Patients were considered to have reliable evidence of tumor
Patients were considered to have reliable evidence of tumor DNA shed for adequate clinical interpretation of plasma findings in the resistance setting (classified as “shedders”) when their known initial driver alteration was detected in ctDNA .
We annotated pathogenic genomic alterations detected after TKI resistance in 3 levels of actionability according to OncoKB precision oncology knowledge SC 560 : Level R1, standard of care resistance alterations that predict sensitivity to FDA or EMA-approved drugs in that indication (EGFR T790 M mutations); Level R2, clinically described resistance alterations with compelling clinical evidence for drug response, but neither the alteration nor the drug is standard or care in the resistance setting (e.g. MET amplifications); Level R3, resistance alterations with biological but not clinical evidence for drug response (e.g. PI3KCA mutations). In the case of MET amplifications, only high-level amplifications (reported as 3+, [supplementary methods]) were considered therapeutically actionable. Variants not clustering within any of these actionable subgroups were annotated as non-actionable genomic alterations .
We used t-test to compare the mean number of genomic alterations between two groups. Progression-free survival (PFS) was defined as the time interval between the date of TKI initiation and the date of radiological and/or clinical disease progression or loss of follow-up. All hypothesis testing was performed at a two-sided significance level of α = 0.05.
Results We included 53 patients that were divided in three cohorts: 31 EGFR-mutant NSCLCs with resistance to first/second-generation EGFR TKIs (cohort 1), 15 EGFR T790M + NSCLCs with osimertinib resistance (cohort 2), and 7 ALK/ROS1-rearranged NSCLCs with resistance to crizotinib and/or next-generation ALK/ROS1 TKIs (cohort 3). The baseline characteristics of these patients (at the time of plasma sequencing) are summarized in Table 1. The median time interval between TKI progression and plasma collection was 18 days (range 0–481), and the median turnaround time for ctDNA results was 12 days (range 8–19). Besides Guardant360, post-progression tumor biopsies or alternative plasma-based genotyping methods (supplementary table S1) were concurrently (within 1 month) performed in 17 patients (55%) and 10 patients (32%) respectively in cohort 1 (Fig. 1a). NGS of ctDNA (Guardant360) was the only method for resistance assessment in cohorts 2 and 3.
Discussion For the purposes of this study, we considered patients as tumor DNA shedders only when their known initial driver alteration was detected in plasma. Although the detection of any ctDNA alteration could potentially indicate tumor DNA release, in patients with oncogene-driven NSCLCs the detection of the ubiquitously present driver alteration is considered as the strongest indicator of the presence of tumor DNA , and this is perhaps a more appropriate definition for sufficient ctDNA to assess for resistance alterations at TKI progression. Thus, in this context, the detection of actionable alterations in plasma can be considered informative and reliable to correctly inform subsequent therapies in clinical practice . Using this criterion, about 65% of the EGFR-mutant NSCLCs and 57% of the ALK/ROS1-rearranged NSCLCs in this study had reliable evidence for tumor DNA shed after TKI resistance. These proportions, particularly in the case of EGFR-mutant NSCLCs, are somewhat lower than those reported in other series or clinical trial cohorts (≈ 70–80 % of shedders in EGFR-mutant NSCLC) [, , , , ]. Of note, three patients (5%) in our study had isolated central nervous system (CNS) progression when they underwent plasma-based NGS, a clinical situation where the performance of ctDNA analysis has been shown to be suboptimal . In addition, plasma-based NGS was not performed immediately after TKI progression in some of the patients included in our study (n = 9 patients [17%] underwent plasma sequencing beyond 3 months after TKI progression), which could have potentially underestimated the proportion of tumor DNA shedders. Alternatively, these results might truly reflect the sensitivity of plasma-based NGS to detect known driver alterations after TKI resistance in the real-world clinical setting.