P1-07-19: Analysis of HER2−Status in Breast Cancer by Mass Spectrometry in Archival, Formalin-Fixed Tissues.

Abstract

Abstract HER2 (ERBB2) is overexpressed in about 25% of breast cancers and predicts clinical benefit from trastuzumab, as well as response to anthracycline-based chemotherapy. Fluorescence in situ hybridization (FISH) to detect HER2 gene copy number and immunohistochemistry (IHC) to detect HER2 protein levels are approved by the FDA to identify HER2−positive (H2) tumors. However, the 2007 ASCO/CAP report concluded that approximately 20% of HER2 testing may be inaccurate. Further, the available data did not clearly demonstrate clear superiority of either IHC or FISH as a predictor of benefit from anti-HER2 therapy. Discordance between these methods is as high as 5%. Thus, novel complementary quantitative methods for interrogating HER2 expression in tumors are needed. Targeted protein analysis by multiple reaction monitoring mass spectrometry (MRM-MS) offers a powerful approach to configure assays for specific proteins without using antibodies. Our studies using this platform have demonstrated applicability to formalin-fixed, paraffin-embedded (FFPE) specimens. In the current studies, we used this approach to measure signals from two tryptic peptides specific to HER2, one each from the extracellular and intracellular domains, selected from among 28 candidates based on their signal intensity and sharpness of their chromatographic profiles. Preliminary studies with a HER2−overexpressing BT474 xenograft in mice demonstrated quantitation and detected previously reported HER2 ectodomain shedding. Subsequent analysis of FFPE tissue from five H2 and five triple-negative (TN) tumors yielded measurement of at least 1 femptomole of receptor for H2 tumors and less than 0.2 femptomole of receptor for TN tumors per microgram of digest analyzed. If we assume 200 picograms of protein per cell, the results suggest 110,00 to 468,000 receptors per cell in the H2 tumors and only 2,000 to 14,000 receptors per cell in the TN tumors. Despite significant biological variability in receptor levels measured among the specimens of each type, a clear separation of the H2 and TN tumors was achieved based on the peptide quantitation. This preliminary study demonstrates the potential of MRM-MS in FFPE tissue to provide an alternate approach to IHC-based protein analysis. MRM-MS offers the potential for more, accurate and robust HER2 quantification in clinical breast cancer tissues. The next phase of this work will encompass a larger sample set, including tumors with equivocal and negative FISH and/or IHC test results. Correlation with response to anti-Her2 therapy will be performed in samples with available follow-up data. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-19.