Finding chinks in the osimertinib resistance armor

Introduction

The use of small molecule inhibitors directed at specific oncogene targets, including epidermal growth factor receptor (EGFR) mutations, has improved outcomes and defined precision medicine for non-small cell lung cancers (NSCLC). However, despite the impressive responses and improvements in survival seen with these agents, they are rarely curative, and even in patients who initially achieve complete responses to therapy, resistance inevitably develops, and disease progression occurs.

For EGFR mutant NSCLC, targeting the T790M resistance mutation defined the role of osimertinib (1), an agent with substantial efficacy but limited toxicities, and resulted in increasing efforts to define resistance mechanisms in the hope that these too could be targeted with further gains in survival.

While initially clonal heterogeneity may be limited early, selection pressures induced by treatments enables the growth of preexisting or new tumor cell clones that are resistant to therapy (2). The varied mechanisms by which resistance can occur, coupled with the co-occurrence of multiple resistance mechanisms within one patient, constitute a major challenge in developing an efficient treatment strategy to counteract tumor progression. The clonal evolution of oncogene-addicted NSCLC can give rise to different molecular aberrations both spatially (between primary and metastasis) and temporally (after treatment failure), further contributing to the complexity of the molecular resistance.

Resistance to EGFR TKIs

Resistance to EGFR TKIs broadly falls into ‘EGFR-dependent’ and ‘EGFR-independent’ mechanisms. The first group results in EGFR alterations, such as the T790M and C797S mutations; the latter addresses other methods that divert signaling dependence, such as activation of other downstream pathways such as RAS, gene fusions, BRAF, or even histologic transformation. Resistance to first and second generation EGFR TKIs are most often EGFR-dependent, with the commonest mechanism being the EGFR T790M occurring in 50–60% (3,4). Other EGFR-dependent mutations such as EFGR D761Y have been described but occur much less frequently (5). EGFR-independent mechanisms most frequently involve MET (5% to 20%) and HER2 amplification (8%) (3,4). Histologic transformation to small cell is another cause of resistance (5–14%) (3,4) and de novo RB1/TP53 mutations enrich for tumors likely to develop such transformation (6).

The spectrum of resistance to osimertinib is distinct to that seen with first or second generation EGFR TKIs and varies according to whether the drug is used in the first-line or later-line context. When used to treat acquired T790M mutation, between 20% and 30% develop resistance via an EGFR-dependent tertiary EGFR change, most commonly in the specific binding site of osimertinib, EGFR C797 (7,8). Mutations at other sites of the EGFR such as G724, L718, G719 and L792, and EGFR amplification have also been described (7-10). Resistance due loss of T790M develops in around half the cases, but remarkably in most of these cases is also associated with the emergence of other bypass mutations such as KRAS mutations, MET amplification, gene fusions or small-cell transformation, thus EGFR-independent (7,8). Therefore, in contrast with earlier generation TKIs, around 60% of Osimertinib treated T790M positive cases develop resistance in an EGFR-independent fashion, namely amplification of MET (6% to 26%) and HER2 (up to 8%). Other less frequent, but still EGFR-independent and potentially targetable mutations have been described: HER2 insertions, KRAS and BRAF V600E mutations, NTRK, RET, ALK and FGFR fusions, and MET exon 14 alterations (7,9,10). Similarly to resistance to earlier-generation TKIs, histologic transformation (to small-cell or squamous) is recognized to occur in between 4% and 15% of osimertinib-resistant cases in the later-line setting (7,10).

Resistance patterns to first-line osimertinib, as observed in the FLAURA trial occurred via EGFR-independent mechanisms in 32%, but surprisingly only 8% developed EGFR C797S mutations (11). More frequent EGFR-independent mechanisms included MET amplification (15%), cell cycle gene alterations (10%), PIK3CA mutations (7%), BRAF or KRAS mutations (3% each) and HER2 amplifications (2%) (11). In later-line osimertinib trials, it is possible that addiction to the EGFR pathway through T790M development is predicated, which may explain why more EGFR-dependent resistance pathways are activated. Since the resistance data from FLAURA comes only from plasma genotyping, histologic transformation could not be identified; moreover, other resistance mechanisms could be higher, due to underestimation of gene amplification in plasma. This may explain why a large proportion (40–50%) have unknown resistance mechanisms (10); tissue biopsies may still be important to clarify osimertinib resistance mechanisms in the first-line context.

