. The Mann-Whitney U test was used to compare z score values between the cases and controls. All of the statistical computations were performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) and STATA computer software programs (STATA Corporation, College Station, TX). A P < 0.05 was considered statistically significant.

The characteristics of the women patients who provided specimens for this study are shown in Table 1. The majority of patients in the case and control groups were white. The cases and controls were similar with respect to median age at diagnosis, menarche, and at first childbirth, and with respect to the numbers of grades 1, 2, and 3 DCIS lesions. We observed no significant differences in focal and extensive necrosis between cases and controls, although controls had more (both focal and extensive) necrotic lesions than did cases. About half of the case DCIS lesions did not have necrosis. The median quantitative morphometric nuclear grades (z scores) were 8.42 and 7.67 for cases and controls, respectively.

HER-2/neu gene amplification was less common in DCIS associated with invasive breast cancer (cases) than DCIS alone (controls). The odds ratio for HER-2/neu gene amplification was 0.35 (95% confidence interval, 0.17–0.72) after adjustment for pathologic and morphometric nuclear grade (Table 2). The odds ratio for pathologic nuclear grade (grades 1 and 2 versus grade 3) was 1.09 (95% confidence interval, 0.51–2.32) and for quantitative morphometric nuclear grade (z score ≤ 2.67 versus z score > 2.67) was 1.81 (95% confidence interval, 0.85–3.83). Furthermore, the odds ratio for HER-2/neu (0.35) did not change substantially after adjusted for necrosis (odd ratio 0.40; 95% confidence interval, 019–0.84). HER-2/neu gene amplification correlated with nuclear grade among both cases and controls: HER-2/neu gene amplification occurred in 44% of grade 3 versus 2% of grade 1 and 2 DCIS with invasive cancer (n = 97; P < 0.00001), and in 56% of grade 3 versus 19% of grade 1 and 2 DCIS alone (n = 98; P < 0.0001). As indicated by these data, HER-2/neu gene amplification was more pronounced in high-grade DCIS alone than in high-grade DCIS with invasive cancer. Examples of HER-2/neu gene amplification are shown in Fig. 1.

We found that HER-2/neu gene amplification was inversely associated with the risk of invasive progression in DCIS patients, even though it correlated with high-grade lesions in both cases (DCIS-associated invasive cancer) and controls (DCIS alone). The inverse risk association was independent of pathologic and quantitative image-analyzed morphometric (z score) nuclear grade.

Several studies (5, 6, 7) have found that normal breast tissue or benign lesions (e.g., hyperplasia with or without atypia) generally do not overexpress HER-2/neu protein but that HER-2/neu overexpression is more common in DCIS than in invasive cancer (5, 6, 7, 8, 13, 14, 15). Most of these studies are case-series in which the HER-2/neu protein was assayed immunohistochemically. Furthermore, none of the studies used a case-control design to compare HER-2/neu protein expression in DCIS alone with this expression in DCIS associated with invasive cancer. We used the FISH technique instead of immunohistochemical assay to evaluate HER-2/neu gene amplification and a case-control study design for our comparative analysis of HER-2/neu gene amplification. Although our results of HER-2/neu gene amplification in DCIS are consistent with earlier reports of HER-2/neu protein overexpression (5, 6, 7, 8, 13, 14, 15), a recent report indicated that ∼20% of breast tumors exhibited overexpression of c-erbB-2 in the absence of c-erbB-2 gene amplification (16). The biological basis of the higher frequency of HER-2/neu gene amplification in DCIS alone than in DCIS-associated invasive cancer remains unexplained.

There is the suggestion that increased cell proliferation and enhanced cell motility associated with overexpressed HER-2/neu may contribute to the development and progression of DCIS (17). Another speculation is that the expression of HER-2/neu may be down-regulated in a significant proportion of DCIS lesions as they progress to invasive cancer or that a subset of invasive cancer may develop de novo by mechanisms independent of HER-2/neu(7, 18, 19). Previous reports (20, 21, 22, 23) suggested that malignant tissues overexpress HER-2/neu while having diploid copies of the gene, indicating that mechanisms other than gene amplification contributed to the overexpression. Therefore, it is possible that HER-2/neu stimulates growth during the in situ phase only, and promotes differentiation and growth inhibition as the lesion progresses to invasive cancer. Although this seems counterintuitive, experimental overexpression of epidermal growth factor receptors may induce growth inhibition (24, 25, 26, 27) independently of differentiation. This growth inhibition by epidermal growth factor receptors is known to result from induction of the cyclin-dependent kinase inhibitor p21 (28). Furthermore, the transfection and overexpression of HER-2/neu in MCF7 human breast cancer cells results in reduced proliferation, induction of p21, Rb hypophosphorylation, and differentiation (29). These regulatory proteins (e.g., p21 and Rb) have not been fully evaluated with respect to their mechanistic relationship with HER-2/neu in DCIS patients.

Our continuing studies may help to additionally define the role of HER-2/neu in the biology of DCIS, including its associations with p21, Rb, and other biomarkers (30). The potential roles of HER-2/neu and other biomarkers, including p21 and Rb, as markers of the risk of DCIS patients for invasive breast cancer and as molecular targets of chemoprevention in breast intraepithelial neoplasia also warrant future studies (31).

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

This work was supported by a contract from the National Cancer Institute N01-CN-65004.

3

The abbreviations used are: DCIS, ductal carcinoma in situ; FISH, fluorescence in situ hybridization; Rb, retinoblastoma.

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