Abstract No. 
2012 San Antonio Breast Cancer Symposium
December 4-8

Protein pathway activation mapping of the I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with triple negative tumors

Wulfkuhle JD, Wolf D, Gallagher RI, Yau C, Calvert V, Espina V, Illi J, Wu Q, Boe M, Yan Y, I-SPYTrial1Investigators, Liotta LA, van'tVeer L, Esserman, L, Petricoin, E F

Background: Identification of new therapies and predictive biomarkers is needed for patients with triple negative (TN) breast cancer. Elucidating pathway activation in cancer is critical as these proteins represent targets for many of the new molecular therapeutics and companion diagnostic markers. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and molecular and clinical/pathological characterization.

Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens collected 1–4 days after first chemotherapy treatment (T2). Reverse Phase Protein Microarray was used to measure the activation of 39 and 100 signaling proteins in the T1 and T2 biopsies, respectively, including drug targets for Phase I-III and FDA cleared therapeutics. Associations between pathway activation and response to chemotherapy (RCB 0/1 vs. 2/3) and relapse-free survival (RFS) were evaluated for the triple-negative (TN) patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p < 0.05; Cox proportional hazard model, likelihood ratio p < 0.05.

Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1)


We observed systemic activation of receptor tyrosine kinases such as MET and ALK in the T2 biopsies from patients that recurred, and VEGFR activation was seen in the T1 relapsed tumors. Activation in the AURORA-PLK1 cell cycle signaling network was seen with increased activation in the T2 biopsy from patients who did not respond to therapy and had poor RFS. Dynamic changes in ERK activation were revealed with decreased ERK activation between T1 and T2 time points associated with recurrence.

Conclusions: Our analysis has revealed activation and increased expression of proteins involved in proliferation (Ki67), DNA repair (phosphorylated p53) and protein kinases in patients with TN disease who had poor clinical response. These events, if validated, point to potential treatment options that could be considered for non-responding TN breast cancer patients. VEGFR, ALK and MET, have FDA cleared therapeutics that could be rationally proposed for rapid clinical investigation, and there are numerous investigational agents that target AURORA and PLK1 in the clinic. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with TN tumors.

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