HER2D16: a potential novel resistance pathway

In this context, Hsu et al. (12) describe a patient who developed resistance to osimertinib after multiple other therapies. The resistance mechanism detected was complex; defined by T790M loss plus two EGFR-independent causes: a HER2 amplification and a novel HER2 ex 16 skipping (HER2D16). The latter has been previously reported in breast cancer only and constitutes one of the three splice variants of HER2, but results in addiction to HER2 signaling (13). Its clinical significance warrants further clarification, but is thought to explain some of the variability in the response to HER2 blockade (13). In breast cancer, the oncogenic properties of HER2D16 are mediated through direct coupling with Src kinase (13,14).

Interestingly, in the article by Hsu et al. (12) the HER2D16 mutation was detected in the plasma prior to commencing Osimertinib, but the allelic fraction increased with disease progression, leading to the hypothesis that this novel mutation mediated resistance. To further this, the authors use an EGFR -T790M/L858R positive cell line (H1975), to stably express HER2D16 and demonstrate elegantly that this novel protein cooperates with the EGFR, in both wild-type (WT) and mutant cells to allow constitutive activation despite osimertinib inhibition. Remarkably, Src levels did not alter after treating cells with dasatinib (a known Src inhibitor), and therefore failed to suppress cellular proliferation, with or without the presence of osimertinib. However, in an attempt to better suppress HER2 signaling in vitro, the authors combined osimertinib with afatinib (a known pan-HER2 TKI). The combination of afatinib and osimertinib was indeed synergistic in vitro, but this was in a construct where both mutations are present in the one cell. Whether this is what actually occurs in vivo is difficult to know.

Discussion

HER2 alterations include amplifications and mutations, but are most commonly in-frame exon 20 insertions and occur de novo in about 1% to 5% of lung adenocarcinomas (15). They have been previously reported as osimertinib resistance mechanisms; in FLAURA, HER2 amplifications were detected in 2%, and HER2 mutations in 1% (11); in the later-line context, up to 5% of HER2 amplifications have been described, but no HER2 mutations (8,10,15). A recent publication described the in vitro use of trastuzumab-emantisine (TDM-1) combined with osimertinib to overcome HER2 amplification-mediated resistance in EGFR-T790M-positive NSCLC cell lines, another example that combination strategies could be used to overcome resistance (16). A phase I–II trial is testing this combination in patients with EGFR-mutant NSCLC, progressing after standard EGFR treatment who developed a HER2 bypass track mechanism of resistance (NCT03784599).

The real question from these data is whether this novel resistance mechanism and the in vitro targeting will translate in the in vivo context. Afatinib binds to Cys797, and preclinical evidence suggested effective inhibition in several EGFR activating mutations including T790M (17), but its clinical performance in patients with erlotinib-resistant cancers harboring T790M was minimal (18). Afatinib is equally potent against WT EGFR and EGFR T790M, so the toxicity resulting from inhibiting WT EGFR precludes the use of doses that would be needed to effectively suppress T790M. This same caveat was reported by the authors (12), since the drug concentration of afatinib which is effective with osimertinib may not be deliverable due to toxicity. Moreover, there is very limited experience with the combination of afatinib-osimertinib in literature, with debatable benefit (19). Similarly, while there was clear preclinical evidence suggesting activity of afatinib on HER2 mutant NSCLC, the largest prospective trial that attempted to test its efficacy was stopped due to futility (20).

In the same context, preclinical data in a T790M-positive cell line suggested that the configuration of the T790M and C797S affected the response to therapy (21): if the two EGFR mutations were in cis (same DNA strand), the cells were refractory to combination first and third-generation TKIs; on the contrary, when the two mutations were in trans (on different DNA strands), a combination of EGFR inhibitors showed clear evidence of in vitro response (21). While this combination has shown some efficacy, unfortunately it is limited, making routine C797S/T790M testing less clinically meaningful (22).

The patient detailed by Hsu et al. (12) was heavily pre-treated, with systemic treatment including chemotherapy, TKIs and even with radiofrequency ablation. The exposure to several treatments is likely to have imposed different selection pressures, leading to multiple different clones. The mixed response to osimertinib, is suggestive of tumoral heterogeneity and is known to be poorly prognostic (23). Since these mutations were found in separate blood samples and the response was heterogeneous, raises the question whether these mutations coexisted in the one site or is the result of spatial heterogeneity. This then draws into challenge whether targeting this resistance pathway would be likely to be broadly effective.

