Abstract
One of the most frequent genetic changes in sporadic breast cancer is amplification of the HER2 gene, usually resulting in protein overexpression on the cell membrane and growth activation of the cells. Breast cancer patients that have this HER2 amplification have a worse prognosis but can be treated with an
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antibody (trastuzumab) directed against the HER2 receptor which results in a survival benefit. However, this treatment is expensive and is associated with potentially severe side effects such as cardiotoxicity and should thus only be administrated to patients that are likely to show a response. It is therefore very important that a reliable diagnostic test exists that selects patients for this targeted treatment. Although many efforts have been made to standardize current diagnostic tests, there is still a lot of variability in procedure and interpretation. In this thesis we evaluated the suitability of a new fully-automatic HER2 protein staining system for use as an aid in a more stable determination of eligibility for trastuzumab therapy, by comparing it with other already established methods, and we explored a new PCR-based technique called MLPA (multiplex ligation-dependent probe amplification) as a low cost, technically uncomplicated and quantitative method to detect amplifications of HER2 in breast cancer in comparison with other techniques used for HER2 amplification or overexpression detection (IHC, CISH). Concordance between MLPA and CISH was 94% and between MLPA and IHC 90%. It is however not very likely that analysis of a single marker like HER2 will be sufficient for proper therapeutic choice-making and perhaps multiplex assays (being able to analyze several prognostic and predictive genes simultaneously) may facilitate oncologists in clinical decision making allowing a more personalized treatment. Furthermore, several processes such as polysomy 17 and genetic variation in breast cancer are still not yet fully understood. Therefore, in the second part of this thesis, we explored MLPA as a multiplex technique: first, we analyzed a large set of patients for both HER2 and TOP2A copy number by MLPA and CISH and found a concordance of 91% for TOP2A and 96% for HER2. MLPA is thus able to simultaneously detect breast cancer HER2 and TOP2A copy number in small quantities of DNA extracted from paraffin blocks, and thereby a good alternative or supplementary technique to other gene amplification detection methods like CISH. Secondly, we investigated the presence of polysomy 17 in breast cancer by simultaneously analyzing 17 chromosome 17 genes in 111 patients using MLPA. None of these patients showed a true polysomy 17; chromosome 17 usually showed a complex pattern of gains and losses, rather unrelated to the copy number status of the centromere (CEP17). And finally, we investigated the frequencies of amplifications, co-amplifications and losses of multiple important or potential breast cancer (onco)genes (MYC, CCND1, ESR1,…) and studied their association with each other and with clinicopathological parameters such as tumor size, age etc. We found a significant correlation between the number of amplified genes per tumor and the histological grade and mitotic index of the tumor.
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