Abstract Lung cancer represents the leading cause of cancer-related deaths worldwide. Despite great advances in its management with the recent emergence of molecular targeted therapies for non-small-cell lung cancer (NSCLC), relapse of the metastatic disease always occurs within approximately one year. Epidermal growth factor receptor (EGFR) mutant tumours are the prime example of oncogene addiction and clonal evolution in oncology, regarding the emergence of resistance to first- and second-generation EGFR inhibitors. Multiple studies have revealed that the EGFR-T790M gatekeeper mutation is the main cause of tumour escape. Recently, irreversible pyrimidine-based EGFR inhibitors especially designed for this particular setting have shown robust clinical activity. However, similar to first- and second-generation inhibitors, the development of a diversified set of resistance mechanisms in response to these new compounds is an emerging issue. To date, clinical management of this growing number of patients has not been clearly established, even if anecdotal responses to subsequent molecularly guided therapies have been observed. By exhaustively reviewing and classifying all the preclinical and clinical data published on resistance to third-generation EGFR inhibitors in NSCLC, this work reveals the heterogeneity of the mechanisms that a tumour can develop to evade therapeutic pressure. Strategies currently being tested in clinical trials are discussed in light of these findings. T790M mutation, EGFR mutation, EGFR-TKI, Nonsmall cell lung cancer, Osimertinib Introduction Lung cancer is the leading cause of cancer-related death worldwide . Despite the emergence of promising therapies in the last decade, curative treatment is not an option for the majority of patients diagnosed with metastatic disease . In the Caucasian population, epidermal growth factor receptor (EGFR) activating mutations are found in approximately 10%–15% of non-small-cell lung cancers (NSCLC) [3, 4]. First- and second-generation EGFR-tyrosine kinase inhibitors (TKIs) are approved as a first-line treatment for these molecularly selected patients in line with the concept of ‘oncogene addiction’. However, disease progression always occurs after approximately 9–12 months of treatment with these targeted therapies [5, 6]. Clinical strategies have been proposed to deal with TKI resistance, such as their continuation beyond progression in the case of slow and pauci-symptomatic malignancies , or the application of local treatments in oligometastatic diseases [8, 9]. Nevertheless, almost invariably, EGFR-mutated NSCLC patients require further systemic treatment. Given the better outcomes generated by tailored agents, overcoming specific resistance mechanisms with additional targeted compounds is of major clinical significance. Among the mechanisms responsible for acquired resistance to first- and second-generation EGFR-TKIs, the EGFR T790M mutation in exon 20 accounts for about 50% of the cases [10–12]. Other mechanisms responsible for acquired resistance to EGFR-TKIs encompass MET and HER2 amplifications, PIK3CA mutations, AXL overexpression and histological transformations (small-cell lung cancer transformation and epithelial-mesenchymal transition), that, taken together, account for the other half of the cases . New pyrimidine-based compounds competing with the ATP-binding in the kinase domain of EGFR have recently been synthetized to overcome the steric clash exerted by the gatekeeper T790M mutation. Osimertinib (AZD9291), rocelitinib (CO-1686), EGF816, HM61713 and AC0010MA are the molecules in clinical trials to date. Initially developed to target single and double mutant EGFR in NSCLC , osimertinib (AZD9291) was found to bind irreversibly and covalently to the Cysteine-797 residue in the ATP binding site of the EGFR kinase . Compared with first-generation TKIs, osimertinib showed equivalent phosphorylation inhibition against sensitizing mutant EFGR in vitro, but superior activity towards T790M-mutant EGFR . A 200 times greater potency against L858R/T790M than wild-type (WT) EGFR and a minimal off-target kinase activity were observed in preclinical studies suggesting an optimal clinical activity profile. In vivo, this compound led to prolonged regression of both sensitizing mutant and T790M-mutant