Liquid-Biopsy-Based Identification of EGFR T790M Mutation-Mediated Resistance to Afatinib Treatment in Patients with Advanced EGFR Mutation-Positive NSCLC, and Subsequent Response to Osimertinib

For patients with advanced non-small-cell lung cancer (NSCLC) and activating epidermal growth factor receptor (EGFR) mutations, EGFR tyrosine kinase inhibitors (TKIs) are standard first-line treatment [1]. First- (gefitinib, erlotinib), second- (afatinib, dacomitinib), and third-generation (osimertinib) EGFR TKIs are currently approved in this setting and have demonstrated robust clinical activity. Recent head-to-head data have demonstrated that the second-generation irreversible ErbB family blockers, afatinib and dacomitinib, and the third-generation irreversible EGFR wild-type sparing TKI, osimertinib, achieve superior clinical outcomes over reversible first-generation TKIs [2‐6]. Nevertheless, progression inevitably occurs with all EGFR TKIs. It is therefore important that tumors are characterized for further molecular aberrations at the time of acquired resistance, so subsequent therapy can be tailored. Liquid biopsy is a strategy for tumor genotyping that is now commonly used to assess the presence of aberrations at the time of progression [7, 8]. Although various underlying mechanisms of acquired resistance to first- and second-generation EGFR TKIs have been described, the ‘gatekeeper’ EGFR T790M mutation, which arises from acquisition of a single recurrent missense mutation within exon 20, is the most common mechanism [9, 10].

Osimertinib is selective for both EGFR-sensitizing mutations and the EGFR T790M-resistance mutation. Based on the phase III AURA3 trial, in which 94% of patients had received first-line treatment with a first-generation EGFR TKI and 7% had been pretreated with afatinib, osimertinib was approved for the second-line treatment of patients with an EGFR T790M mutation following progression on or after EGFR TKI therapy [1, 11]. Consequently, it is imperative that patients are screened for the EGFR T790M mutation at the point of acquired resistance. Furthermore, it is important to assess the relative frequency of acquired EGFR T790M resistance mutations in patients treated with first- and second-generation EGFR TKIs, as this information may drive the selection of first-line treatment.

To date, analyses of the frequency of acquired EGFR T790M mutation have mainly been conducted in patients who received first-generation EGFR TKIs, with prevalence rates of 49–69% [9, 10, 12]. However, available data from studies undertaken in Taiwanese [13] or Japanese patients [14], and a predominantly Asian population [12] suggest that development of the EFGR T790M mutation may also be the major mechanism of resistance to afatinib (43–68% of patients). Data on the prevalence of the EGFR T790M mutation after afatinib failure, and subsequent response to osimertinib, are lacking in Caucasian patients. As the mechanism of action of afatinib differs from that of reversible EGFR TKIs, evidence of similar resistance mechanisms across these agents is essential. Additionally, it is important to demonstrate that osimertinib is as effective a treatment option after afatinib failure as it is after erlotinib and gefitinib. We therefore conducted a retrospective analysis in patients who progressed on afatinib, with the aim of identifying the prevalence of EGFR T790M mutations in this setting, and the subsequent responses of these patients to osimertinib.

To our knowledge, this study represents the largest real-world analysis of EGFR T790M prevalence following progression on afatinib in predominantly Caucasian patients with EGFR mutation-positive NSCLC. The results demonstrate that the EGFR T790M mutation is the most common mechanism of acquired resistance in the 54 patients who received first-line afatinib (66.7% of cases). These findings are largely consistent with the rates of EGFR T790M reported following treatment with first-generation EGFR TKIs (49–73%) [8‐12, 15, 16] or afatinib in predominantly Asian patients (43–68%) [12‐14]. In our overall cohort of 67 patients who received afatinib in the first-, second-, or third-line, the rate of acquired EGFR T790M mutation was 73.1%; emergence of the mutation did not appear to correlate with baseline characteristics. However, it was not possible to define when the resistance mutations emerged in the 18.4% of patients who had received prior treatment with erlotinib or gefitinib, as T790M testing took place only after afatinib failure had occurred, and not before initiation of afatinib treatment in the majority of patients. Consequently, these mutations could have arisen at the time of failure of first-generation EGFR TKI therapy.

Of note, the droplet digital PCR method used in this study has the advantage of higher sensitivity compared to alternative methods. In 21 T790M-positive patients, the number of identified T790M copies/ml was below 10 and therefore well below the limit of detection of the widely-used cobas EGFR Mutation Test version 2.0. The higher rate of T790M positivity following afatinib in the current study (73%) compared with previous studies (43–68%) [12‐14] might be attributable to the higher sensitivity of droplet digital PCR compared with other methods. Droplet digital PCR may, for example, detect cases where the T790M allele is present in only a small proportion of tumor cells. Importantly, T790M allele frequency did not appear to influence response rate, indicating that patients with subclonal T790M-positive tumors may still benefit from second-line treatment with osimertinib. We did not monitor other molecular resistance mechanisms to afatinib therapy due to the lack of approved drugs targeting these mechanisms.

