ALK-Is have dramatically improved outcomes in NSCLC patients as well as in several other hematological and solid malignancies (17). However, despite the impressive responses they elicit, patients invariably relapse due to acquired resistance mutations. Solid biopsies remain the gold standard for biomarker testing. However, logistics for obtaining repeat tumor biopsies are complicated and seldom feasible since many patients are unable to endure an invasive procedure, which at the end of the day leads to an empirical prescription of sequential ALK-Is. Nevertheless, blinding sequential strategies might have a deleterious effect on patient’s survival due to the incompletely overlapping ALK mutation coverage of different ALK-Is. In this exploratory analysis, we show, as proof of concept, that plasma NGS is feasible, enabling the detection of resistance mechanisms in patients with ALK-positive NSCLC upon progressive disease. We also provide an algorithm capable of retrieving somatic mutations in the ALK locus that would otherwise be discarded by the commercial bioinformatic pipeline. Remarkably, the developed algorithm performs well in terms of discarding samples with no mutations. Measuring the abundance of DNA molecules in a given sample by NGS is subjected to PCR amplification bias, as not all targeted amplicons are amplified with the same efficacy during library preparation. This limitation can be, at least partly, alleviated by ensuring that all molecules are distinguishable before amplification using unique molecular identifiers (UMIs)(18, 19). With this approach, instead of counting reads, reads are grouped by UMIs, where each distinct UMI identifies the original molecule. In this scenario, parameters such as molecular coverage and molecular counts are pivotal, however free bioinformatic tools, based on the optimization of these parameters remains lacking. As presented in Table 2, the commercial pipeline only detected 3 out of 12 mutations. MAFs of variants detected by the commercial pipeline were 2.8%, 2.1% and 0.4%. According to the manufacturer’s specifications, the limit of detection, in terms of MAF, for mutations is 0.1%. However, in our hands, mutations with a MAF below 0.5% are seldom detected by the commercial pipeline. By using the VALK pipeline, some mutations that would otherwise have been missed can be rescued. Yet, confirmation using an alternative technique such as dPCR would be required to rule out false-positive calls.
Regarding acquired mutations in the ALK locus, our results are consistent with those of previous studies. Specifically, secondary mutations were detected in the plasma samples of 4 of the 11 (36%) patients treated with first-line crizotinib, with the G1269A mutation being detected in two cases. In this regard, mutation detection rate after crizotinib failure might vary from 60% (20) to 24% (21), G1269A being the most prevalent mutation. In this subset of patients we also detected the L1196M and S1206Y mutations, which have been reported to occur in 7% and 2% of cases, respectively, of ALK-positive NSCLC patients treated with crizotinib (11). Finally, we detected the A1200V mutation after crizotinib failure in one patient. This mutation is also known to appear upon crizotinib progression (20). On the other hand, we found that the G1202R mutation was identified in 3 of the 10 patients (30%) progressing on alectinib. This mutation is known to arise mainly after treatment with second-generation ALK-Is (11). Recently, Johannes N et al reported a 53% ALK mutation detection rate in samples obtained post-progression on alectinib (22) in which G1202R was the most frequent mutation. In our cohort, more than one mutation in ALK locus was detected in two samples collected during second- and third-generation ALK-I treatment. Likewise, it has been described that ALK resistance mutations become more frequent with each successive generation of ALK-I as sequential treatment may promote the appearance of resistance mutation at the ALK locus (23).
A reduced number of studies analyzing samples collected upon progression to an ALK-I by NGS have so far been conducted (11, 19, 20), and the molecular mechanisms underlying treatment failure remains poorly understood. To our knowledge, we are the first group to evaluate the feasibility and clinical utility of the Oncomine™ Pan-Cancer Cell-Free Assay, which is a relatively inexpensive panel. This panel detected somatic mutations in 14 genes: TP53, ALK, BRAF, PIK3CA, MAP2K1, FGFR2, FGFR3, EGFR, MYC, MET, IDH2, CCND3, CCND1 and SMAD4. Similarly, mutations in TP53, FGFR2, PIK3CA, MET have been identified in the tumor biopsy of patients progressing on ceritinib (11). The E545K and E545A mutations, which are two of the most common oncogenic mutations in PIK3CA, have also been detected upon progression in advanced EGFR-positive NSCLC patients (24). On the other hand, the IDH2 R140Q detected in our cohort is known to transform cells in vitro and induces myeloid and lymphoid neoplasms in mice (25, 26). The R149Q mutation in IDH2 is frequent in angioimmunoblastic T-cell lymphoma (27). In NSCLC, IDH1/2 mutations are rarely detected in primary tumors but it has been suggested that they could be branching drivers leading to subclonal evolution, based on the MAFs at which these mutations are detected (28). It is therefore not surprising that we found them upon treatment failure.
