Limited Expression of C-erbB-2 in Node Negative Breast Cancer Patients.

Manubai S. Patel [1]

Steven A. Bigler [2]

Charles F. Streckfus [3]

Stephanie F. Elkins [1]

J. Tate Thigpen [1]

Ralph B. Vance [1]

Lenora G. Bigler [1,4]

Departments of Medicine [1], Pathology [2], and Dental Research [3], University of Mississippi Medical Center, Jackson, MS 39216-4505

Forty-three cases of node-negative breast cancer were treated at the University of Mississippi Medical Center from 1986-1992. Patient profiles were as follows: average age was 53 years; 86% of the cases were diagnosed as infiltrating ductal carcinoma; 64% of the population were African American and 36% were Caucasian. The average tumor size was 2.92 cm ([+or-]2.22 cm). In nine cases the tissue was unacceptable for immunohistochemical staining, therefore, only 34 tumors were examined for c-erbB-2 staining. We found that 19 (56%) of the tumors examined exhibited some degree of c-erbB-2 expression (5 were strongly positive, 14 were weakly positive), while 15 (44%) of the tumors examined were negative for c-erbB-2 expression. Of the patients that were erb positive, only 4 had recurrence of disease after a 5-year follow-up and 4 were deceased from disease progression. Therefore, there appears to be no statistical evidence that c-erbB-2 is a marker for survival in node-negative breast cancer patients in the popula tion examined in this study. Key Words: c-erbB-2, inimunohistochemistry, node-negative breast cancer

Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of death in women in the United States today. The American Cancer Society estimates that in 2000, approximately 185,000 new cases of female breast cancer will be diagnosed in the United States, and 43,500 women will die of the disease. With the advent of screening mammography, more breast cancer is being detected at earlier stages, it is usually confined to the breast and has no nodal involvement. However, 30% of these cases will recur due to micrometastases present at the time of diagnosis (Allred et al., 1992). Chemotherapy has been shown to improve disease-free survival in axillary nodenegative patients, but overall survival has not been affected by treatment (Allred et al., 1992; Hayes, 1996). Therefore, the choice of adjuvant therapy for node negative patients is difficult and often times controversial.

Some of the key decisions in the current management of primary breast cancer involve the need for prognostication. Currently, the single most important prognostic factor is the number of positive axillary lymph nodes. Another valuable pathologic factor is tumor size, which has independent prognostic significance and is of particular importance in the prognosis for node-negative-patients (Allred et al., 1998). Other significant pathologic features include poor cellular differentiation, histological and nuclear grade, and the presence of lymphatic and/or vascular invasion (Allred et al., 1998). These factors are consistently associated with worse patient outcome although there appears to be considerable interobserver variation in assessing these features.

Of the biochemical measurements currently available, the most important is the presence or absence of estrogen and progesterone receptors in the tumor (Alfred et al., 1998; Richner et al., 1990). However, much attention has recently been focused on the amplification or overexpression of the c-erbB2 oncogene and its gene products. The gene for cerbB-2 encodes a 185kD glycoprotein which is structurally homologous to the epidermal growth factor receptor (Maguire and Greene, 1989). Antibodies have been developed to peptides in the region of the C-terminus of the c-erbB-2 protein which will generally react with paraffin-embedded tissue following deparaffinization and permeabilization of the membrane with methanol. Therefore, retrospective examination of archival tissues is possible. A number of studies have indicated that c-erbB-2 is amplified and overexpressed in approximately 30% of primary adenocarcinomas of the breast, however, data evaluating the prognostic significance of c-erbB-2 in node-negative patients have yielded conflicting results (Maguire and Greene, 1989; Torre et al., 1997; Hartmann et al., 1994). This study was designed to evaluate the prognostic significance of c-erbB-2 expression in node-negative breast cancer patients treated at the University of Mississippi Medical Center from 1986 to 1992 whereby a reasonable follow-up period would have passed for patient evaluation.

MATERIALS AND METHODS

Patient Population--A total of 43 patients with node-negative breast cancer were treated at our institution between 1986 and 1992. Patients were excluded from the study if the paraffin blocks were not available, they had ductal carcinoma in situ, lobular carcinoma in situ or Paget's disease. This left a total of 34 patients with tissue acceptable for examination in this study.

Primary treatment of the patients consisted of modified radical mastectomy or lumpectomy, followed by radiation treatment. Complete axillary lymph node dissection was performed on all patients. Clinical information and patient characteristics are presented in Table 1. Histological grade and c-erbB-2 staining was independently reviewed and evaluated by a certified pathologist following the criteria of Bloom and Richardson (1957).

