Kristen rat sarcoma (KRAS) protein is a membrane-associated guanosine triphosphate (GTPase) signal transducer that helps regulate cell growth, proliferation and survival. Many mutations occurring at key amino acid positions (e.g. G12, G13, and Q61) render KRAS constitutively active and drive oncogenesis in patients across a wide range of indications. Therapeutic targeting of KRAS mutants was unsuccessful for several decades due to the apparent lack of suitable drug binding pockets. However, Ostrem et al. discovered compounds that covalently bind to the cysteine in the KRASG12C mutation with high specificity and reignited the pursuit for therapeutic inhibitors [1]. The prevalence of KRASG12C mutations is highest in non-small cell lung cancer (NSCLC) patients with a prevalence of 9–14% [2, 3]. Recently, there has been widespread efforts to develop KRASG12C inhibitors as monotherapies and in combinations [4, 5].

Mass spectrometry assays offer multiplexed protein quantification in complex clinical samples (e.g. plasma, urine, and tissue) [6, 7]. These MS assays overcome several challenges of antibody-based technologies, which often suffer from non-specific binding issues. In addition, mass spectrometry assays can be developed rapidly (weeks to months) while technologies relying on monoclonal antibodies may require years for production and characterization of suitable reagents. The gold-standards for MS-based protein quantification are targeted proteomics assays, including selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), with stable isotope-labeled peptides as internal standards [8]. For irreversible covalent inhibitor drugs, methods may be designed to measure the bound drug-protein complex or the free protein target. Measuring the free target protein is advantageous in early drug discovery where numerous compounds are generated, optimized, and evaluated in preclinical cellular and animal models. In this way, a single assay can be deployed to evaluate target engagement across multiple candidate compounds. In clinical samples, measuring the free target enables a single assay that evaluates target engagement and also compares the target expression levels within the studied population. Several targeted proteomics assays have been developed to quantify RASG12C protein expression in cell line models and frozen tissue samples at baseline and/or after administration of RASG12C inhibitors [9,10,11,12,13,14,15,16].

These studies have proven the utility of mass spectrometry for KRASG12C target engagement from frozen tissues; however, formalin-fixed, paraffin-embedded (FFPE) preservation is the most common storage approach for clinical tissues. Therefore, the FFPE is the standard tissue format available for both patient diagnostics and researchers developing therapeutic interventions and biomarker strategies for solid tumors. In addition, the amount of biopsied tissue samples available for clinical studies are often limited to thin sections. However, there have been a few reports of RAS mutations quantified by targeted proteomics in FFPE tissues. We previously demonstrated that a targeted data independent acquisition method enabled relative quantification of 4 KRAS mutant proteins (G12A, G12D, G12V, G13D) in FFPE tumor biopsies acquired at baseline [17]. However, this proof-of-concept work did not intend to quantify target engagement of G12C nor provide detailed assay evaluation. Hansen et al. reported an assay for FFPE tissues to evaluate a RASG12C inhibitor (ARS-1620), requiring sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) to enrich for proteins in the 15–25 kDa range before in-gel trypsin digestion and PRM analysis [18]. However, methods that require SDS-PAGE are not well-suited to clinical environments due to sample low throughput, difficultly in automation, and large input sample requirements. Finally, we recently reported a multiplexed assay using FAIMS-PRM to quantify 5 proteins of clinical interest (EGFR, ESR1, HER2, KRAS, MET) in FFPE tissues, including the RASG12C mutation [19]. The spiked standard curves demonstrated excellent sensitivity for the RASG12C specific peptide spiked into formalin-fixed tissue samples. However, the study was focused on quantification of HER2 in breast cancer FFPE tissues and the clinical cohort did not include patients with a RASG12C mutation.

In this manuscript, we demonstrate a targeted proteomics mass spectrometry-based assay for precise quantification of RASG12C in preclinical and clinical FFPE tissues without a protein enrichment step (e.g. antibody pull-down or SDS-PAGE). Removing the requirement for isolating the KRAS protein before digestion and LC–MS reduces the sample input requirement for precious clinical samples and enables the panel to be expanded with additional protein biomarkers. We demonstrated the specificity of this assay by quantifying baseline RASG12C levels in FFPE samples from NSCLC patients with and without a KRASG12C mutation. We then evaluated the reproducibility for RASG12C quantification across consecutive tissue slices from the same FFPE block and among adjacent tumor regions within the same FFPE tissue section. And finally, we deployed this assay to quantify target engagement of a covalent binding RASG12C inhibitor (AZD4625) [20] in FFPE tissues from 2 xenograft models.