Comprehensive plasma genotyping reveals mutational signatures that confer resistance to tyrosine kinase inhibitors (TKI) in patients with advanced lung cancer.
Authors: Bharde Atul , Behel Vichitra , Bhonde Mandar , Bose Chirantan , Raut Nirmal Vivek , D'Souza Alain , Jadhav Bhagwat , Prajapati Sangeeta , Kad Trupti , Ratnaparkhi Mrunal , Joshi Sujit Krishnarao , Jadhav Vikas Balasaheb Leelavati , Hariramani Kanchan , Mobeen Fauzul , Vasudevan Aravindan , Uttarwar Mohan , Khandare Jayant , Prabhash Kumar , Shafi Gowhar 
Published: June 2023

e20505 Background: Targeting actionable mutations with TKI has altered the treatment paradigm for progressive lung cancers. Significant fraction of patients remain unresponsive to TKI due to cooccurrence of activating EGFR mutations (mEGFR) with other driver mutations. Biopsy of the advanced patients is challenging and is often inconclusive due to tumor heterogeneity. Plasma genotyping using a comprehensive gene panel poses a viable alternative to obtain a broad mutational landscape for Tx decisions. Here we demonstrate the clinical utility of our comprehensive gene panel for detecting actionable mutations and novel alterations from advanced lung cancer patients. Methods: Mutational landscape of 82 advanced-stage lung cancer patients was monitored longitudinally using target-hybridization enrichment of ctDNA and FFPE derived genomic DNA (tDNA). Libraries prepared using a custom designed comprehensive gene panel OncoIndx, targeting 600 cancer relevant exons including MSI and HRR pathway genes, sequenced on Illumina platform. Variant calling was performed using an in-house developed bioinformatics pipeline. Results: Cumulatively, 551 mutations from ctDNA (n = 70) and tDNA (n = 12) samples with pathogenic, likely pathogenic and unknown significance were detected. This included SNVs (31.64 %), truncations (44.73%), splice variants (8.73%), frameshift (6 %), CNVs (4.18 %) and indels (2.9 %). Genome-wide alterations conceded low TMB (10-18) and high average HRD (43-45%) scores in ctDNA and tDNA respectively. mEGFR was the most frequently detected alteration (45.12 %), and had comparable occurrence in ctDNA and tDNA. 28 % ctDNA samples with mEGFR co-occurred with targetable ATM, RAD54 or TP53 mutations and had high HRD scores. Other actionable variants of RAS, ALK, ROS, BRAF and MET were primarily detected in ctDNA samples, albeit at low frequency. Surprisingly, 10 % patients with MET alterations that potentiate TKI resistance were concomitant with mEGFR, and showed high genome instability. Single or multiple mutations in HRR pathway genes were detected in 45 % ctDNA samples. Alterations likely to lead therapy resistance were principally observed in ctDNA samples suggesting that liquid biopsy provides elaborate cancer mutation landscape. Conclusions: Monitoring patients longitudinally for the Tx response is highly implicated. Targeted gene profiling of ctDNA detected multiple variants that were inconspicuous in tDNA. We observed a significant enrichment of variants co-occurring with mEGFR, a new finding on ctDNA mutational analysis. Further investigation into this may suggest better treatment strategies to benefit from TKI resistance. Comprehensive plasma genotyping suggests that patients positive for TKI blunting MET, RAS, BRAF mutations with or without mEGFR co-occurrence may benefit from HRD inhibitors in combination with TKI. Clinical trial information: CTRI/2016/08/007149 .