Project description:The Estrogen Receptor alpha (ERa) is the key transcriptional regulator in luminal breast cancer and the main target for adjuvant treatment. Luminal gene signatures are dictated by the transcriptional capacities of ERa, which are a direct consequence of the receptors binding preference at specific sites on the chromatin. The identification of ERa binding signatures on a genome-wide level has greatly enhanced our understanding of Estrogen Receptor biology in cell lines, but the technique has its limitations with respect its applicability in limit amounts of tumor tissue. Here, we present a refinement of the ChIP-seq procedures to enable transcription factor mapping on limited amounts of tissue culture cells and illustrate the applicability of this refined technology by mapping the ERa genome-wide chromatin binding landscape in core needle biopsy material from primary breast tumors.
Project description:Breast cancer is the most frequently diagnosed female cancer accounting for 23 % of the total cases and the second leading cause of cancer mortality in the world, particularly in western countries. Since GEPARDUO trial reported the therapeutic benefit of combined doxorubicin and cyclophosphamide regimen in sequential administration with docetaxel, the combination regimen has become a standard therapeutic strategy in neoadjuvant systemic therapy for patients with operable breast cancers regardless of an intrinsic subtype. Although approximately 70% of entire patients are currently receiving the chemotherapy regimen, pathologic complete response (pCR) rate is still low, ranging from 23% to 32.7% due to the high heterogeneity of breast cancers. Therefore, the need for a marker predictive of response to a particular cytotoxic regimen, especially before neoadjuvant chemotherapy, is becoming all the more necessary to optimize therapeutic efficacy and to avoid unnecessary complications caused by systemic therapy. In the study, here we generated the first high-coverage proteomic data for needle biopsy FFPE sample being characterized with identical clinical conditions including chemotherapeutic regimens and the stage classification.
Project description:Most human tumor tissues that are obtained for pathology and diagnostic purposes are forma-lin-fixed and paraffin embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to prote-olytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accel-erated sample preparation of FFPE tissues based on paraffin-removal with hot water. Com-bined with tissue homogenization using disposable micropestles and a modified protein aggre-gation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label free quantitation of FFPE cores from human ductal breast carcinoma in-situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2=0.992) with a median %CV of 16.9%. Im-portantly, this small volume is amenable to tissue micro array (TMA) cores and core needle bi-opsies, while our results and the easy-of-use indicate that a further downsizing is feasible. Fi-nally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.
Project description:We used the Human Transcriptome Array 2.0 expression data to examine quality control, reproducibility, and insights on differential gene expression inflammatory breast cancer biopsy specimens prior to systemic treatment.
