Original Articles
Loss of Nuclear BRCA1 Expression in Breast Cancers Is Associated with a Highly Proliferative Tumor Phenotype

https://doi.org/10.1016/S0165-4608(97)00267-7Get rights and content

Abstract

Recent studies have suggested that BRCA1-associated hereditary breast cancer may be a more aggressive form of disease than sporadic breast cancer. BRCA1-associated breast cancer has been reported to be significantly associated with grade 3 disease. Because grade 3 disease indicates a poor prognosis, this implies that women with a germ-line mutation in BRCA1 who develop breast cancer may have a poorer prognosis than those with sporadic disease. However, little is known about the association of BRCA1 expression with biological markers of prognosis. The present study examined the expression of BRCA1 in a total of 40 archival breast tumor specimens from three patient cohorts (sporadic, familial, and early onset breast cancer) to determine localization of the protein. Furthermore, BRCA1 staining was compared with expression of markers of tumor biology. We found that BRCA1 is generally located in the nucleus and the cytoplasm of normal and malignant breast tissue. Nuclear staining for BRCA1 was observed in most sporadic tumors, but nuclear BRCA1 was reduced or absent in the majority of familial and early onset breast tumors. Although no correlation was found between nuclear BRCA1 expression and estrogen and progesterone status, a significant inverse correlation was found between nuclear BRCA1 and expression of the proliferation marker Ki-67 (P = 0.01). Our findings suggest that tumors associated with a germ-line mutation in one of the breast cancer genes may be highly proliferative and support the view that loss of BRCA1 expression in breast tumors may lead to a more aggressive tumor phenotype.

Introduction

The breast cancer susceptibility genes, BRCA1 and BRCA2, were cloned in 1994 and 1995, respectively 1, 2. Germ-line mutations in these highly penetrant cancer predisposition genes may account for 5–10% of all breast cancer 3, 4. An estimated 1 in 200 to 1 in 800 women may carry mutations in BRCA1, conferring a significantly increased risk of breast and breast–ovarian cancer 3, 5. The frequency may be even greater in Ashkenazi Jews, with approximately 1 in 100 estimated to carry a specific germ-line mutation in BRCA1 [5].

Studies have suggested that BRCA1-associated hereditary breast cancer may be a more aggressive form of disease than sporadic breast cancer [6], with high rates of nuclear polymorphism, mitotic activity, and tubular differentiation suggesting that BRCA1-associated tumors may be highly proliferative [7]. In addition, BRCA1-related breast cancer is more frequently aneuploid and has higher tumor cell proliferation rates [8]. BRCA1 mRNA levels are reduced in the progression of in situ carcinoma to invasive breast cancer [9]and the retroviral transfer of wild-type BRCA1 inhibits growth in vitro of breast and ovarian cancer cell lines [10]. Furthermore, intraperitoneal treatment with BRCA1 as a retroviral construct inhibits the growth of MCF-7 breast cancer cell tumors in nude mice [10]. All of this evidence supports the role of BRCA1 as a tumor suppressor gene, with the presence of BRCA1 causing growth inhibition and loss of BRCA1 associated with cellular proliferation. Until now, there has not been a study of the expression of BRCA1 protein in relation to markers of tumor biology and cellular proliferation in breast cancers.

The present study examined the expression of BRCA1 in a total of 40 archival breast tumor specimens from three patient cohorts (sporadic, familial, and early onset breast cancer) to determine localization of the protein. To examine the relation, if any, between BRCA1 expression and markers of tumor biology, BRCA1 staining was compared with estrogen and progesterone receptor (ER and PR) status, which potentially indicates good prognosis, and the proliferation marker Ki-67 indicating poor prognosis.

Section snippets

Patients and Tumor Specimens

Archival tumors were examined from three different patient cohorts as follows:

  • 1.

    Sporadic breast cancer (n = 28): Patients with breast cancer diagnosed at ⩾60 years (unlikely to be linked to BRCA1/BRCA2).

  • 2.

    Familial breast cancer (n = 7): Patients with breast cancer and a strong family history indicative of a dominantly inherited cancer predisposition (including patients with known BRCA1 mutations). Two separate tumors from the same breast were examined from one patient. Both of these tumors were

Expression of BCRA1 in Normal Breast Tissue

In normal breast tissue, the D-20 BRCA1 antibody revealed both cytoplasmic and nuclear staining (Fig. 1) that was restricted largely to epithelial cells. Staining was noted in all glandular structures within a section, and the intensity of cytoplasmic immunoreactivity was homogeneous. The intensity of nuclear staining varied, with strongly staining nuclei being found adjacent to nuclei with little or no immunoreactivity (Fig. 1).

Expression of BRCA1 in Sporadic Breast Cancer (⩾60 Years)

Tumors from 28 patients with breast cancer diagnosed at the age of

Discussion

Currently, the subcellular localization and function of the BRCA1 protein, its role in normal cells, and its role in oncogenesis are under active investigation. In the present study, BRCA1 was found in both the nucleus and cytoplasm of normal breast epithelial cells. Cytoplasmic staining was a consistent feature in the breast cancers examined from all three cohorts, with cytoplasmic BRCA1 detected in 39 of 40 cases. The only exception was one case in which a tumor from a patient in the familial

Acknowledgements

This study was supported by the National Health and Medical Research Council of Australia and the University of Sydney Cancer Research Fund. Pathological advice was kindly provided by Dr. Rosemary Balleine. ER and PR determinations were performed by Ms. Jane Milliken, Department of Anatomical Pathology, Westmead Hospital, in the course of routine patient management.

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      Our findings predict enhanced cytoplasmic staining of BRCA1 in familial breast cancers that carry similar BRCT mutations. There are a few published immunohistochemical reports observing increased cytoplasmic BRCA1 in tumors from patients with BRCA1 germ-line mutations (e.g. Refs. [14,17,18,39]); however, most have lacked suitable controls for antibody cross-reactivity and/or classification of the mutations involved. Furthermore, it is important to distinguish between full-length BRCA1 and the exon 11-splice variant form, as we have done here by using the exon 11-specific antibody Ab4 (see Fig. 3), because the shorter splice variants often lack the NLSs and are therefore intrinsically more cytoplasmic.

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