EGFR tumours in xenograft or transgenic models . In the clinical setting, recent reports from phase I/II trials evaluating osimertinib showed an objective response rate of 63%–70% in patients with EGFR T790M-positive NSCLC [17, 18]. In a randomized phase III study, osimertinib was superior to chemotherapy in EFGR-T790M patients who progressed on a first EGFR inhibitor, achieving a median progression-free survival of 10.1 months, with an impressive activity on central nervous system metastases . However, all patients eventually developed a secondary resistance to treatment. This phenomenon is well described for first- and second-generation EGFR-TKIs, and numerous resistance mechanisms have been discovered to date [11, 20–24]. Evidence of new resistance mechanisms in the context of EGFR T790M-mutated lung cancer are now emerging in the literature, with multiple preclinical and clinical studies reported to date. The aim of this review is to provide an overview of resistance mechanisms to EGFR third-generation TKIs and emphasize the importance of deciphering the tumour clonal and biologic evolution to elaborate further successful therapeutic strategies. Preclinical data on acquired resistance to third-generation EGFR-TKIs The first evidence of resistance mechanisms to third-generation TKIs were initially assessed by in vitro preclinical studies . Secondary resistance was artificially induced by prolonged exposure of PC9, HCC827 and H1975 cell lines (all derived from human EGFR-mutant lung adenocarcinomas) to gradually increasing concentrations of third-generation EGFR inhibitors. Of note, the N-ethyl-N-nitrosourea agent was used to promote mutagenesis in most experimental procedures . Published mechanisms of molecular resistance to osimertinib, rocelitinib and WZ4002 (irreversible pyrimidine-based EGFR-TKI not in clinical development) described in preclinical models are summarized in Table 1. Table 1. Preclinical studies reporting resistance mechanisms to third-generation EGFR inhibitors Study Therapy Cell linea T790M status Method Resistance mechanism Reference Ercan et al. 2012 WZ4002 PC9 + FISH MAPK1 amplification  H1975 + qRT-PCR Downregulation of DUSP6, DUSP5, SPRY4 and SPRED2 (negative regulators of ERK1/2) PC9 xenograft + Downregulation of DUSP6 Cortot et al. 2013 WZ4002PF00299804 PC9 – WB Non prolonged exposure: IGFR1 activation then ERK activation (decreased level of IGFBP3)  Prolonged exposure: DUSP6 down-regulation Walter et al. 2013 Rocelitinib H1975 + RNA-seq Epithelio-mesenchymal transition  Ercan et al. 2015 WZ4002 Ba/F3 ND cDNA sequencing EGFR mutations: L718Q; L844V; C797S  Eberlein et al. 2015 Osimertinib H1975 + NGS NRAS gain; KRAS gain; NRAS mutation Q61K;  PC9 – NRAS mutations: G12V; G12R; E63K; NRAS gain; MAPK1 gain; CRKL gain PC9 + KRAS gain WZ4002 PC9 – NRAS mutation Q61K; KRAS gain Niederst et al. 2015 WZ4002 MGH121 + Sanger sequencing EGFR mutation C797S  Ortiz-Curan et al. 2016 Osimertinib Rocelitinib PC9 + Exogenous expression ERBB2 overexpression  HCC827 + MET amplification PC9; HCC827 – KRAS G12C mutant Shi et al. 2016 Osimertinib HCC827 + qPCR MET gain (primary resistance)  Mizuuchi et al. 2016 CNX-2006 HCC827 + qPCR MET amplification; MET amplification and loss of amplified EGFR mutant allele  Nukaga et al. 2017 Rocelitinib PC9 – WESqRT-PCR EGFR WT amplification; EGFR ligand overexpression  Osimertinib PC9 – KRAS mutation G13D Rocelitinib Osimertinib H1675 + WES; WB PI3KCA mutation G118D (LOH); epithelio-mesenchymal transition (Src-AKT pathway activation) Della Corte et al. 2017 Osimertinib HCC827 – WB Epithelio-mesenchymal transition (activation of Hedgehog pathway)  Study Therapy Cell linea T790M status Method Resistance mechanism Reference Ercan et al. 2012 WZ4002 PC9 + FISH MAPK1 amplification  H1975 + qRT-PCR Downregulation of DUSP6, DUSP5, SPRY4 and SPRED2 (negative regulators of ERK1/2) PC9 xenograft + Downregulation of DUSP6 Cortot et al. 2013 WZ4002PF00299804 PC9 – WB Non prolonged exposure: IGFR1 activation then ERK activation (decreased level of IGFBP3)  Prolonged exposure: DUSP6 down-regulation Walter et al. 2013 Rocelitinib H1975 + RNA-seq Epithelio-mesenchymal transition  Ercan et al. 2015 WZ4002 Ba/F3 ND cDNA sequencing EGFR mutations: L718Q; L844V; C797S  Eberlein et al. 2015 Osimertinib H1975 + NGS NRAS gain; KRAS