Abstract 936: Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC)
Authors: Kelly William J. , Nuttall Barrett , Rebelatto Marlon , Schick Marcus , Meier Armin , Sweet Steve , Kim Beom-Jun , Chain David , Kim Yeoun Jin , Lai Zhongwu , Wang Ying , Creasy Todd H. , Gurrapu Ramya , Gulati Sakshi , Barkell Alice , Dougherty Brian , Carroll Danielle , Cecchi Fabiola 
Published: March 2024

Abstract
Background: Multi-omics analysis is widely used in cancer diagnosis, prognosis and prediction of treatment response. Here we introduce the use of targeted proteomics coupled with NGS as tools to assess oncogenic alterations along with quantitative protein expression in patients with mCRC. Our proteomics panel included proteins that are antibody drug conjugates (ADCs) targets (ERBB 1-4, TROP2, CEACAM5 etc.) as well as proteins involved in sensitivity to ADC payload (SLFN11, drug efflux pump, TOP1 etc.).
Methods: The HORIZON III trial (NCT00384176) evaluated FOLFOX + bevacizumab or cediranib as first-line treatment in patients with mCRC (Schmoll J Clin Oncol 2012). In the current analysis, a subset of samples from the HORIZON III trial were assessed for molecular alterations using NGS and proteomics. Pre-existing HER2 IHC scores were used as previously referenced (Cecchi J Clin Oncol 2023). Samples were processed (n=135) with an automated extraction and library preparation method, then sequenced with both a Whole Exome and targeted-custom gene panel. NGS analysis had a success rate of 116/135 samples (87.3%). Absolute quantitation of protein targets was performed using parallel reaction monitoring (PRM) mass spectrometry after collecting tumor epithelial cells by laser microdissection and punch biopsies (n=60) followed by trypsin digestion.
Results: The NGS analysis showed that patients had mutated RAS (50%), BRAF (10%), PIK3CA (16%), and TMB-H (18%) and MSI-H (5%) status. A distinguished pattern of mutual exclusivity among KRAS, NRAS, and BRAF mutations is evident. The HER2-positive patients were wildtype RAS, BRAF, and PIK3CA, MSS and TMB-L. Samples that were IHC HER2-positive matched those with NGS ERBB2 amplification. Using PRM, nearly all the evaluable samples had detectable HER2. HER2 IHC 3+ sample showed the highest protein expression at 30,000 amol/ug (equivalent to 1M receptors/cell). Within the HER2-negative patients, HER2 protein expression showed broad dynamic range. CEACAM5 and HER2 are the only ADC targets that reached the level of 1M receptors/cell. Highest TROP2 protein expression is 5 times lower than the highest HER2 value.
Conclusions: Our preliminary findings for this cohort indicate the benefit of the use of multi-modal methodologies to inform better strategies for patient stratification and precision medicine, though further assessment of proteogenomic characteristics is needed. This data may inform future clinical development and guide patient eligibility to clinical trials and possible treatment sequencing for ADCs.
Citation Format: William J. Kelly, Barrett Nuttall, Marlon Rebelatto, Marcus Schick, Armin Meier, Steve Sweet, Beom-Jun Kim, David Chain, Yeoun Jin Kim, Zhongwu Lai, Ying Wang, Todd H. Creasy, Ramya Gurrapu, Sakshi Gulati, Alice Barkell, Brian Dougherty, Danielle Carroll, Fabiola Cecchi. Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 936.