P5-06-03: Gene Expression Associated with Breast Cancer Primary and Secondary Resistance to Neoadjuvant Chemotherapy.
Authors: FK Fujiki, MLH Katayama, H Brentani, DM Carraro, APS Abreu, Filho MC Barros, CT Oliveira, JRF Caldeira, JCS Goes, MM Brentani, MAK Folgueira
Journal: Cancer Research (2011)

Abstract In breast cancer, neoadjuvant chemotherapy may provide valuable information on tumor resistance mechanisms, as variable degrees of tumor response, from pathological complete response until progressive disease, may be observed. To have a better insight of the resistance process to chemotherapy, we have compared resistant pre chemotherapy samples (representing primary or intrinsic resistance) and residual post chemotherapy from partially responsive tumors (secondary or acquired resistance), by means of their transcriptional profile.Tumors from 36 patients submitted to neoadjuvant chemotherapy (4 cycles doxorrubicin and cyclophosphamide) were collected (12 pre-chemotherapy from primary resistant samples and 24 post chemotherapy from residual samples) and analyzed by cDNA microarray (training set, n=25, 9 resistant; 16 residual) and/or by qRT-PCR (technical validation set, n=11, and biological validation set, n=11; 8 resistant; 14 residual). In samples from the training set analysed using a cDNA microarray platform with 4608 genes (Student's t test; p &amp;lt;0,01; FDR &amp;lt;10), 138 genes were differentially expressed between resistant and residual samples, which correctly classified tumors in unsupervised hierarchical clustering analysis, with high confidence. Genes were mainly involved in regulation of cell cycle, regulation of cell growth, cell division, protein modification and DNA dependent DNA replication. Among genes involved in cell growth and cell division, 12 were selected to be further evaluated, using qRT-PCR: MORF4L2, NOTCH2, HTRA1, DLC1, CYR61, MLH1, CHFR, CDC16, CDKL1, NOTCH2, HRAS, CDK2, in technical and biological validation sets of samples. In the technical validation group, a significant positive correlation was observed for the expression of five genes (as evaluated by microarray and qRT-PCR, Spearman correlation, p&amp;lt;0.05), which were then tested in discriminant analysis to detect the best combination to classify resistant and residual tumors. The best combinations included all the five selected genes, CYR61/DLC1/CDKL1/NOTCH2/HTRA1, as well as three of them, CYR61/DLC1/HTRA1, which correctly classified 100% and 91% of tumors from the technical and biological validation groups (100% and 73% in leave one out cross validation, respectively). CYR61 was more expressed in residual samples, however no other main individual gene expression differences were detected. Our data indicate that primary and secondary chemoresistance may present some differences, involving mainly cell growth and cell proliferation processes. CYR61, a CCN growth factor family member, may be synthesized by tumor cells as well as by adjacent fibroblasts, exhibiting autocrine and paracrine actions, stimulating proliferation, migration and chemoresistance. CYR61 enhanced expression in post chemotherapy samples suggests that epithelial stromal interactions may be active players in the chemoresistance process, deserving more detailed analysis for targeted therapies. Supported by FAPESP. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-03.