gain; NRAS mutation Q61K;  PC9 – NRAS mutations: G12V; G12R; E63K; NRAS gain; MAPK1 gain; CRKL gain PC9 + KRAS gain WZ4002 PC9 – NRAS mutation Q61K; KRAS gain Niederst et al. 2015 WZ4002 MGH121 + Sanger sequencing EGFR mutation C797S  Ortiz-Curan et al. 2016 Osimertinib Rocelitinib PC9 + Exogenous expression ERBB2 overexpression  HCC827 + MET amplification PC9; HCC827 – KRAS G12C mutant Shi et al. 2016 Osimertinib HCC827 + qPCR MET gain (primary resistance)  Mizuuchi et al. 2016 CNX-2006 HCC827 + qPCR MET amplification; MET amplification and loss of amplified EGFR mutant allele  Nukaga et al. 2017 Rocelitinib PC9 – WESqRT-PCR EGFR WT amplification; EGFR ligand overexpression  Osimertinib PC9 – KRAS mutation G13D Rocelitinib Osimertinib H1675 + WES; WB PI3KCA mutation G118D (LOH); epithelio-mesenchymal transition (Src-AKT pathway activation) Della Corte et al. 2017 Osimertinib HCC827 – WB Epithelio-mesenchymal transition (activation of Hedgehog pathway)  a EGFR mutations harboured by cell lines: PC9: EGFR exon19Δ ; H1975: EGFR L858R & T790M; HCC827: EGFR exon19Δ and MET gain; MGH121: EGFR exon19Δ. EGFR, epidermal growth factor receptor; ND, non-determined; FISH, fluorescence in situ hybridization; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; MAPK1, mitogen-activated protein kinase 1; DUSP, dual specificity protein phosphatase; SPRY4, Sprouty homolog 4; SPRED2, sprout-related EVH1 domain containing 2; IGFR1, insulin growth factor receptor 1; ERK, extracellular signal-regulated kinases; WB, western blot; IGFBP3, insulin growth factor binding protein 3; NGS, next-generation sequencing; CRKL, CRK like proto-oncogene; ERBB2, erb-b2 receptor tyrosine kinase 2; PI3KCA, phosphoinositide 3-kinase. Acquired tertiary EGFR resistance mutation to TKI As the cysteine residue 797 in the exon 20 of EGFR forms a covalent bond with all third-generation TKIs , it was predicted as a hotspot for amino-acid substitutions leading to resistance to these agents. As expected, the emergence of the EGFR C797S mutation was observed in vitro under prolonged exposure of a patient derived EGFR-mutant cell line to WZ4002 [27, 28]. Additionally, in Ba/F3 (murine pro-B cell line) exogenously expressing EGFR activating mutation, T790M and C797S mutations conferred markedly less sensitivity to third-generation EGFR-inhibitors compared with the ones lacking C797S . Another study of Ba/F3 cells harbouring common EGFR-activating mutations, using a mutagenesis screen after chronic exposure to WZ4002, identified two new potential EGFR tertiary resistance mutations: L718Q and L844V . These mutations are thought to be responsible for steric interference leading to decreased compound affinity for the EGFR kinase domain. Supporting this hypothesis, further functional exploration revealed that triple-mutant cells Ex19del/T790M/L718Q exhibit a cross-resistance to all third-generation TKIs. Activation of by-pass RTK signalling Increased signalling through tyrosine kinase receptors other than EGFR have been observed in response to third-generation EGFR-TKIs, notably through insulin-growth factor 1 receptor (IGF1R) activation and MET amplification. In the PC9 cell line which is resistant to third-generation TKIs, two independent studies observed that the use of IGF1R inhibitors could restore TKI sensitivity [30, 31]. Authors noted a decrease of IGF-binding protein 3 (IGFBP3) expression in resistant cells compared with parental cells. They suggested that ligand (IGF-1, IGF-2) availability increased with the loss of IGFBP3 expression and could explain the sustained IGFR1 phosphorylation. However, the mechanism involved in IGFBP3 down-regulation remains unclear. As described for the first-generation EGFR-TKI resistance , MET amplification has been observed after prolonged exposure of HCC827 cell lines to third-generation EGFR-TKIs (osimertinib or CNX-2006) [32, 33]. Consistently, a recent publication using exogenous expression of EGFR T790M-mutant in HCC827 cells demonstrated that the presence of MET amplification decreased the sensitivity to third-generation EGFR inhibitors . Moreover, MET inhibitors were able to restore activity of third-generation TKIs in cell culture and in xenograft models . Equivalent results were obtained with xenograft mice models transplanted with PC9 tumour cells and treated