Discovering genetic profiles by array-CGH in familial breast tumors

Background

We have recently shown that BRCA1 breast tumors can be identified on the basis of their somatic genetic aberrations detected by comparative genomic hybridization (CGH) profiles with high performance (sensitivity: 96%) [1]. Also, BRCA2 show some specific alterations, but are more similar to sporadic breast tumors [2]. These results illustrate that breast tumors from different genetic backgrounds (BRCA1 and BRCA2) develop different genomic instabilities, and therefore genomic profiles. We hypothesize that this may also be true for BRCAx (BRCA3, BRCA 4, etc.) tumors. We therefore applied CGH to familial breast cancer cases from families without BRCA1/2 mutations.

Aims

To produce high-resolution profiles for various types of familial breast cancer, including BRCA1, BRCA2 and BRCAx. To build classifiers based on aCGH profiles. We further aim to optimize class discovery by parallel data analysis of continuous and discrete data as obtained by 'amplicon-finding' algorithms [3]. We also compare BRCA1/2 murine breast tumors with human tumors in an attempt to extract maximal biological meaning from the ploody changes observed in both species [4].

Methods

Array-CGH was performed on genomic DNA isolated exclusively from formalin-fixed paraffin-embedded archival breast cancer specimens. Prior to hybridization, multiplex PCR was performed to assess DNA quality. Then, genomic DNA samples were hybridized to a 3500 BAC array [4] representing one clone for each 1 Mb across the human genome.

Results

We produced array-CGH profiles for 24 BRCA1 tumors, 16 BRCA2 tumors, 19 control (unselected) tumors and 50 tumors from high-risk families (BRCAx, no BRCA1/2 mutations identified) and show, first, that they reproduce metaphase-CGH profiles. Pronounced alterations included 1p-loss (including the DNA damage response protein FRAP1) in 40% of tumors of all classes. An extensive region on 1q (including MUC1) shows gain in many tumors but most frequently so (up to 70%) in BRCA1 tumors. In a region on 3p (including the tumor suppressor RASSF1), loss was observed in >40% of BRCA1 tumors. 3q (including Evi1) was amplified in all tumors classes but most frequently in BRCA1/2 (70%) compared with controls (20–25%). 4p loss is significantly more frequent in BRCA1 (45%) than in either BRCA2 or controls (10–20%) and contains a BRCA1 interacting gene, CtBP1. The centromeric region of chromosome 5 shows loss in 10% of BRCA2, 25% of CONTR and 45–50% of BRCA1 tumors studied. Preliminary analysis of the array-CGH results for the familial breast tumor series, designated BRCAx, show that this is not a homogeneous group. Generally, BRCAx profiles present with fewer gains and losses compared with BRCA1, BRCA2 and sporadic breast tumors. This is a finding that needs further quantification and confirmation.

Conclusions

Array-CGH can be successfully applied on archival formalin-fixed tumor samples. Array-CGH profiles prove useful in the classification of hereditary (BRCA1) breast tumors. Further data analysis should reveal whether BRCAx can be classified is this manner. We propose the use of array-CGH profiles in clinical genetic counseling and are currently working towards this goal.