In addition, we found the E746_A750del mutation in one patient who did not benefit from treatment with ALK-Is. In this way, some researchers have found that mutations in EGFR in some NSCLC tumors coexist alongside ALK rearrangements (29) which may lead to primary resistance to ALK-I (30). Likewise, a non-V600 BRAF mutation was detected after 3 months of treatment with second-line ceritinib treatment, suggesting that resistance of the tumor to the ALK-I could be due to the acquisition of the BRAF mutation. It has been reported that ceritinib enhances the efficacy of trametinib, a MEK inhibitor, in BRAF/NRAS-wild type melanoma cell lines (31), which makes it plausible that ceritinib wouldn’t have any effect in BRAF-mutated cells. Finally, two patients in whose plasma sample the F129L-activating mutation in MAP2K1 (MEK1) was detected, exhibited marked resistance to second- and third-generation ALK-Is. This mutation has been identified as the molecular mechanism underlying MEK/ERK pathway activation in resistant clones of human HT-29 colon cancer cells (32). Moreover, the activation of this downstream pathway is critical to the survival of ALK-positive NSCLC cells (33, 34). Indeed, the combination of ALK and MEK inhibition was highly effective at suppressing tumor growth in a preclinical model of EML4-ALK NSCLC (35). Taken together, it is plausible that the F129L-activating mutation in MAP2K1 is an acquired mutation that leads to tumor resistance to ALK-Is.
Mutations in the FGFR2 and FGFR3 genes were detected in two patients progressing on ALK-Is, suggesting sensitivity to fibroblast growth factor receptor inhibitors. It has been reported that alectinib, despite being a potent ALK-I, has limited inhibitory activity against other protein kinases such as FGFR2 (36). It may therefore be worth confirming whether the appearance of mutations in FGFR genes is a recurrent event after treatment failure with an ALK-I. If this proved to be the case, clinical trials evaluating the efficacy of combinations of ALK-Is with FGFR inhibitors would be of particular interest. Nevertheless, with respect to the FGFR2 mutation encountered, it is important to point out that although G305R has been identified in tumor samples (37, 38), it has not been biochemically characterized so, in this study, it was classified as being of unknown clinical significance.
Three CNVs in c-MYC, CCND1 and FGFR3 were detected upon disease progression in one patient, who was being treated with crizotinib. Remarkably, c-MYC amplification determines many oncogenic effects, including cell growth and proliferation (39) and it has been identified as a potential mechanism of primary resistance to crizotinib in ALK-rearranged NSCLC patients (40). It has been previously suggested by Alidousty et al that co-occurrence of early TP53 mutations in ALK + NSCLC can lead to chromosomal instability. Specifically, authors reported that, in a subset of 53 ALK + tumors, up to a quarter of TP53-mutated tumors showed amplifications of known cancer genes such as MYC or CCND1 (41). Consistent with this, we detected the P92A and V157F mutations in the TP53 gene in the same plasma sample of this patient.
Our results suggest that the P92A mutation in the TP53 gene could be of prognostic significance, although this observation should be interpreted with caution. Our data are consistent with those of previous studies. First of all, the median PFS was 11.6 months (95%CI: 6.5–20.9 months) for ALK-positive NSCLC patients treated with first-line crizotinib (N = 11) or alectinib (N = 2), which was very similar to that of the 10.9 months reported in the PROFILE 1014 trial, which included 172 patients randomized to crizotinib (42). When stratifying these patients according to the P92A mutation status, the median PFS was 7.7 months, for patients with tumors harboring the P92A mutation compared with 14.7 months for those in whom this mutation was not detected. Although this difference was not statistically significant. Similarly, Aisner et al recently reported that concomitant mutations in TP53 are associated with poorer survival among ALK + NSCLC patients (43). Likewise, TP53 mutations were identified as poor prognosis biomarkers in the phase 3 ALTA-1L trial (44).