The importance of identifying resistance mechanisms is based on the principle that further specific targeting may evoke durable benefit and minimal toxicity. Considering that resistance to osimertinib usually involves combined mechanisms, such as the activation of alternative cellular pathways and/or aberrant downstream signaling, osimertinib-based combination therapies are currently being investigated (10). Moreover, several case reports and small clinical series including novel combinations with other TKIs against EGFR, RET, ALK, ROS, MET and BRAF inhibitors have been reported (10,22,24-26). However, again, all this initial evidence requires further confirmation. An interesting approach is the recently launched ORCHARD Phase II trial (NCT03944772) which will explore treatment options after disease progression on first-line osimertinib according to the onset of acquired resistance mechanisms. In this innovative platform trial, patients will be allocated to a biomarker-matched study treatment: osimertinib plus gefitinib - osimertinib plus savolitinib (a novel MET inhibitor)—osimertinib plus necitumumab or platinum-based doublet plus durvalumab—within each group based on tumour molecular profile.

Conclusions

Since no clear mechanism of resistance is identified in between 30–40% of patients treated with later-line osimertinib and up to 50% of patients treated with first-line osimertinib (10), studies like Hsu et al. (12) are key to identify targetable alterations. The combination TKI approach is familiar and easy to implement although toxicity would need to be considered and needs confirmation of benefit. But more importantly, this study shows us that resistance is a complex process. It may incorporate both de novo clonal heterogeneity and clonal selection, but also may be the result of mutagenesis with single cells developing multiple resistance mechanisms.