Immunohistochemistry--Immunohistochemical staining for c-erbB-2 was performed using a standard avidin-biotin-peroxidase technique (Vector Labs, Burlington, CA). Briefly, 5 [micro]m paraffin sections were placed on charged microscope slides, baked in a convection oven at 57[degrees]C, deparaffinized in xylene for 10 minutes and rehydrated through graded alcohol. Prior to immunostaining, the sample slides were processed as directed by the manufacturer to recover antigenicity using the antigen retrieval CITRA system (Biogenex, San Ramon, CA). Following antigen retrieval, the sections were washed in phosphate-buffered saline and incubated with normal serum for 10 minutes. Samples were then incubated with a 1:40 dilution of c-erbB-2 antibody (clone NCL-CB11, Novocastra Laboratories, UK) for 1 hour at room temperature. After washing with Tris-buffered saline, the slides were incubated with secondary biotinylated antibody for 10 minutes, washed, and incubated with the avidin-biotin complex for 5 minutes. Following a series of washes, the peroxidase reaction was completed using diaminobenzidine with a nickel solution being added to achieve a black color. Sections were then dehydrated through graded alcohol and mounted. Only membrane staining was considered and scored as strongly positive, weakly positive or negative.

Statistical Methods--The statistical analyses were performed using the SPSS statistical software package and conducted in two phases. The first phase was descriptive which included means and standard deviations where applicable. Bivariant frequency distributions were performed on all dichotomous variables that were associated with the outcome variable of current status (living, deceased) and recurrence (tumor recurred, tumor did not recur). Chi squares were performed to determine if associations existed between the predictor and outcome variables. The alpha level was p=[less than]0.05. The second phase employed multiple Pearson product-moment correlation coefficients among variables with interval data.

RESULTS

A total of 43 patients with node-negative breast cancer were treated at the University of Mississippi Medical Center from 1986-1992. Of these, 34 had tissue suitable for immunostaining. The clinical history and general statistics regarding these patients are presented in Table 1.

The estrogen receptor (ER) status was positive in 71.9% of the patients and 28.1% were ER negative. Thirty-eight point seven percent of the patients were progesterone receptor negative, with 61.3% being positive. Twenty-seven patients had received adjuvant treatment and 7 had no adjuvant therapy. Of the 27 receiving chemotherapy, 12 received a combination of cyclophosphamide, methotrexate and 5fluorouracil (CMF), 12 received tamoxifen and 3 received a combination of CMF and tamoxifen. With a minimum of 5 years follow-up, 6 patients had recurrence of their disease and 6 had died from disease progression.

Figures 1 and 2 demonstrate the representative staining patterns of c-erbB-2 in two of the patients in this study. Of the 34 patients tested for over-expression of c-erbB-2, fifteen were negative, 5 were strongly positive and 14 were weakly positive. It was interesting to note that of the strongly positive group, 4 of the 5 were African-American. The Caucasian patients equally expressed some levels of c-erbB-2 or were negative (6-6); while only 9 African-American patients were totally negative. Overall, there was no statistically significant association between the expression of c-erbB-2 and recurrence of disease or overall survival. Furthermore, there was no significant association between expression of c-erbB-2 and ERIPR status, size of the tumor or nuclear grade. However, more recurrent tumors did express c-erbB-2 than non-recurrent tumors (4 vs 2).

DISCUSSION

c-erbB-2 is an oncogene that encodes a transmembrane glycoprotein receptor that, when overexpressed, can predict for a poor prognosis in a number of cancers (Maguire and Greene, 1989). Recent studies have demonstrated that c-erbB-2 expression plays a role in the prediction of responsiveness to adjuvant therapy with those having overexpression being less responsive than those with normal expression (Carlomagno et al., 1996; Ravdin and Chamness, 1995). Patients with ovarian cancer and node-positive breast cancer have shown a correlation between overexpression of c-erbB-2 and prognosis (Meden and Kuhn, 1997), but the studies which have examined the importance that c-erbB-2 may play in node-negative patients have proven to be ambiguous. Some studies have shown no correlation with the expression of c-erbB-2 and outcome while others claim to demonstrate an association with prognosis and c-erbB-2 expression (Allred et al., 1992; Richner et al., 1990; Hartmann et al., 1994). One retrospective analysis of c-erbB-2 exp ression in node-negative patients reported that estrogen receptor positive, c-erbB-2 negative patients had the longest overall and disease-free survival rates (Richner et al., 1990). In contrast, low risk patients (i.e., ER positive and tumor size [less than] 3cm) that had tumors that overexpressed c-erbB-2 had worse overall and disease-free survival when compared to c-erbB-2 negative patients. Thus, there may be a subset of node-negative patients that have otherwise positive prognostic factors (ER status, size and grade) who may benefit from adjunct chemotherapy if they overexpress c-erbB-2 (Allred et al., 1992).

In our study there was no statistical significance associated with c-erbB-2 expression and overall survival, however the number of patients examined was small and the overall survival was so great to begin with that it was difficult to determine significance. Therefore, until a larger population of node-negative patients is analyzed, the test for c-erbB-2 should still be utilized to aid in the prognosis of node-negative cancer patients.

ACKNOWLEDGMENTS

The authors are grateful to the staff in the Department of Pathology Immunology laboratory for assistance with the immunohistochemistry analyses and the staff of the Cancer Registry for patient history information.

(4.) Author for correspondence. Division of Medicine/Oncology, telephone: [phone]

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