Abstract P4-11-29: Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy
Authors: Nuciforo Paolo , Thyparambil Sheeno , Aura Claudia , Garrido-Castro Ana , Peg Vicente , Jimenez Jose , Hoos William , Burrows Jon , Hembrough Todd , Perez-Garcia Jose , Cortes Javier , Scaltriti Maurizio 
Published: May 2015

Abstract
Introduction
Approximately 20% of breast cancer patients overexpress HER2 and are treated with anti-HER2 therapies. However, there is a great deal of disparity of HER2 levels in the patients that are classified as HER2 positive (IHC3+). Techniques like FISH or IHC do not allow for HER2 quantification and a significant proportion of patients are wrongly classified as HER2 positive. Liquid Tissue-Selected reaction monitoring (LT-SRM) is a multiplexed mass spectrometric technique that can objectively quantify levels of Her2 and other targets from formalin fixed paraffin embedded (FFPE) sections. Given the different available anti-HER2 therapies (trastuzumab, TDM1, lapatinib and pertuzumab) with different modes of action, it would be beneficial for a clinician to understand the levels EGFR and HER3 so as to personalize the therapy. In this work, we have used LT-SRM to quantitate HER2, EGFR and HER3 from FFPE samples (one slide) of patients treated with anti-HER2 agents and correlated the levels of these proteins to clinical outcome.
Methods
FFPE sections from 60 HER2 positive (IHC3+) primary breast cancer patients were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue® buffer. Of the 60 samples, 24 were from metastatic setting and 36 from adjuvant setting. After trypsin digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. In addition to LT-SRM, FISH for HER2 was also conducted.
Results
HER2 quantitation by LT-SRM revealed receptor level ranges from 283 to 14938 amol/µg. ROC analysis was conducted and a cut-off of 2758.75 amol/µg gave the optimal sensitivity and specificity. Survival analysis revealed statistically significant DFS (4.40 years vs 3.38 years; p=0.013) and OS (4.43 years vs 4.03 years; p= 0039) in patients expressing ≥ 2758.75 amol/µg in the adjuvant setting and also statistically significant OS (5.51 years vs 3.37 years; p=0.037) in the metastatic setting. Correlation of HER2 FISH and levels of HER2 is ongoing. Approximately 41% of samples expressed EGFR (range 45 to 2317 amol/µg) and similarly 51% of the samples expressed HER3 (range 84 to 360 amol/µg) with 18% of samples expressing all three targets. Correlation of EGFR/HER3 expression with clinical outcome is ongoing.
Conclusion
We used an objective multiplex non-antibody based method to quantify multiple targets from FFPE tissue. Clinical correlation analysis of HER2 revealed improved OS and DFS in samples with high HER2 protein levels. Currently, we are expanding these studies to a larger set of samples and taking into account also the expression of other markers such as EGFR and HER3. This approach can potentially identify those tumors that are more dependent on these receptors for survival and also those patients that are exquisitely sensitive to anti-HER2 therapy.
Citation Format: Paolo Nuciforo, Sheeno Thyparambil, Claudia Aura, Ana Garrido-Castro, Vicente Peg, Jose Jimenez, William Hoos, Jon Burrows, Todd Hembrough, Jose Perez-Garcia, Javier Cortes, Maurizio Scaltriti. Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-29.