with rocelitinib after erlotinib failure, where MET amplification was the only somatic abnormality acquired in mice bearing rocelitinib-resistant tumours  and concurrent use of a MET inhibitor restored sensitivity to third-generation EGFR-TKIs. These results suggest that MET inhibition could be an option to overcome this resistance. ErbB gene family amplification is another resistance mechanism cited in the literature in the context of first-generation EGFR inhibitors . Exogenous expression of ERBB2 in PC9 gefitinib-resistant cells is associated with decreased sensitivity to third-generation EGFR inhibitors , suggesting a role for HER2 activity as a potential bypass mechanism of resistance. However, to our knowledge, no observation of ERBB2 amplification was reported in preclinical models in response to third-generation EGFR-TKIs. Moreover, a recent report shows that EGFR WT gene amplification associated with EGFR ligand overexpression could emerge in PC9 cells exposed to rocelitinib not developing the T790M resistance mutation . These results suggest that tumour cells may take advantage of mutant selectivity of third-generation EGFR-TKIs through WT receptor bypass signalling, also exploiting autologous feedbacks. However, it remains to be elucidated whether it is ligand overexpression or gene amplification that mainly promotes resistance. Aberration in downstream signalling pathways Alterations involving downstream signalling actors can also result in resistance to EGFR-TKIs. Mutations in the RAS genes, amplification of MAPK genes and inactivation of ERK negative regulators are of special interest in the context of third-generation TKI resistance. Based on the restored sensitivity of EGFR-resistant cancer cell lines to first- and second-generation EGFR-TKIs when associated with MEK inhibitors, some authors investigated the role of the RAS–MAPK signalling pathway in resistance to third-generation inhibitors. Systematic analysis of genetic alterations harboured by osimertinib-resistant PC9 and H1975 cell lines uncovered multiple anomalies in the RAS family genes: KRAS mutation (G13D) ; KRAS amplification and NRAS mutations (Q61K; G12V; G12R; E63K) . Among these cells resistant to third-generation EGFR-TKIs, the EGFR T790M mutation was lost in one case. In addition, copy-number gains of NRAS, MAPK1 and CRKL were detected across the resistant populations. Of note, in EGFR-mutant cells the amplification of CRKL (coding an adaptator kinase protein of RAS signalling pathway)-induced resistance to gefitinib  and amplification of MAPK1 (encoding ERK2)-induced resistance to WZ4002  by activating ERK and AKT signalling. Molecular in vitro and in vivo studies of tumours bearing NRAS mutations and KRAS gain after osimertinib treatment showed that an association with a MEK inhibitor could restore third-generation TKI efficacy, suggesting a functional impact of these alterations in driving resistance. Consistent with these results, another group observed that the stable expression of the KRAS G12C mutation in PC9 cells induced a decreased sensitivity to osimertinib . In addition, prolonged exposure of PC9 cells to third-generation EGFR-TKIs-induced down-regulation of dual-specific phosphatase (DUSP) family genes acting as negative regulators of ERK1/2 [25, 30]. Genetically engineered mouse models harbouring the EGFR T790M mutation confirm these results in vivo, showing the emergence of robust ERK1/2 phosphorylation associated with decreased DUSP6 expression in drug-resistant tumours promoting cell proliferation and aggressiveness. However, the mechanisms of DUSP6 silencing have not yet been elucidated. Altogether, these data support the emergence of RAS/MAPK-dependent resistance mechanisms when lung cancer cell lines are treated with third-generation EGFR inhibitors, as it has been observed with primary resistance to first-generation therapy . Histological transformation Epithelial-mesenchymal transition (EMT) is a known resistance mechanism to first-generation TKIs , along with small-cell lung cancer transformation . In the initial publication related to rocelitinib discovery, cell lines becoming resistant to this compound showed independency towards EGFR signalling . These cells acquired spindle-like morphology and a mesenchymal-like gene signature (up-regulation of