Abstract 4719: Dynamic changes in gene expression after one dose of trastuzumab (T) reveal novel biomarkers of response to combination therapy in HER2 positive early stage breast cancer
Authors: Harris Lyndsay N. , Sprecher Emmett , Wei-Gu Lisa , Lezon-Geyda Kimberly , Sakar Sudipa , Krop Ian , Winer Eric P. , Tuck David P. 
Published: June 2012

Abstract
Introduction: The use of a ‘brief exposure’ to single agent T allows the measurement of dynamic changes in the transcriptome that may predict response to T-based combinations. We have shown that AKT and IGF pathway signatures are significantly perturbed in HER2 positive tumors treated with T, only in tumors that ultimately respond to therapy (ASCO 2010). We now demonstrate that transcriptional changes in responding tumors are unique, and uncover unanticipated mechanisms of action of T in early stage breast cancer.
Patients/Methods: 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&gt;T+C. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina HT12v3 Beadchip and Illumina 610QUADv1 SNP arrays. RNA was also processed for sequencing using the Ovation® RNA-Seq System (NuGEN Technologies Inc). The Illumina RNA-Seq Library Preparation and sequencing was performed at the Sequencing Core of the Yale Center for Genome Analysis (West Haven, CT) using the Genome Analyzer IIx sequencer. Gene expression was analyzed in Bioconductor using LIMMA analysis. Gene expression levels from RNA-Seq data was computed using Cufflinks taking mapping results from tophat in SAM format using the Human hg18 RefSeq reference gene set. Genomic biomarkers were compared pathologic complete response (pCR) vs objective response (CR+PR=OR) and non-response (SD+PD=NOR) by RECIST criteria at the surgical timepoint. Pathway analysis was performed using pre-defined gene signatures (IGF-1, AKT) and using DAVID bioinformatics resource.
Results: Gene expression before and after one dose of trastuzumab showed significant changes only in tumors that ultimately achieved pCR. Specifically, AKT and IGF-1 pathways were downregulated in pCR tumors compared with NOR and OR tumors (p=0.04 and p=0.01 respectively). In addition, HER2 and GRB7 were significantly downregulated only in pCR tumors (p=0.003) by both Illumina HT12 arrays and by RNA-sequencing for 17q12 amplicon genes. Analysis of differential expression showed significant changes only in pCR tumors(adjusted pvalue &lt;0.05), compared with NOR and OR tumors. Functional annotation clustering showed enrichment for SH3 domain genes (Enrichment score 2.24) apoptosis pathway genes (ES 1.85;) and differentiation genes (ES 1.3).
Conclusions: Early changes in gene expression can predict pathologic complete response to trastuzumab-containing chemotherapy regimens. Changes in HER2 amplicon genes, AKT, IGF-1 pathways, and genes associated with both apoptosis and differentiation may predict pCR. Brief exposure to trastuzumab may uncover novel mechanisms of action of this life-saving therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4719. doi:10.1158/1538-7445.AM2011-4719