aglogo.gif (1177 bytes)The role of BRCA1 in non-inherited breast cancer


Background of research project

Germline mutations in BRCA1 strongly predispose to breast and ovarian cancer. Breast tumors in mutation carriers almost invariably show loss of the wildtype allele, indicating that BRCA1 acts as a tumor suppressor gene. However, somatically acquired BRCA1 mutations in non-inherited breast cancer have very rarely been reported, shedding doubt on the role of BRCA1 in sporadic breast cancer.

Research questions

In this project we want to address the possibility that somatic mutations haven't been detected more frequently than they have because they occur only in a small proportion of tumors with a certain phenotype and genotype, as predicted by current knowledge on BRCA1 cellular function.

Project description

There are four ways to explain the absence of sporadic mutations in BRCA1: 1. The mutation mechanism in tumors differs strongly from what has thus far been observed for germline mutations. Hence, these mutations may have been missed by technology that would succesfully screen out germline mutations. An example is epigenetic silencing of gene expression (by promotor-methylation, without changing the primary DNA sequence). 2. BRCA1 function may be critical in certain stages of mammary gland development. Loss of BRCA1 function after this stage may not provide a selective advantage in tumorigenesis anymore. Hence only a very small proportion of cases - presumably the early-onset cases - may harbor somatic mutations. 3. Loss of BRCA1 may lead to a very specific cancer morphology, of which an insufficient number of cases have thus far been analysed for the presence of mutations. Recent pathology studies of BRCA1-related breast tumors have indeed defined such morphological characteristics. 4. Since BRCA1 is probably involved in repair of DNA damage, it has been proposed that loss of BRCA1 leads to a mutator phenotype. However, a mutator phenotype can only lead to cancer when certain other cell cycle checkpoints, such as p53, are also defect. There is indeed an increased incidence of p53 mutation in BRCA1-related breast cancer. Hence, somatic BRCA1 mutations may only be relevant against a certain background of other mutations. We will here test these hypotheses by performing a mutation screening of BRCA1 in a defined subset of non-inherited breast cancer, namely early-onset cases with a defined morphological phenotype, with loss of heterozygosity (LOH) at the BRCA1 gene locus, and in which p53 is mutated. The presence of a germline BRCA1 mutation has been excluded.

Experimental design

From previous work, we have already identified about 25 breast tumors that comply with the criteria set forth above. In addition, we have developed a comprehensive mutation screening assay, based on PCR and conformation-sensitive gel electrophoresis (CSGE). BRCA1 will be completely analysed for mutations in the tumor DNA from the 25 cases by CSGE. We will also perform immunostaining with antibodies against BRCA1 to examine protein expression. When a mutation is found, we will examine the normal DNA from the patient (from blood lymphocytes) to exclude the possibility that it is a germline mutation. Finally, we shall analyse the methylation status of the promotor region of BRCA1, and correlate this with immunohistochemical expression patterns and LOH.

Techniques to be applied

PCR; polymorphic marker analysis (LOH); DNA sequencing; immunohistochemistry (in collaboration with the Department of Pathology)

Statistical methods

Fisher's exact test

Plan of work and time schedule

The initial stage will entail the checking of the eligibility of the cases. This means examining the morphology of the tumor (grading, scoring of mitoses, lymphocytic infiltrate, together with a pathologist), demonstrating loss of heterozygosity with intragenic polymorphic markers, and immunohistochemistry with anti-BRCA1 antibodies. Then depending on the length of the project, we will either screen genomic DNA of the tumors by CSGE, and/or determine the methylation status of the promotor region.

Equipment to be used

PCR Thermocycler, light microscope, automated DNA sequencer

Patients involved

Yes, 50

Laboratory animals involved

No

Clinical/Non-clinical

Non-Clinical

Approval required from the Committee on medical ethics

No

Approval required from the committee on the use of laboratory animals

No

Updated:  19-02-2003
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