Abstract P2-06-13: Pathway Profiling of Signal Transduction Proteins in Paired Tumor and Adjacent Normal Tissues Obtained from Breast Cancer Patients
Authors: Kim PS , Von Ahsen O , Schmitz A , Schatz C , Magonova K , Lee T , Harvie G , Barham R , Leesman G , Kuller A , Lin F , Gong H , Krahn T , Singh S. 
Published: April 2011

Background: Targeted therapeutic strategies are currently limited to patients with hormone receptors and/or HER2 positive disease in breast cancer (BCA) treatment. However, patients often develop resistance to these therapies. The ability to functionally profile a whole spectrum of pathway proteins (and their variants) in tumor may provide valuable information about the potential mechanism for drug resistance and evidence for rational selection of suitable targeted therapies. Here we report a comprehensive profile of HER1, HER2, p95HER2, HER3, cMET, IGF1R, PI3K, Shc, AKT and other signal transduction pathway proteins in BCA tissues and their matched adjacent normal tissues (ANTs). Methods: A multiplexed Collaborative Proximity ImmunoAssay (COPIA), antibody-microarray platform requiring co-localization of 2 detector antibodies on captured biomarker proteins has been used for comprehensive pathway analysis. Channeling events between 2 detector enzymes (glucose oxidase & horse radish peroxidase) in proximity enabled the profiling of the target biomarkers with extreme sensitivity and specificity, and a direct comparison to electrochemiluminescence based immunoassay platform (MSD) was performed for pathway proteins in tumor vs. ANTs for their expression and activation in samples collected from 20 BCA patients. Results: Three dilutions of lysate (10ug, 1ug, 0.1ug) were analyzed for quantitative differential pathway modulation for COPIA.

- Substantially higher cytokeratin (CK) levels were found in 16/20 tumor samples when compared to paired-ANT; 3/20 samples showed high levels of CK in ANTs. Substantial levels of HER3 and IGF1R expression was detected in 9 and 5 tumor samples respectively.

- Over-expression of HER2 with high degree of activation was found in 2 patients. In one of the HER2-overexpressing patients, HER3 was also highly expressed and moderately phosphorylated. Co-expression of cMET and IGF1R was evident as well.

- A significant degree of HER2 phosphorylation was found in many patients with low level HER2 expression; this may be due to co-expression of high level of HER3 and other RTKs with trans-activational potential. Evidence of activated PI3K complex will be reported.

- In direct comparison to MSD, COPIA detected activated pathway proteins in samples that were not detectable with MSD. MSD was sensitive enough to detect the very extreme cases. COPIA appeared to be a more desirable method for detection of protein expression and activation for samples with limited availability. The distinct pathway modulation in each patient (detected by COPIA) will be reported.

Discussion: COPIA was used to detect the differential expression and phosphorylation of HER2, other RTKs and pathway proteins in 20 paired tumor and matched ANTs. As this platform requires magnitudes lower amounts of specimen, it can be used to profile tumors at different metastatic sites and could provide comprehensive metastatic profiles. The comprehensive functional pathway profiling of tumor specimen may provide insightful information for potential drug-resistant mechanisms and may guide appropriate selection of targeted drug-combinations or drug-sequencing.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-13.