Clinicopathological characteristics of patients with EGFR mutation
Fifty-nine advanced EGFR-mutant NSCLC patients were enrolled in this study, comprising 28 men and 31 women with a median age of 56 years (range, 38-79 years) at diagnosis. Among these patients, 98.3% were diagnosed with adenocarcinoma and 94.9% had an Eastern Cooperative Oncology Performance Status (ECOG-PS) score of 0 or 1. Nineteen of the patients had brain metastases and 32.2% were at stage ⅣA at baseline. Thirty-four patients harbored an EGFR exon 19 deletion mutation and the remaining 25 patients harbored an EGFR exon 21 L858R mutation. Using ddPCR, T790M mutation in the ctDNA samples from all 59 patients was assessed before treatment. Based on the ddPCR results, the patients were stratified into two groups: 17 patients had an EGFR-activating mutation with low-frequency de novo T790M mutation and the other 42 patients were T790M-negative pretreatment. Most of the baseline clinicopathological features such as gender, age, ECOG-PS score, smoking status, stage, and brain metastases were similar between the two groups. However, the composition of the EGFR-activating mutation differed between the groups. As shown in Table 1, the low-frequency de novo T790M mutation may coexist with EGFR 19del mutation (p=0.020).
Low-frequency de novo T790M mutation is not rare in NSCLC patients with EGFR-activating mutation
T790M mutation in the ctDNA samples from all 59 patients was evaluated with ddPCR before treatment. The results showed that the overall incidence of low-frequency de novo T790M mutation was 28.81% (17/59). The MAF for T790M mutation ranged from 0.01% to 0.28%, with an ultra-low allele frequency between 0.01% and 0.11% in approximately 88.24% (15/17) of patients (Figure 1).
MAF of plasma T790M mutation showed an upward trend during treatment with first-generation EGFR-TKIs
Serial plasma samples were collected every 2 or 3 months during treatment with first-generation EGFR-TKIs, although plasma samples could not be collected at every visit for all patients. A total of 214 plasma samples were collected for T790M detection using ddPCR, among which 59 plasma samples were obtained pretreatment and 155 plasma samples were obtained during the first-generation EGFR-TKI treatment. A downward trend in the MAF of T790M was found in most patients with a low-frequency de novo T790M mutation at the early stage of EGFR-TKI treatment, but an upward trend was observed when the tumor progressed. After disease progression, nine patients retained T790M mutation in their ctDNA, which was detected by ddPCR. As shown in Figure 2, the MAF of T790M resistance mutation ranged from 0.04% to 1.75%. Furthermore, in the group without de novo T790M mutation pretreatment, the MAF of T790M was negative at the early stage of EGFR-TKI treatment. However, the MAF of T790M in most patients without de novo T790M mutation pretreatment also showed an upward trend over the course of tumor development. ddPCR results showed T790M mutation in nine patients after disease progression and the MAF for T790M resistance mutation ranged from 0.034% to 12.9% (Figure 3).
Most cases of T790M resistance mutation emerge due to the selection of the low-frequency de novo T790M clone
In this study, EGFR T790M mutation in the plasma ctDNA of the patients was monitored with ddPCR every 2 to 3 months until disease progression during treatment with first-generation EGFR-TKIs. In the group with the low-frequency de novo T790M mutation, 15 patients exhibited disease progression and 66.67% (10/15) of them retained the T790M mutation, which was detected with NGS in biopsy specimens from one patient and with ddPCR in ctDNA samples from nine patients. The resistance model for the above-mentioned cases was defined as “selection” resistance. In the group without T790M pretreatment, 26 patients exhibited disease progression and 42.3% (11/26) of them were found to have developed T790M resistance mutation. T790M mutation was detected in ctDNA samples from nine of these patients using ddPCR and in biopsy specimens from the other two patients using ARMS-PCR. The resistance model for these patients was defined as “acquisition” resistance. Taken together, the probability of T790M mutation was 51.22% (21/41) in all patients after disease progression (Figure 4).
There may be no difference in the occurrence time of T790M resistance mutation with “selection” and “acquisition” origins in clinical practice
As shown in Figure 5, among the patients with the T790M resistance mutation after disease progression during the first-generation EGFR-TKI treatment, those with low-frequency de novo T790M mutation had a median PFS1 of 11.0 months (95% confidence interval [CI], 9.141-12.859 months). Patients without the T790M mutation before treatment had a median PFS1 of 11.5 months (95% CI, 9.989-13.011 months; p = 0.649). The median PSF1 for both groups was similar, suggesting there may be no difference in the occurrence time of T790M resistance mutation between patients with “selection” and “acquisition” resistance mutations in clinical practice.
Low-frequency de novo T790M mutation may not affect the efficacy of first-generation EGFR-TKIs
The median follow-up period for the patients in this study was 21.3 months (range, 1.2-36.2 months). At the end of the follow-up period (Mar 23, 2020), 88.2% (15/17) of patients with a low-frequency de novo T790M mutation experienced disease progression, with an objective response rate (ORR) of 82.4%. In the group of patients (42 cases) without pretreatment de novo T790M mutation, six patients were lost to follow-up and 72.2% (26/36 cases) of the remaining patients developed disease progression, with an ORR of 83.3%. As shown in Figure 6, patients with low-frequency de novo T790M mutation had a median PFS2 of 12.0 months (95% CI, 9.781-14.219 months), which was comparable to that of patients without pretreatment T790M (median PFS2, 11.4 months; 95% CI, 9.342-13.458 months; p = 0.451).
The “selection” model of T790M resistance mutation may be treated more effectively with third-generation EGFR-TKIs
In the group of patients with the low-frequency de novo T790M mutation, 10 patients retained the T790M mutation after disease progression. Seven of these patients were further treated with Osimertinib. The optimal efficacy in this group was estimated and indicated a partial response (PR) in six patients (85.7%) and stable disease (SD) in one patient (14.3%), with an ORR of 85.7%. In this group, four patients experienced disease progression at the end of the follow-up period and the PFS3 for five patients was more than 8 months. In the group of patients without T790M mutation before treatment, nine patients further received Osimertinib when T790M mutation was discovered after disease progression. The optimal efficacy in this group was estimated as PR in two (25.0%) patients and SD in six (75.0%) patients, with an ORR of 25.0%. Six patients experienced disease progression at the end of the follow-up period and the PFS3 for five patients was less than 6 months in this group (Figure 7,8).