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Updated: Jul 12, 2025
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue
Andrew G Chambers1, David C Chain1, Steve M Sweet1
1Early Oncology, AstraZeneca, One MedImmune Way, Gaithersburg, MD, 20878, USA.
Background:
Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings.
Methods:
Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section.
Results:
Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625).
Conclusions:
This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.
Insights
This study developed a mass spectrometry assay for formalin-fixed, paraffin-embedded (FFPE) tissues, enabling precise quantification of drug-target engagement in oncology clinical trials. The assay successfully measured RAS and RASG12C proteins in non-small cell lung cancer tumors.
Area of Science:
- Oncology
- Proteomics
- Biomarker Discovery
Background:
- Quantifying drug-target binding is crucial for understanding drug mechanisms and dose-response relationships.
- Covalent inhibitors' target engagement is assessed by measuring free target levels.
- Mass spectrometry assays are established for frozen tissues but not widely for FFPE tissues in clinical settings.
Purpose of the Study:
- To develop and validate a targeted mass spectrometry assay for quantifying drug-target engagement in FFPE tissues.
- To enable the application of mass spectrometry-based proteomics in clinical oncology drug development.
Main Methods:
- A targeted mass spectrometry assay using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) was optimized for FFPE tissues.
- Internal standards were used for precise quantification of wild-type RAS and RASG12C.
- Technical reproducibility and biological variation were assessed in non-small cell lung cancer tumor samples.
Main Results:
- The assay quantified wild-type RAS (622-2525 amol/µg) and RASG12C (127-2012 amol/µg) in FFPE non-small cell lung cancer tumors.
- Measurement variation was low (0-18% CV across sections, 5-20% CV among regions).
- Quantitative target engagement was demonstrated in FFPE xenograft models treated with a RASG12C inhibitor.
Conclusions:
- Mass spectrometry-based proteomics can be expanded to analyze FFPE tumor biopsies.
- This approach facilitates preclinical and clinical oncology drug development.
- FFPE tissue analysis by mass spectrometry holds potential for broader clinical applications.
