To explore the molecular mechanisms for the similarities between inherited and noninherited forms of breast cancer, we tested the hypothesis that inactivation of BRCA1 by promoter hypermethylation is associated with reduced gene copy number and chromosome 17 aneusomy as observed in tumors from BRCA1 mutation carriers. Using a combination of methylation-specific PCR analysis and fluorescence in situ hybridization, we observed varying degrees of promoter methylation in 39 of 131 (29.8%) primary tumors. Despite significant tumor heterogeneity, mean copy numbers of BRCA1 and CEP17 per cell were lower in methylated cases compared with unmethylated cases [1.78 versus 2.30 (P = 0.001) and 1.85 versus 2.29 (P = 0.005), respectively]. Methylation was more frequently observed in younger women (P = 0.05) with high-grade (P = 0.001), estrogen receptornegative (P = 0.04), and progesterone receptor-negative (P = 0.01) tumors. Moreover, methylation was associated with reduced or absent BRCA1 transcripts, which was reversible in the heavily BRCA1-methylated cell line UACC3199 following treatment with 5-aza-2V-deoxycytidine and trichostatin A. We identified five CpGs at positions À533, À355, À173, À21, and +44 as critical in the reexpression of BRCA1. We conclude that BRCA1 methylation contributes to a subset of sporadic breast cancers with the resulting molecular and clinicopathologic phenotype similar to that of hereditary BRCA1-associated breast cancers. Our data support a model of carcinogenesis in which BRCA1 promoter methylation may serve as a ''first hit,'' much like an inherited germ line mutation, and promote tumor progression down a restricted set of molecular pathways. (Cancer Res 2005; 65(23): 10692-9)

Inherited mutations in the BRCA1 and BRCA2 genes are the strongest genetic predictors of breast cancer and are the primary causes of familial breast/ovarian cancer syndrome. The frequency, spectrum and penetrance of mutant BRCA1/BRCA2 alleles have been determined for several populations, but little information is available for populations of African ancestry, who suffer a disproportionate burden of early onset breast cancer. We have performed complete sequence analysis of all BRCA1 and BRCA2 exons and intron-exon boundaries for 434 Nigerian breast cancer patients from the University College Hospital in Ibadan, Nigeria. In contrast to previous suggestions that BRCA1/BRCA2 mutation frequencies are low or undetectable in African American populations, we find that Nigerian breast cancer patients have an exceptionally high frequency of BRCA1 and BRCA2 mutations (7.1 and 3.9%, respectively). Sixteen different BRCA1 mutations were detected, seven of which have never been reported previously, while thirteen different BRCA2 mutations were seen, six of which were previously unreported. Thus, our data support enrichment for genetic risk factors in this relatively young cohort. To improve breast cancer outcomes, we suggest that family-based models of risk assessment and genetic counseling coupled with interventions to reduce breast cancer risk should be broadly disseminated in Nigeria and other underserved and understudied populations.Breast cancer patients of African ancestry have increased likelihood of early onset of disease and high frequency of hormone receptor negative tumors. While overall breast cancer incidence is lower among populations of African ancestry than other groups, mortality rates are higher. These high mortality rates are observed in both African and African American populations for reasons that are poorly understood and yet remain understudied.

Purpose: Germ-line mutations in the BRCA1 tumor suppressor gene predispose to early onset breast cancers with a distinct phenotype characterized by high tumor grade, aneuploidy, high proliferation rate, and estrogen receptor-negativity. The molecular mechanisms and cooperative oncogenes contributing to multistep tumor progression in cells lacking BRCA1 are not well defined. To examine whether C-MYC (MYC), a transforming oncogene associated with genetic instability, contributes to multistep tumor progression in BRCA1-associated breast cancer, we have analyzed tumors from women with hereditary BRCA1-mutated and sporadic breast cancers.Experimental Design: We performed fluorescence in situ hybridization using a MYC:CEP8 assay on formalinfixed paraffin-embedded tumor tissues from 40 women with known deleterious germ-line BRCA1 mutations and 62 sporadic cases, including 20 cases with hypermethylation of the BRCA1 gene promoter.Results: We observed a MYC:CEP8 amplification ratio >2 in 21 of 40 (53%) BRCA1-mutated tumors compared with 14 of 62 (23%) sporadic tumors (P ‫؍‬ 0.003). Of the 14 sporadic cases with MYC amplification, 8 (57%) were BRCA1-methylated. In total, MYC amplification was found in a significantly higher proportion of tumors with BRCA1 dysfunction (29 of 60, 48% versus 6 of 42, 14%; P ‫؍‬ 0.0003). In a multivariable regression model controlling for age, tumor size, and estrogen receptor status, BRCA1-mutated tumors demonstrated significantly greater mean MYC:CEP8 ratio than sporadic tumors (P ‫؍‬ 0.02).Conclusions: Our data indicate that MYC oncogene amplification contributes to tumor progression in BRCA1-associated breast cancers. Thus, we conclude that the aggressive histopathological features of BRCA1-associated tumors are in part due to dysregulated MYC activity.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p2l-22, we have constructed a long-range physical map of 2.8 megabases for 9p2i by using overlapping yeast artificial chromosome

The molecular mechanism for the occurrence of leukemia in multiple members of a family has not been fully elucidated but data support the contribution of highly penetrant mutations in leukemia susceptibility genes. We have investigated the genetic etiology of an unusual three‐generation family with apparent autosomal dominant transmission of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) accompanied by somatic loss of the long arm of chromosome 5 and/or loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) of leukemia cells have been performed, confirming acquired hemi‐ and homozygous deletion of the long arm of chromosome 5. However, the chromosome lost in the observed LOH event is from the affected parent, in contradiction to the expectation for a two‐hit hypothesis involving a tumor suppressor gene. Furthermore, genetic linkage has been performed at 5q31–33 as well as other loci (21q22 and 16q21–23.2) previously implicated in familial leukemia. In this family, linkage analysis excludes loci at 5q31–33 and 21q22, but localization to 16q21–23.2 cannot be excluded. We observed a maximum multipoint LOD score of 1.19 between marker D16S265 and D16S503 at 16q22 (P = 0.03), suggesting possible linkage to this locus. Considering this family and the previous 16q‐linked family together, the linkage of a leukemia susceptibility gene to 16q22 achieved an LOD score of 3.63 at D16S265 with Θ = 0. Thus, somatic deletion of the long arm of chromosome 5 appears as a necessary but surprisingly noncausative event for onset of AML and MDS in this family, thereby confirming a multistep etiology in which chromosome 5 plays an important secondary role. Genes Chromosomes Cancer 28:164–172, 2000. © 2000 Wiley‐Liss, Inc.