VIM, down-regulation of CDH1). Accordingly, recent reports confirmed these observations in HCC827 and H1975 resistant cell lines, showing activation of the Hedgehog pathway  and the Src-AKT pathway , respectively. To date, no in vitro reports of small-cell lung cancer transformation after EGFR-TKI chronic exposure is available, highlighting the potential role of the tumour microenvironment in promoting this phenomenon. Resistance mechanisms to third-generation EGFR-TKI in the clinical setting As osimertinib has been recently approved for EGFR T790M-mutated NSCLC, an increasing number of cases with acquired resistance to this third-generation TKI are being reported. Moreover, although the clinical development of rocelitinib has been interrupted, evidence concerning mechanisms of resistance to this compound are already available and can support upcoming data on other third-generation inhibitors. Interestingly, by comparing the molecular characteristics of samples (solid or liquid) collected during the course of treatment to initial pre-treatment specimens, numerous EGFR-TKI resistance mechanisms identified from preclinical studies have been confirmed in patients (Table 2). Table 2. Studies reporting resistance mechanisms to third-generation EGFR inhibitors in the clinical setting Study Therapy Source N T790M pre-TKI Method Resistance mechanism Allelic pos./T790M T790M post-TKI References Kim et al. 2015 Osimertinib Tissue 1 + FISH; qRT-PCR WT EGFR gain; MAPK1 overexpression; AKT3 overexpression –  1 + qRT-PCR FGFR1 gain; FGF2, AXL overexpression – 1 + qRT-PCR EGF overexpression + 1 + IHC Small cell carcinoma transformation – Yu et al. 2015 Osimertinib Tissue 1 + NGS EGFR mutation C797S In cis +  Planchard et al. 2015 Osimertinib Tissue 1 + CGH; FISH HER2 amplification –  1 + FISH; FISH MET amplification – Piotrowska et al. 2015 Rocelitinib Tissue 2 + IHC; NGS Small-cell lung cancer transformation –  3 + EGFR amplification + 6 + Loss of T790M – Song et al. 2015 HM61713 Tissue 1 + NGS EGFR mutation C797S +  Ham et al. 2016 Osimertinib Tissue 2 + IHC Small cell carcinoma transformation –  Ortiz-Curan et al. 2016 Rocelitinib Pl. eff. 1 + FISH NGS ERBB2 amplification +  Osimertinib Tissue 1 + MET amplification + 1 + MET amplification; HER2 amplification + 1 + EGFR mutation C797S + Tissue Plasma 1 + KRAS mutation G12D EGFR mutation C797S – Bersanelli et al. 2016 Osimertinib Tissue 1 + NGS EGFR mutation L718Q +  Ou et al. 2017 Osimertinib Tissue 1 + CGH MET amplification + (3%)  Knebel et al. 2016 Osimertinib Plasma 1 + ddPCR EGFR mutation C797S; amplification of EGFR exon19Δ-mutant allele +  Thress et al. 2016 Osimertinib Plasma 6/15 + ddPCR EGFR mutation C797S cis (5); trans (1) +  4/15 + ddPCR Loss of EGFR mutation T790M – Chabon et al. 2016 Rocelitinib Plasma 17/43 + CAPP-seq MET gain (11), ERBB2 gain (4), EGFR gain (4) T790M loss: 28/43  18/43 EGFR: C797S(1) L798I(1) L692V(1) E709K(1); PI3KCA: E542K(3); E545K(3) E81K(1); KRAS: G12A(1) Q61H(1) A146T(1); CDKN2A: D74A(2); RB1: R787Q(1); ALK: R1061Q(1); KIT: L576P(1) In cis (C797S; L798I) Li et al. 2017 Osimertinib Tissue 1 + IHC Small cell carcinoma transformation –  Ho et al. 2017 Osimertinib Tissue 1 + NGS BRAF mutation V600E +  Chen et al. 2017 Osimertinib Plasma 1 + NGS EGFR mutations: C797S/G/N; In trans +  L792F/H; L718Q In cis Plasma 1 + NGS EGFR mutations: C797S; In trans + L792F/H/Y In cis Pl. eff. 1 N.D NGS EGFR mutation: C797S In trans + L792F In cis Ou et al. 2017 Osimertinib Plasma 1 + NGS EGFR mutations: C797S/G; G796S/R; L792F/H In cis +  Study Therapy Source N T790M pre-TKI Method Resistance mechanism Allelic pos./T790M T790M post-TKI References Kim et al. 2015 Osimertinib Tissue 1 + FISH; qRT-PCR WT EGFR gain; MAPK1 overexpression; AKT3 overexpression –  1 + qRT-PCR FGFR1 gain; FGF2, AXL overexpression – 1 + qRT-PCR EGF overexpression + 1 + IHC Small cell carcinoma transformation – Yu et al. 2015 Osimertinib Tissue 1 + NGS EGFR mutation C797S In cis +  Planchard et al. 2015 Osimertinib Tissue 1 + CGH; FISH HER2 amplification –  1 + FISH; FISH MET amplification – Piotrowska et al. 2015 Rocelitinib Tissue 2 + IHC; NGS Small-cell lung cancer transformation –  3 + EGFR amplification + 6 + Loss of T790M – Song et al. 2015 HM61713 Tissue 1 + NGS EGFR mutation C797S +  Ham et al. 2016 Osimertinib Tissue 2 + IHC Small cell carcinoma transformation –  Ortiz-Curan et al. 2016 Rocelitinib Pl. eff. 1 + FISH NGS ERBB2 amplification +  Osimertinib Tissue 1 + MET amplification + 1 + MET amplification; HER2 amplification + 1 + EGFR mutation C797S + Tissue Plasma 1 + KRAS mutation G12D EGFR mutation C797S – Bersanelli et al. 2016 Osimertinib Tissue 1 + NGS EGFR mutation L718Q +  Ou et al. 2017 Osimertinib Tissue 1 + CGH MET amplification + (3%)  Knebel et al. 2016 Osimertinib Plasma 1 + ddPCR EGFR mutation C797S; amplification of EGFR exon19Δ-mutant allele +  Thress et al. 2016 Osimertinib Plasma 6/15 + ddPCR EGFR mutation C797S cis (5); trans (1) +  4/15 + ddPCR Loss of EGFR mutation T790M – Chabon et al. 2016 Rocelitinib Plasma 17/43 + CAPP-seq MET gain (11), ERBB2 gain (4), EGFR gain (4) T790M loss: 28/43  18/43 EGFR: C797S(1) L798I(1) L692V(1) E709K(1); PI3KCA: E542K(3); E545K(3) E81K(1); KRAS: G12A(1) Q61H(1) A146T(1); CDKN2A: D74A(2); RB1: R787Q(1); ALK: R1061Q(1); KIT: L576P(1) In cis (C797S; L798I) Li et al. 2017 Osimertinib Tissue 1 + IHC Small cell carcinoma transformation –  Ho et al. 2017 Osimertinib Tissue 1 + NGS BRAF mutation V600E +  Chen et al. 2017 Osimertinib Plasma 1 + NGS EGFR mutations: C797S/G/N; In trans +  L792F/H; L718Q In cis Plasma 1 + NGS EGFR mutations: C797S; In trans + L792F/H/Y In cis Pl. eff. 1 N.D NGS EGFR mutation: C797S In trans + L792F In cis Ou et al. 2017 Osimertinib Plasma 1 + NGS EGFR mutations: C797S/G; G796S/R; L792F/H In cis +  ddPCR, digital droplet PCR; FISH, fluorescence in situ hybridization; NGS, next-generation sequencing; CAPP-seq, cancer personal profiling by deep sequencing; IHC, immunohistochemistry; CGH, comprehensive genomic hybridization; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; ND, non-determined; EGF, epidermal growth factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor. Acquired tertiary EGFR resistance mutation to TKI As expected from preclinical data, the exon 20 EGFR C797S mutation was identified in the clinical setting as an acquired resistance mechanism to osimertinib, rocelitinib and HM61713 treatment [35, 44, 45]. This mutation confers cross-resistance to all third-generation TKIs as it prevents their binding to the EGFR active site. In accordance with cell line studies, emergence of the EGFR L718Q resistance mutation was also reported in the clinic [46, 47]. Additional insights come from a study carried out with circulating tumour DNA (ctDNA), in which 16 genes were sequenced on 115 serial plasma samples collected from 43 patients treated with rocelitinib during a phase I/II trial [35, 48]. Among the identified single nucleotide variations identified in this study were new mutations in the EGFR gene with allele frequency increasing over the course of rocelitinib treatment in four patients: E709K, L692V, C797S and L798I . Concerning the L798I mutation, in silico prediction suggested that this mutation has the potential to affect covalent bond formation of rocelitinib with the C797 residue of EGFR. The potential of E709K or L692V mutations to drive resistance remains to be further explored. Similarly, taking advantage of ctDNA sequencing (with a 20-gene panel), a recent study revealed that patients included in a phase I osimertinib trial developed the C797S resistance mutation in 40% (6/15) of the cases . Such occurrences can be found in the same (in cis) or different (in trans) T790M-mutated alleles of the genome and in some cases the T790M mutation was lost in the tertiary resistant tumour cells. As the EGFR C797S mutation does not induce resistance to first-generation TKIs, re-challenging these T790M-negative tumours with first-generation EGFR-TKIs appears to be an attractive therapeutic opportunity . Interestingly, two recent publications observed an important intra-patient heterogeneity of resistance mechanisms to third-generation TKIs through longitudinal monitoring of ctDNA [47, 49]. In these reported cases, osimertinib resistance was associated with emergence of a C797S mutation along with G796S/R (the homologous amino acid residue with known resistant mutations in others kinases: ALK G1202R or ROS1 G2032R) and/or with L792F/H/G mutations (predicted to induce allosteric interference with osimertinib fixation site ). Noteworthy, these mutations were systematically observed in