Acquired resistance remains a major challenge in the treatment of advanced stage NSCLC, limiting long-term efficacy of targeted therapies. As the number of targeted agents increases, the ability to dynamically monitor tumour evolution is critical for optimising patient management. Liquid biopsies provide a minimally invasive approach to assess treatment response and detect markers of resistance in real time.

Using digital PCR (dPCR), we conducted a retrospective analysis of ctDNA, aiming to: (i) compare oncogene driver variant allele frequency (VAF) at baseline and disease progression, and (ii) evaluate dPCR as a tool for real-time assessment of treatment response dynamics. Samples were collected prior to first-line advanced or adjuvant treatment for NSCLC, and at subsequent disease progression/relapse. Analysis was conducted on these paired samples from NSCLCs known to harbour EGFR or KRAS mutations (n =18 and 5 respectively). Mutational status was determined via National Health Service (NHS) molecular testing, using an NGS panel on tissue. ctDNA was extracted (QIAamp MinElute ccfDNA kit) and analysed by digital PCR (dPCR) (QIAcuityDx), reporting wild-type and mutant allele abundance

Testing by dPCR detected mutations in 21/23 cases, offering 100% concordance with the NHS molecular results for KRAS (G12C 3, G12D 1, and G12V 1) and 88% EGFR (ex19 deletion 5, L858R 6 and T790M 5). This included detection in baseline samples with EGFR (13/16) and KRAS (5/5) mutations, as well as progression/relapse samples (15/16 and 5/5, respectively). EGFR ctDNA VAF increased at progression relative to baseline in 11 of 16 cases (69%), including all cases tested for EGFR T790M (n=5), and most EGFR ex19 cases (4/5). VAF of L858R was more commonly reduced at disease progression (4/6, 67%), though this group was most heterogenous with respect to treatments received (Gefitinib 3, Erlotinib 1, Osimertinib 2). Overall, this analysis revealed a mixed response in KRAS ctDNA dynamics where 3 cases showed increase at disease progression (G12D, and 2 G12C), and 2 decreasing (G12V and G12C). Ongoing clinical correlation will be reported, detailing these differences in the context of overall tumour burden.

Our findings confirm the feasibility of ctDNA monitoring in advanced-stage NSCLC and demonstrate strong concordance between dPCR and NHS molecular testing by NGS. The high accuracy of dPCR supports its potential as a complementary tool for detecting resistance mechanisms and refining treatment strategies. Crucially, ctDNA analysis is derived from minimally invasive liquid biopsies, enabling frequent monitoring at a low patient burden but with potentially high clinical benefit. A prospective study is planned to further investigate ctDNA dynamics stratified by mutation and treatment type.