Browsing by Author "Sonntag, Johanna Sigrid Nelly"

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Open Access
Biomarker discovery and pathway activation profiling in breast cancer using protein microarrays
(2013) Sonntag, Johanna Sigrid Nelly; Kontermann, Roland (Prof. Dr.)
Breast cancer, the most frequent cancer entity among women, is nowadays widely recognized as a heterogeneous disease in terms of histopathology as well as on the molecular level. Over the last few years, gene expression profiling studies have improved our understanding of the underlying molecular mechanisms associated with the very heterogeneous outcomes of breast cancer patients. The existence of intrinsic molecular subtypes, which are linked to unique biological and prognostic features, was repeatedly demonstrated and points to the need of tailored therapy options. However on the functional level, breast cancer is not only a genomic but mainly a proteomic disease and gene expression profiling might provide only limited insights. Following the hypothesis that intrinsic biologic features of breast tumors affect prognosis and also therapy response, the general aim of this thesis was to further explore breast cancer heterogeneity with protein microarrays on the functional proteomics level. Around 70 – 80% of all breast cancer patients belong to the luminal intrinsic molecular subtype, characterized as a surrogate marker by overexpression of hormone receptors. An improved classification of this subtype is crucial for therapy decision as part of the patients are at higher risk of recurrence requiring chemo-endocrine treatment, whereas the other part is at lower risk and does not benefit from chemotherapy. However, accurate definition of low and high risk hormone receptor-positive breast cancer has remained a challenge so far. Thus, the first objective of this thesis was the identification of a robust and quantitative protein biomarker signature to facilitate risk classification of hormone receptor-positive breast cancer. To approach this aim reverse phase protein arrays were used to screen across over 120 breast cancer relevant proteins and a novel bioinformatics workflow for biomarker hit selection was applied. Using this approach, a biomarker signature consisting of caveolin-1, NDKA, RPS6, and Ki-67, was identified as most promising to distinguish between low and high risk hormone receptor-positive breast cancer. Since genomic and transcriptomic profiling alone cannot sufficiently predict protein pathway activation, it is important to explore and define the heterogeneity of hormone receptor-positive breast cancer on the proteome level. Especially as protein signaling pathways present the direct targets of new classes of therapeutics. Thus, the second thesis objective addressed the question of whether hormone receptor-positive breast cancer can be further categorized according to similar signaling pathway activation patterns and whether these patterns reflect common molecular mechanisms. Therefore, comprehensive protein pathway activation profiles of breast cancer specimens were obtained using reverse phase protein arrays. To complement this analysis, a microspot immunoassay was developed, which enabled the simultaneous quantification of eight different growth factors in tumor lysate as well as blood plasma of matching patient samples. Four subgroups were identified, based on differential expression of 90 cancer-relevant signaling proteins. Each subgroup showed unique characteristics which were also related to established clinicopathological features as well as growth factor expression. One subgroup, for example, was characterized by high expression levels of almost all analyzed proteins. In addition, VEGF tumor lysate levels were significantly higher in this subgroup and an enrichment of poorly differentiated tumors was observed underlining the aggressive phenotype. In contrast, another subgroup was characterized by weak signaling activity. Interestingly, this subgroup was mostly associated with invasive lobular carcinoma, the second most common histologic type of breast cancer, reflecting a link between histopathology and underlying molecular mechanisms. In summary, the reverse phase protein array based pathway activation profiling of hormone receptor-positive breast cancer, presented in this thesis, provides a comprehensive snapshot of the heterogeneity of this subtype on the proteomic level. Insights obtained can serve as basis to refine the concept of clinically relevant subtypes towards an improved definition of patient-tailored therapy options.