Characterization of mutational signatures demonstrating adaptive mutability post anti-EGFR therapy in a real-world metastatic colorectal cancer cohort.
Authors: Eluri Madhulika , Sun Ryan , Bubie Adrian , Alshenaifi Jumanah , Zhang Nicole , Drusbosky Leylah , Parseghian Christine Megerdichian , Raghav Kanwal Pratap Singh , Kopetz Scott 
Published: June 2024

3545 Background: Clinical benefit from anti-EGFR therapy (EGFRi) for RAS/RAF WT metastatic colorectal cancer (mCRC) is limited by secondary mutations in mitogen-activated protein kinases. The source of these new clones has been debated. In contrast to the outgrowth of pre-existing rare subclones, a new model, termed adaptive mutability, proposes the transient loss of DNA repair genes and concurrent induction of error-prone polymerase repair mechanisms (Russo et al Science, ’19). However, clinical evidence for this effect in a large patient cohort is lacking. Using longitudinal circulating tumor DNA (ctDNA) in a real-world cohort, we sought to explore mutational signatures and assess the presence of adaptive mutability in mCRC patients after EGFRi treatment. Methods: 288 patients from Guardant Health’s GuardantINFORM database with mCRC who received EGFRi and underwent longitudinal ctDNA NGS testing by Guardant360 (Redwood City, CA) between December 2014 and March 2023 were included. We extracted de novo single-nucleotide variant mutational signatures from the somatic ctDNA mutations per-cohort level, comparing pre- to post-treatment mutational profiles. Extracted mutational signatures were compared against COSMIC catalogue. Results: Of the 288 patients identified, 99.7% (287/288) were MSS/MSI-L prior to treatment. Pre-treatment, patients had a mean and median of 3 reportable variants per sample, with 30.2% (87/288) samples with >5 variants. Post-treatment, patients had a mean of 5 and a median of 4 reportable variants, with 64.6% (186/288) of patients with >5 variants. Overall mean shift in reportable variants between pre- and post-EGFRi was 5 variants, with a median of 4 changes. In comparing pre- and post-EGFRi cohorts for new mutations, three mutational signatures emerged: SBS17b, SBS15, and SBS3. SBS15 is associated with mismatch repair (MMR) deficiency and classically seen in MSI-H tumors, though notably this cohort was overwhelmingly MSS. SBS15 has been associated with polymerase epsilon exonuclease (POLE) defects and likely reflects repair by alternate error-prone polymerases such as Polι, Polλ, and Polκ. SBS3 has been associated with homologous recombination (HR) deficiency. SBS17b has been associated with exposure to 5-FU based chemotherapy in CRC patients, which was co-administered in many of the EGFRi treated patients. Conclusions: New mutations arising with EGFR inhibition demonstrate strong evidence for adaptive mutability, showcasing loss of mismatch repair and HR fidelity, and reliance on error-prone polymerases. This is the first large cohort to indirectly demonstrate evidence for this genomic mechanism underlying secondary acquired resistance. Further validation of the adaptive mutability signatures may support therapeutic strategies leveraging this vulnerability to extend benefit from targeted therapies.