trans with the C797S-mutated allele and in cis with the T790M-mutated allele. Whether these mutations and their allelic position could influence therapeutic efficacy and strategy in EGFR-mutated NSCLC is an important unanswered issue. Activation of bypass RTK signalling In the clinic, evidence of MET gene amplification has been observed in lung tumours with acquired resistance to third-generation EGFR-TKIs [51, 52]. Experimental data on lung cancer cell lines support the hypothesis that this alteration drives resistance through MAPK/ERK pathway activation, independently of EGFR signalling . MET amplification seems to occur with a relatively high frequency, as it has been identified in 26% (11/43) of cases using ctDNA longitudinal cohorts of patients treated with rocelitinib . EGFR amplification was reported as a classic resistance mechanism to rocelitinib (9%) [35, 53] and a recent publication reported the emergence of a C797S mutation associated with EGFR amplification in an osimertinib-treated patient . Similarly, HER2 amplification was detected at the same rate (9%; 4/43). Two reports of HER2 amplification after osimertinib treatment have been reported [34, 51]. Interestingly, in the longitudinal ctDNA analysis from the rocelitinib phase I/II study, one patient displayed MET plus HER2 gain and another patient harboured MET gain and a C797S EGFR mutation on two distinct tumour lesions . This brings to light the important intra- and inter-tumour heterogeneity of resistance mechanisms that could occur during therapy with third-generation TKIs . Moreover, a publication mentioned the emergence of FGFR1 focal amplification in an osimertinib-resistant tumour after 9 months of treatment . The overexpression of the FGF2 gene was observed in the same sample suggesting occurrence of an autocrine loop-mediated resistance to osimertinib. Taken together, and consistent with preclinical reports, these data suggest that independently of EGFR blockade, bypass RTK signalling activating ERK pathway can occur in NSCLC exposed to third-generation TKIs in the clinical setting. Aberration in downstream signalling pathways Consistent with resistance mechanisms discovered in vitro, multiple cases of RAS/MAPK pathway alteration have been described in patients treated with third-generation EGFR-TKIs. As revealed by recent publications, KRAS mutations can occur in patients treated with rocelitinib and osimertinib: KRAS G12C, G12A, G12S, Q61H and A146T substitutions have been reported [34, 35]. Functional preclinical studies using exogenous expression of a KRAS mutant in EGFR-mutated NSCLC cell lines support this finding . In addition, a publication reported the emergence of a BRAF V600E mutation in response to osimertinib exposure in tumour cells isolated from a pleural effusion, resulting in RAS signalling dependency . Of note, anecdotal observation of MAPK gene overexpression was also reported , but whether such genomic abnormalities could drive resistance to third-generation inhibitors remains to be elucidated. Histological transformation To our knowledge, transformation of non-small cell to small-cell lung cancer in patients manifesting failure of third-generation EGFR-TKI treatment has been reported in four studies [53, 55, 57, 58]. In all studies, biopsies of the tumour progressing after an initial response to osimertinib or rocelitinib reveal expression of neuroendocrine differentiation markers (e.g. chromogranin A, CD56). In most of these cases, good responses were reported with standard chemotherapy approved for small-cell lung cancer. Surprisingly, loss of tumour suppressor gene RB1, recognized as a fundamental step in small-cell lung cancer oncogenesis , was not found in these patient samples . Further mechanistic studies are needed to better understand such specific tumour clonal evolution. Concerning EMT, although no clear evidence has been reported thus far in the clinic for novel EGFR inhibitors, this is a common feature of ALK-addicted lung tumours exposed to new ALK-inhibitors  and we speculate that it may be involved in EGFR-mutant NSCLC too. Understanding the preclinical and clinical data on resistance to third-generation EGFR-TKIs The availability of third-generation EGFR inhibitors represents a major therapeutic advance for the management of EGFR mutant NSCLC. This