Aberrations of breast cancer susceptibility genes occur early in sporadic breast tumors and in acquisition of breast epithelial immortalization.

In the search for early deletion targets in sporadic breast cancer, the analysis of TP53, BRCA1, BRCA2, and ATM revealed loss of heterozygosity (LOH) in tumor cells at 1 or more genes in 18 of 24 cases examined. Notably, in more than 60% of such tumors, LOH was detectable in morphologically normal terminal ductal lobular units (TDLUs) adjacent to carcinoma (LOHint). At BRCA2 and ATM, LOHint was most frequent, particularly in TDLUs of women

Genome-wide allelotyping of a new in vitro model system reveals early events in breast cancer progression.

Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.

Biallelic inactivation of the thyroid hormone receptor beta1 gene in early stage breast cancer.

Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in several types of solid tumors. In breast cancer, 3p loss of heterozygosity occurs in invasive tumor cells as well as in morphologically normal terminal ductal lobular units adjacent to carcinoma in some cases [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996.]. The most frequent region of allelic loss at 3p24.3 in morphologically normal terminal ductal lobular units encompasses the thyroid hormone receptor beta1 (TRbeta1) gene. Here we have observed a variable degree of TRbeta1 promoter hypermethylation in all 11 cases of primary breast cancer examined. Moreover, hypermethylation occurred at the same CpG sites in nonmalignant tissue peripheral to carcinoma in 4 of 11 cases. The lack of TRbeta1 nuclear staining, a likely result of biallelic gene inactivation, was observed in 25% (22 of 85) of primary tumors. This is a first demonstration of promoter hypermethylation and a concurrent reduction of TRbeta1 transcripts in breast cancer cell lines, although specific CpG sites targeted for gene silencing remain to be determined. Gene expression was restored by treatment with 5-aza-deoxycytidine in such cases. The observation of early, frequent, and multiple mechanisms of TRbeta1 inactivation suggests a potential role for this gene in the suppression of breast tumorigenesis.

Increased risk of local recurrence is associated with allelic loss in normal lobules of breast cancer patients.

Allelic losses characteristic of tumor cells, when displayed by morphologically normal terminal ductal lobular units (TDLUs) adjacent to carcinoma [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996], may indicate an extended field of increased cancer susceptibility within the affected breast tissue. We investigated this possibility by asking whether the presence of loss of heterozygosity (LOH) at chromosome 3p11-26 in histologically normal TDLUs (3pLOHn) could lead to an increased risk of local tumor recurrence. We assessed LOHs in normal TDLUs adjacent to 48 informative cases of early-stage invasive breast cancer samples and found 3pLOHn in approximately 25% (13 of 48) of patients whose tumors had 3pLOH in this region. Our analyses suggest that the most frequent region of LOH is localized at 3p24.3. We also demonstrate, using a Cox proportional hazards regression model, that the presence of 3pLOHn was the only variable significantly related to local tumor recurrence, leading to a 3.9-5.2-fold increase in the hazard ratio (P < 0.05). The time to recurrence was longer in such cases than in those without 3pLOHn, suggesting de novo tumor development. These data provide a strong rationale to assess histologically normal breast tissue at the margins of surgically excised cancers for molecular predictors of local recurrence after breast-conserving treatment.