The data from the cell line construct suggest that the HER2D16 mutation is targetable and the authors argue that it should be included as standard testing for reversible mechanisms of osimertinib resistance. However, it is important to acknowledge that while it is useful to identify potential mediators of resistance, the main impetus to implementing such testing is if this mutation is targetable in vivo with an effective yet non-toxic regimen. Until then, while these results are fascinating and important in better understanding the biology of this disease, they remain primarily of academic interest.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Lung Cancer Research. The article did not undergo external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-20-579). JLL has served as a compensated consultant or received honorarium from Merck Sharpe and Dohme (MSD), Roche, Boheringer-Ingelheim, Pfizer, AstraZeneca, Bristol Myers Squibb (BMS), Sanofi, Tecnofarma and Eli Lilly. BS has served as a compensated consultant or received honorarium from Amgen, Loxo Oncology, Roche/Genentech, Pfizer, Novartis, AstraZeneca, Merck and BMS. TJ has served as a compensated consultant or received honorarium from Ignyta, Roche, AstraZeneca, Novartis, Pfizer, BMS and Merck.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Mok TS, Wu YL, Ahn MJ, et al. Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer. N Engl J Med 2017;376:629-40. [Crossref] [PubMed]
2. McGranahan N, Swanton C. Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future. Cell 2017;168:613-28. [Crossref] [PubMed]
3. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med 2011;3:75ra26. [Crossref] [PubMed]
4. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res 2013;19:2240-7. [Crossref] [PubMed]
5. Balak MN, Gong Y, Riely GJ, et al. Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin Cancer Res 2006;12:6494-501. [Crossref] [PubMed]
6. Offin M, Chan JM, Tenet M, et al. Concurrent RB1 and TP53 Alterations Define a Subset of EGFR-Mutant Lung Cancers at risk for Histologic Transformation and Inferior Clinical Outcomes. J Thorac Oncol 2019;14:1784-93. [Crossref] [PubMed]
7. Oxnard GR, Hu Y, Mileham KF, et al. Assessment of Resistance Mechanisms and Clinical Implications in Patients With EGFR T790M-Positive Lung Cancer and Acquired Resistance to Osimertinib. JAMA Oncol 2018;4:1527-34. [Crossref] [PubMed]
8. Papadimitrakopoulou V, Wu Y, Han J, et al. Analysis of resistance mechanisms to osimertinib in patients with EGFR T790M advanced NSCLC from the AURA3 study. Ann Oncol 2018;29:LBA51. [Crossref]
9. Yang Z, Yang N, Ou Q, et al. Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients. Clin Cancer Res 2018;24:3097-107. [Crossref] [PubMed]
10. Leonetti A, Sharma S, Minari R, et al. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br J Cancer 2019;121:725-37. [Crossref] [PubMed]
11. Ramalingam SS, Cheng Y, Zhou C, et al. LBA50Mechanisms of acquired resistance to first-line osimertinib: Preliminary data from the phase III FLAURA study. Ann Oncol 2018;29:LBA50. [Crossref]
12. Hsu CC, Liao BC, Liao WY, et al. Exon 16-Skipping HER2 as a Novel Mechanism of Osimertinib Resistance in EGFR L858R/T790M-Positive Non-Small Cell Lung Cancer. J Thorac Oncol 2020;15:50-61. [Crossref] [PubMed]
13. Castagnoli L, Ladomery M, Tagliabue E, et al. The d16HER2 Splice Variant: A Friend or Foe of HER2-Positive Cancers? Cancers (Basel) 2019.11. [Crossref] [PubMed]
14. Castagnoli L, Iezzi M, Ghedini GC, et al. Activated d16HER2 homodimers and SRC kinase mediate optimal efficacy for trastuzumab. Cancer Res 2014;74:6248-59. [Crossref] [PubMed]
15. Li BT, Ross DS, Aisner DL, et al. HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers. J Thorac Oncol 2016;11:414-9. [Crossref] [PubMed]
16. La Monica S, Cretella D, Bonelli M, et al. Trastuzumab emtansine delays and overcomes resistance to the third-generation EGFR-TKI osimertinib in NSCLC EGFR mutated cell lines. J Exp Clin Cancer Res 2017;36:174. [Crossref] [PubMed]
17. Li D, Ambrogio L, Shimamura T, et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 2008;27:4702-11. [Crossref] [PubMed]
18. Miller VA, Hirsh V, Cadranel J, et al. Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol 2012;13:528-38. [Crossref] [PubMed]
19. Zhang P, Nie X, Wang B, et al. Combined therapy with osimertinib and afatinib in a lung adenocarcinoma patient with EGFR T790M mutation and multiple HER2 alterations after resistance to icotinib: A case report. Thorac Cancer 2018;9:1774-7. [Crossref] [PubMed]
20. Dziadziuszko R, Smit EF, Dafni U, et al. Afatinib in NSCLC With HER2 Mutations: Results of the Prospective, Open-Label Phase II NICHE Trial of European Thoracic Oncology Platform (ETOP). J Thorac Oncol 2019;14:1086-94. [Crossref] [PubMed]
21. Niederst MJ, Hu H, Mulvey HE, et al. The Allelic Context of the C797S Mutation Acquired upon Treatment with Third-Generation EGFR Inhibitors Impacts Sensitivity to Subsequent Treatment Strategies. Clin Cancer Res 2015;21:3924-33. [Crossref] [PubMed]
22. Arulananda S, Do H, Musafer A, et al. Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non-Small Cell Lung Cancer. J Thorac Oncol 2017;12:1728-32. [Crossref] [PubMed]
23. Dong ZY, Zhai HR, Hou QY, et al. Mixed Responses to Systemic Therapy Revealed Potential Genetic Heterogeneity and Poor Survival in Patients with Non-Small Cell Lung Cancer. Oncologist 2017;22:61-9. [Crossref] [PubMed]
24. Piotrowska Z, Isozaki H, Lennerz JK, et al. Landscape of acquired resistance to osimertinib in EGFR-mutant NSCLC and clinical validation of combined EGFR and RET inhibition with osimertinib and BLU-667 for acquired RET fusion. Cancer Discov 2018;8:1529-39. [Crossref] [PubMed]
25. Offin M, Somwar R, Rekhtman N, et al. Acquired ALK and RET gene fusions as mechanisms of resistance to osimertinib in EGFR-mutant lung cancers. JCO Precis Oncol 2018.2. [Crossref] [PubMed]
26. Vojnic M, Kubota D, Kurzatkowski C, et al. Acquired BRAF Rearrangements Induce Secondary Resistance to EGFR therapy in EGFR-Mutated Lung Cancers. J Thorac Oncol 2019;14:802-15. [Crossref] [PubMed]