Tiziana Venesio

Cell proliferation of breast cancer evaluated by anti-BrdU and anti-Ki-67 antibodies: Its prognostic value on short-term recurrences

The prognostic value of breast cancer proliferative activity was evaluated in 385 women operated for primary, non-metastasised mammary carcinoma. Cell kinetics was measured using two immunohistochemical techniques. Cells in S-phase of cell cycle were labelled in vitro by incubation of fresh tissue fragments with 5-bromo 2-deoxyuridine (BrdU), a thymidine analogue. Nuclei of cells in active DNA synthesis were stained by an anti-BrdU monoclonal antibody (Mab). Cells in interphase and mitosis were detected with Ki-67, a Mab that is known to react with a nuclear antigen present in G1/S/G2/M phases of cell cycle, but not in resting cells. This reagent provides a means of evaluating the growth fraction of neoplastic cells. BrdU was incorporated in a proportion of tumour cells ranging from 0.1 to 65.5% (median 6.8%). In the panel of tumours presented in this report the median percentage of Ki-67 positive cells (Ki-67 score) was 9.0% (range 0.1-77%). The relationship between disease-free survival (DFS), BrdU labelling index, Ki-67 score and 13 different clinico-pathological variables was investigated by multivariate analysis, using the Cox proportional hazards model. Axillary node status (P = 0.009) and Ki-67 score (P = 0.038) emerged as independent prognostic factors. Nodal status and tumour growth fraction allowed division of patients into groups at different risk of relapse: tumours with a proliferative index below the median value showed a lower recurrence rate than tumours with a high proliferative activity (P < 0.001). In particular, no relapse occurred in pN0 patients bearing carcinomas with a Ki-67 labelling < 9.0% 4 years after surgery. These findings suggest that the evaluation of proliferative activity in breast cancer enhances the probability of correctly predicting outcome after surgery and could be of assistance in the planning of adjuvant therapies.

Genetic and molecular heterogeneity of breast cancer cells

We have undertaken a systematic study of primary human breast tumor DNAs to identify and characterize frequently occurring somatic mutations. Loss of heterozygosity (LOH) was found on chromosomes 1p (37%), 1q (20%), 3p (30%), 7 (41%), 13q (30%), 17p (49%), 17q (29%) and 18q (34%) in our tumor DNA panel. Specific subsets of tumors could be defined based on the particular collection of mutations they contained. One goal of these studies has been to determine whether there is a significant association between specific mutations and clinical parameters of the disease. We have found that LOH on chromosome 17p in tumor DNAs is associated with breast tumors having a high proliferative index and that LOH on chromosome 7 is associated with patients having a poor prognosis. Our analysis of chromosome 17 suggests that there may be as many as four tumor suppressor genes affected in primary human breast tumors.

Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas☆

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

Growth Suppression of Normal Mammary Epithelial Cells by Wild-Type p53

We examined the status of the p53 gene in the HC11 normal mammary epithelial cells. Two mutations were identified: a Cys to Trp change at codon 138 and a microdeletion of codon 123 to 130 resulting from mutation of the splice acceptor site. These two mutations were independent, and no wild-type p53 allele was found. Introduction of wt-p53 strongly inhibited growth in monolayer. Thus, the absence of wt-p53 can be sufficient for the immortalization of mammary cells.

Genetic Changes in Breast Cancer

Human breast cancer is a group of neoplastic diseases that vary in histological appearance and biological behavior, including the potential for invasiveness and metastasis (Lipmman and Dickson, 1988). Several epigenetic factors influence the development of breast cancer. These seem to be related to the timing and degree of exposure to hormones and growth factors, such as age of menarche, menopause, first pregnancy, and breast feeding (Lipmman and Dickson, 1988). In addition, approx 5% of all. breast cancers are clustered in families that have a high incidence of early onset of the disease (Hall et al., 1990).