Molecular Resistance Mechanisms to EGFR, ALK, and ROS1 TKIs

The identification of activating EGFR mutations and ALK and ROS1 rearrangements has ushered in an era of precision medicine for advanced NSCLC characterized by swift advances and adaptive drug development. However, most patients with tumors harboring these genomic alterations derive durable, yet not indefinite, benefit from targeted therapies. Molecular and histologic evaluation at baseline and at the time of progression on EGFR, ALK, and ROS1 TKIs has been integral in identifying key molecular mediators of therapeutic resistance. Mechanisms of resistance are characterized as “on-target” for acquired alterations to the driver tyrosine kinase or “off-target” if mediated by driver-independent molecular changes.

Osimertinib, a third-generation EGFR inhibitor, is increasingly becoming the standard for patients with advanced, treatment-naive EGFR-mutant lung cancer, and it is also approved for the treatment of patients with EGFR T790M-mediated acquired resistance to first- and second-generation TKIs. Despite osimertinib’s efficacy in this disease, tumor response is finitely durable, and acquired resistance to osimertinib almost invariably develops. Analyses of several patient cohorts indicate that the mechanisms of osimertinib resistance can be heterogeneous, although off-target molecular resistance mechanisms predominate. These include MET or HER2 amplification, activation of the RAS-MAPK or RAS-PI3K pathways, cell-cycle gene alterations, and secondary oncogenic fusion events involving, for example, RET, NTRK, ALK, or BRAF.1
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 Transformation to squamous or SCLC histology can occur in up to 15% of patients whose disease progresses on first- or later-line osimertinib. Whereas resistance to first- and second-generation EGFR TKIs is frequently driven by the emergence of an acquired EGFR T790M mutation (in approximately 50% to 60% of cases), osimertinib resistance is EGFR-dependent in a smaller fraction of cases ranging from 6% to 38%.1
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On-target resistance mainly occurs via acquisition of additional mutations in exons encoding the EGFR kinase domain, most commonly C797S/G but also G796S/R, L792F/H, L718Q/V, and G724S.1
 

Interestingly, emerging data suggest that some of these mutations retain sensitivity to earlier generation EGFR TKIs, raising the possibility for TKI sequencing or combination therapy approaches to combat resistance.4
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It is also important to note that the therapeutic setting (first line vs. subsequent line) may bear influence on the landscape of acquired resistance to osimertinib.1
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Therefore, identifying molecular drivers of osimertinib resistance is critical for developing up-front combination strategies to delay or prevent common resistance mechanisms (e.g. osimertinib plus first-generation [NCT03122717] or second-generation [NCT03810807] EGFR inhibitors). Finally, profiling acquired resistance to osimertinib allows for the study of personalized post-progression therapy (e.g., ORCHARD trial: NCT03944772) and the development of next-generation TKIs.6

For ALK-rearranged advanced NSCLC, second-generation TKIs, are widely accepted as the front-line standard because of improvement in PFS over crizotinib, impressive central nervous system activity, and favorable toxicity profiles.7
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. Acquired resistance mutations in exons encoding the ALK kinase domain are more common after progression on second-generation inhibitors, causing resistance in over 50% of cases, compared to 20% to 30% with disease progression on crizotinib.9
Moreover, the spectrum of secondary ALK kinase mutations varies with specific TKI exposure. For the second generation TKIs ceritinib, alectinib and brigatinib, ALK G1202R is the most common mutation to emerge at the time of progression; yet tumors harboring this mutation retain sensitivity to the third-generation inhibitor lorlatinib.10
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In fact, a tailored approach to subsequent therapy can be employed when ALK TKI resistance mutations emerge, depending on prior TKI exposure and the specific mutation identified. The ALK Master Protocol is a National Cancer Institute–sponsored trial investigating such a biomarker-driven approach for the treatment of patients whose disease has progressed on second-generation ALK TKIs (NCT03737994).

Off-target pathway activation is implicated less commonly at resistance to second-generation ALK TKIs , but in some situations this may be targetable, such as when it is due to EGFR pathway activation and MET amplification.9
Histologic changes such as epithelial-to-mesenchymal transition, squamous cell carcinoma, and small cell transformation have been reported, but only as isolated cases.10
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Finally, the phase III CROWN trial recently demonstrated superior efficacy of lorlatinib over crizotinib in patients with ALK-rearranged NSCLC who had not received prior systemic therapy, resulting in U.S. Food and Drug Administration approval for this indication.15
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The uptake of first-line lorlatinib remains to be seen, but unique resistance mechanisms have been described with second- and third-line lorlatinib, including novel compound ALK mutations and loss-of-function NF2 mutations.17

Therapeutic resistance in ROS1-rearranged NSCLC has largely been studied in the context of crizotinib therapy, but patterns of resistance to more recently approved type I ROS1 TKIs, namely entrectinib and lorlatinib, are beginning to emerge.18
Secondary mutations in exons encoding the ROS1 kinase domain have been identified clinically in crizotinib-resistant tumors.19
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 The most common of these is G2032R, which causes resistance not only to crizotinib but also to other inhibitors including entrectinib and lorlatinib. Importantly, promising preclinical activity (repotrectinib and taletrectinib) and early clinical efficacy (repotrectinib21) against G2032R have been observed with next-generation ROS1/TRK/ALK inhibitors18
. Other kinase mutations associated with crizotinib resistance can be targeted by lorlatinib, including S1986F, S1986Y, and L1951R, with the latter also retaining sensitivity to entrectinib.18
Type II ROS1 inhibitors (e.g., cabozantinib) may have activity in patients with tumors progressing on type I TKIs, although this “type-switching” strategy requires further investigation. Off-target resistance mechanisms to ROS1 TKIs are less commonly described but include epithelial-to-mesenchymal transition; SCLC transformation; mutations in KRAS, NRAS, BRAF, PIK3CA, CTNNB1, or KIT; MET amplification; and a switch to EGFR-dependent signaling.20
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Actionable findings at the time of resistance can be identified for patients with lung cancers harboring oncogenic drivers. Histologic transformation has now been described as a mediator of resistance to EGFR-, ALK-, and ROS1-targeted TKIs. Therefore, tissue biopsy with DNA-based next-generation sequencing, RNA-based sequencing, and histologic assessment remains the standard for comprehensive resistance mechanism profiling. Though plasma-based next-generation sequencing lacks the ability to capture histologic information and is dependent on sufficient tumor shedding for the detection of circulating DNA, it may effectively reflect the interlesional heterogeneity present in tumors from patients with systemic progression. For all patients with EGFR-mutated or ALK/ROS1-rearranged NSCLC, tissue biopsy with molecular profiling and/or cell-free DNA testing (when a tissue biopsy cannot be performed) should be conducted at the time of progression on TKI, because subsequent management can ideally be tailored to information gained from these analyses. As standards of care evolve for these diseases, it is imperative that we continue to collect patients’ tissue and plasma for clinical and translational investigation and, when possible, enroll patients in clinical trials evaluating management strategies at the time of progression.

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