Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes

Abstract

Several oncogenes and tumor-suppressor genes have been shown to be implicated in the development, progression and response to therapy of invasive breast cancer. The phenotypic uniqueness (and thus the heterogeneity of clinical behavior) among patients' tumors may be traceable to the underlying variation in gene copy number of these genes. To obtain a more complete view of gene copy number changes and their relation to phenotype, we analyzed 20 breast cancer-related genes in 104 invasive breast cancers with the use of multiplex ligation-dependent probe amplification (MLPA). We identified MYC gene amplification in 48% of patients, PRDM14 in 34%, topoisomerase IIa (TOP2A) in 32%, ADAM9 in 32%, HER2 in 28%, cyclin D1 (CCND1) in 26%, EMSY in 25%, IKBKB in 21%, AURKA in 17%, FGFR1 in 17%, estrogen receptor alpha (ESR1) in 16%, CCNE1 in 12% and EGFR in 9% of patients. There was a significant correlation between the number of amplified genes and the histological grade and mitotic index of the tumor. Gene amplifications of EGFR, CCNE1 and HER2 were negatively associated with estrogen receptor status whereas FGFR1, ADAM9, IKBKB and TOP2A revealed a positive association. Amplifications of ESR1, PRDM14, MYC and HER2 were associated with a high mitotic index, and PRDM14 and HER2 amplifications with high histological grade. MYC amplification was detected more frequently in ductal tumors and high-level MYC amplifications were significantly associated with large tumor size. HER2/MYC, HER2/CCNE1 and EGFR/MYC co-amplified tumors were significantly larger than tumors with either of these amplifications. Gene loss occurred most frequently in E-cadherin (CDH1) (20%) and FGFR1 (10%). In conclusion, MLPA analysis with this 'breast cancer kit' allowed to simultaneously assess copy numbers of 20 important breast cancer genes, providing an overview of the most frequent (co)amplifications as well as interesting phenotypic correlations, and thereby data on the potential importance of these genes in breast cancer.

Analysis of copy number changes on chromosome 16q in male breast cancer by multiplex ligation-dependent probe amplification

“…Values between 0.7 and 1.3 were regarded normal. 13,14 Whole arm loss was defined as copy number loss of 475% of all the probes, as defined before using array-comparative genomic hybridization. 15 Partial loss on the long arm of the chromosome was defined as any probe showing copy number loss.…”

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

“…Determination of ERα levels using a ligand binding assay showed that patients with high ERα levels had a shorter relapse-free period than patients with low ERα [21]. Further, fluorescence in situ hybridization of ERα-positive breast tumors has shown that the ERα gene (ESR1) is amplified in about 20% of cases, the amplification being strongly associated with poor disease-free survival [22], whilst multiplex ligationdependent probe amplification-based copy number analysis shows a similar percentage of ESR1 amplification in breast cancer, an association with higher mitotic index and a trend towards higher grade [23]. Other studies have shown similar levels of ERα gene amplification, but by contrast, ESR1 amplification was associated with better response to endocrine therapies [24,25].…”

Expression of EMSY, a Novel BRCA2-link Protein, is Associated with Lymph Node Metastasis and Increased Tumor Size in Breast Carcinomas

“…A recent report on comparing EMSY copy number within breast ductal carcinoma in situ (DCIS) by multiplex ligation-dependent probe amplification (MLPA), showed that EMSY amplification was more frequent in high-grade DCIS than in low/intermediate-grade DCIS, suggesting that in high grade DCIS, EMSY may be potential target for treatment and/or an predictor of progression (Moelans et al, 2010;Moelans et al, 2011). We were unable to investigate EMSY expression separately in DCIS due to small number of cases.…”