P3.02b-108 Assessment of Clinical Usability of a cfDNA-Based Assay Detecting EGFR T790M Mutation in EGFR-TKI Refractory NSCLC Patients: Topic: EGFR RES

Abstract

Assessment of acquired resistant EGFR mutation T790M in circulating free DNA (cfDNA) in the plasma of EGFR-TKI treated NSCLC patients presents several challenges. Furthermore, the feasibility and required sensitivity of cfDNA-based detection methods in second-line therapy are not well elucidated. Here, we examined the cfDNA of patients for T790M and other activating mutations of EGFR to assess the clinical usability of such data for diagnosis purposes. cfDNAs were prepared from the plasma samples of 45 NSCLC patients who were confirmed as harboring activating EGFR mutations (exon19 deletion, n = 20; L858R, n = 23; and minor mutations, n = 2). EGFR mutations in cfDNA samples were detected using highly sensitive methods (NGS-utilizing ultra-deep sequencing, droplet digital PCR) and originally developed assays (BNA-clamped PCR/F-PHFA combined method, and BNA-clamped qPCR) and these results were compared to tissue-based definitive diagnoses. No significant change was observed in amounts of extracted cfDNA among EGFR-TKI naïve (n = 18) and refractory (n = 27) groups. There was a positive significant correlation between the amount of cfDNA and diameter in target regions, suggesting that tumor volume reflects the amount of cfDNA. Significant negative correlation was observed between cfDNA amounts and PFS following EGFR-TKI treatment in the TKI-naïve group. The overall percentage agreement between cfDNA and tissue-based analyses ranged from 89 to100 % in major activating mutations and was approximately 85% in T790M. Detected fragment number of each mutation in cfDNA samples by ultra-deep sequencing suggested that it caused the observed difference in the agreement rates between activating mutations and T790M. We confirmed the strong agreement between the high performance assays and definitive diagnosis when the same tissue samples were tested. Next, cfDNA genotyping results were compared to tissue-based definitive diagnosis. In the case of BNA-clamped qPCR, the positive percent agreement was 63% (26/41) in major activating mutations, whereas the negative percent agreement was 100% (45/45). T790M was detected in 46% (12/26) cfDNA samples derived from the EGFR-TKI refractory group. We performed re-biopsies in a proportion of enrolled patients and investigated the tissue-plasma results concordance in matched samples. The observed overall percent agreement was 63% in case of T790M and 88% regarding exon19 deletions. Due to heterogeneity or other biological features of drug-treated tumors, the cfDNA assay feasibility of detecting T790M was more limited than that of detecting activating mutations. To assess the T790M status in EGFR-TKI refractory patients, tissue-based assay and cfDNA-based assay should be performed complementarily.