The use of tissue biopsy to determine EGFR T790M mutation status in patients progressing following initial treatment with an EGFR TKI can be problematic, as some patients may refuse or be ineligible for biopsy, or progression may have occurred at an inaccessible site. In one analysis of EGFR T790M mutation status among 24 patients who progressed following afatinib, only 14 patients (58%) underwent tissue biopsy at the time of progression, and only 11 samples were sufficient for molecular analysis [17]. Consequently, use of liquid biopsy maximizes the number of patients for whom EGFR T790M mutation status can be determined and who are therefore able to benefit from subsequent treatment with osimertinib. Additionally, liquid biopsy may identify patients with EGFR T790M mutation who would not otherwise be able to access osimertinib because of a false-negative tissue biopsy result, caused by the biopsied site not being representative of all metastatic sites [7]. However, a relative lack of sensitivity for EGFR mutations has been reported for some current liquid biopsy assays compared with tissue biopsy [18]. In our analysis, both liquid biopsy and tissue rebiopsy were used to determine EGFR T790M mutation status, with a concordance rate of approximately 80%. Importantly, all patients included in this analysis received a valid test result for T790M, allowing those who tested positive to receive subsequent treatment with osimertinib. Based on these data, and encouraging results in other studies [7, 15, 19, 20], it appears that liquid biopsy represents an important technical advance in the context of EGFR T790M testing.

In our study, the ORR achieved with afatinib was above 90% in both the overall cohort and the EGFR T790M-positive patient group, with high CR rates (19.4% and 26.5%, respectively); response was independent of baseline characteristics. These high response rates might have been influenced by the single site nature of our study. The ORR achieved with osimertinib after afatinib in patients with an EGFR T790M mutation was also high (75.5%), with 22.4% having CRs and 53.1% having PRs. These findings are similar to those in the AURA3 trial [11], in which an ORR of 71% was achieved; most patients (94%) had received erlotinib or gefitinib as first-line treatment. Although duration of response data is immature in our study (49% of patients were still on treatment at the cutoff date), the median ToT with osimertinib was 14 months overall and not reached in patients who received first-line afatinib.

In patients who progressed on osimertinib and were eligible for subsequent therapy, the most commonly-used option by far was chemotherapy (72.7%). Other studies have reported promising outcomes in patients treated with sequential osimertinib after afatinib. In a retrospective analysis of the LUX-Lung 3, 6, and 7 trials, 37 patients were identified who received subsequent osimertinib following discontinuation of afatinib, mostly in the ≥third-line setting [21]. In these patients, median time on osimertinib treatment was 20.2 months (95% CI: 12.8–31.5) and median OS for osimertinib had not been reached after >4 years’ follow-up. A recent multicenter observational study across ten countries assessed outcomes in 204 patients who received sequential osimertinib after first-line afatinib. In these patients, overall median ToT was 27.6 months, with particularly promising ToT in Asian patients (46.7 months) and patients with an EGFR Del19 mutation (30.3 months) [22]. Finally, a recent phase II trial in 111 T790M-positive patients demonstrated that PFS and response rate were significantly better in patients treated with sequential afatinib and osimertinib versus sequential erlotinib/gefitinib and osimertinib [23].

This study has a number of limitations due to its retrospective nature. First, it was potentially subject to selection bias as only patients who progressed during a defined period of time were included. Second, time points for radiographic assessments were not standardized. For this reason, we assessed ToT rather than the more stringent endpoint of progression-free survival (PFS) as a time-to-event variable, which included patients who were treated beyond progression, a practice that is not proven to provide additional clinical benefit. However, it is noteworthy that no patients received treatment beyond progression in the T790M-positive group. This reflects the availability of an effective subsequent targeted treatment option, osimertinib, in this setting following progression and the molecular confirmation of T790M. Four patients in the T790M negative group were treated beyond progression.

Recent data from the phase III FLAURA study have shown that first-line osimertinib significantly improves PFS compared with first-generation EGFR TKIs, while demonstrating a similar safety profile and lower rate of serious adverse events [4], thus suggesting that osimertinib will be broadly used as first-line treatment. However, as mechanisms of resistance to osimertinib are diverse, and are predominantly independent of EGFR [24], subsequent treatment options post-osimertinib are mostly limited to the chemotherapy at the moment. Therefore, given the high rate of EGFR T790M observed after afatinib treatment, and the high efficacy of osimertinib after afatinib failure observed in this, and other, studies [21], sequential afatinib followed by osimertinib might represent a valid chemotherapy-free regimen that could be offered to patients. Future investigations on the ideal treatment sequence of EGFR TKIs for patients with EGFR mutation-positive NSCLC are clearly warranted.

In patients with stage IV NSCLC and sensitizing EGFR mutations who progress on treatment with the irreversible ErbB family blocker afatinib, the EGFR T790M mutation is present in the majority of cases and is thus the clear main mechanism of acquired resistance. Liquid biopsy can be used to determine T790M mutation status, which maximizes the number of patients who are able to receive subsequent treatment with the third-generation EGFR TKI, osimertinib. Notwithstanding the retrospective nature of this study, osimertinib appears to provide high response rates when administered after afatinib, potentially enabling prolonged chemotherapy-free treatment.