P1-06-23: Changes in Gene Expression after One Dose of Trastuzumab (T) in HER2+ Breast Cancer Cell Lines Predict Novel Pathways of Response in HER2 Positive Early Stage Breast Cancer.

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Abstract Introduction: Trastuzumab is a targeted therapy against the HER2 cell surface receptor and has greatly improved prognosis for HER2+ breast cancer patients. Despite decades of research, the mechanism of action of T remains unclear and mechanisms of resistance have not been adequately defined. We sought to determine if models of response and resistance in vitro could identify biomarkers in vivo in HER2+ early breast cancer. Methods: BT474 and UACC812 cell lines were treated with 10ug/ml T for 0 and 24 hours. Fresh tumor core biopsies were taken at a 2 week timepoint after a single dose of T (8mg/m2) from 80 HER2−overexpressing, early breast cancer patients enrolled on a clinical trial of T>T+C. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina HT12v3 Beadchip arrays. All arrays were processed at the Yale Center for Genome Analysis (West Haven, CT). Clinical response at surgery was defined as pathologic complete response (pCR), objective response (CR+PR=OR) and non-response (SD+PD=NOR) by RECIST criteria. Gene expression was analyzed in Bioconductor using LIMMA analysis. Pathway analysis was performed using the DAVID bioinformatics resource. Results: We identified gene expression signatures of T response and resistance in HER2+ breast cancer cell lines across 24-hour exposure to trastuzumab. 180-genes changed significantly in a T-sensitive HER2+ cell line (BT474) and 58 genes changed significantly across treatment in a T-resistant HER2+ cell line (UACC812). We applied these signatures to gene expression profiles from early stage HER2+ breast tumors treated with a single dose of T. The BT474 T-responsive signature was enriched among the changes in expression across treatment for responsive patients. The expression change for the BT474 signature genes was also able to partially cluster responsive and resistant breast tumors by outcome. A subset of the UACC812 T-resistance signature was also enriched in the differential between responsive and resistant tumors prior to T treatment. Pathway analysis based on the direction of change of genes in pCR and sensitive (BT474) cell lines found gland development/differentiation (enrichment score=4.5), DNA synthesis (ES=4.0), chaperone (ES=2.8) and transcriptional machinery (ES=2.2) to be coordinately downregulated in both sensitive cell lines and tumors. This suggests that the downregulation of differentiation pathways seen in our ‘pCR signature’ (Harris etal: AACR 2011) is not an epiphenomenon of cell loss. There were no significant pathways upregulated in both sensitive tumors and cell lines, however discordant genes were enriched in chromatin regulation pathways (ES=4.1). Of note, our previous findings of amplicon gene downregulation in pCR tumors, yet upregulation in cell lines points to a novel mechanism of chromatin modulation heretofor undiscovered in response to T. Conclusions: These results demonstrate the value of iterative study of in vitro and in vivo response mechanisms in HER2 cell lines and tumors, and the importance of brief exposure studies in understanding the mechanism of response to T, and other targeted therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-23.

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