work reviews numerous studies emphasizing the multiple resistance mechanisms emerging through an EGFR-dependent or independent manner (Figure 1). Resistance mechanisms to third-generation EGFR inhibitors have been approached pooling together the information of the different molecules tested in preclinical and clinical studies. Nevertheless, we acknowledge that the different chemical structure of these compounds  could influence the diversity of tumour strategies to circumvent EGFR inhibition . Figure 1. View largeDownload slide Heterogeneous resistance mechanisms to third-generation EGFR-TKI. The left part of the figure represents EGFR-mediated resistance mechanisms, whereas the right part depicts EGFR-independent ones. Stars illustrate protein mutations, red arrows inhibitory effect and green arrows activating effects. In the pale yellow panels are specified clinicaltrials.gov references of ongoing combination therapy trials (third-generation EGFR inhibitors plus compound in brackets). Every associations are tested with osimertinib. Figure 1. View largeDownload slide Heterogeneous resistance mechanisms to third-generation EGFR-TKI. The left part of the figure represents EGFR-mediated resistance mechanisms, whereas the right part depicts EGFR-independent ones. Stars illustrate protein mutations, red arrows inhibitory effect and green arrows activating effects. In the pale yellow panels are specified clinicaltrials.gov references of ongoing combination therapy trials (third-generation EGFR inhibitors plus compound in brackets). Every associations are tested with osimertinib. The first important observation brought by this review is the crucial added value of preclinical models in predicting resistance mechanisms that can occur in the clinic. For every category of resistance mechanisms identified in preclinical models, tertiary EGFR mutations (C797S, L718Q), bypass (c-MET, HER2) or downstream activation (RAS family mutation and amplification) and histological transformation, the same acquired alterations have been disclosed in the clinic. Indeed, most experimental data of acquired resistance obtained by in vitro prolonged exposure of cell lines to specific inhibitors have been translated into clinical findings . Similarly, several resistance mechanisms identified after first-generation EGFR inhibitors, such as MET and HER2 RTK bypass and histological transformation, have been shown to be relevant mechanisms for third-generation inhibitors. Taken together, the data accumulated on previous generation TKIs and preclinical models can be used to quickly elucidate resistance mechanisms to third-generation EGFR-TKIs and adjust patient treatment. This review also points out the important intra- and inter-tumour heterogeneity observed in patients with acquired resistance to third-generation EGFR inhibitors. Scientists have shown that given an initiating driver event (e.g. EGFR mutations, ALK rearrangements) clones within a tumour do not share the same alterations accounting for resistance, whether in space (between tumour and metastasis) or in time (after subsequent therapy) [61–64], according to a branched evolutionary phylogeny. According to recent works using an in vitro model of high-resolution clones labelling and tracking (ClonTracer), emergence of the resistance to first-generation EGFR-TKIs in EGFR-mutated NSCLC cell lines occur from both types of clones: pre-existing sub-clones selected during the course of therapy and sub-clones acquiring de novo alterations [65, 66]. Early pre-existing resistant clones were predominantly T790M-positive whereas late resistant clones (derived from drug-tolerant cells) acquired mostly EGFR-independent resistance mechanisms. Whether these results could be extrapolated to clonal resistance under third-generation EGFR inhibitors remains to be demonstrated but the variety of escape mechanisms reported in this review strongly outline the need for a better understanding of clonal evolution in T790M-positive tumours. Sequential and multi-site biopsies or liquid biopsies are of particular interest to improve our understanding of the mutational process of clonal evolution. Indeed, an increasing number of studies suggest an expanding value of ctDNA compared with tissue biopsy to monitor tumour evolution under therapy [67–69]. Liquid biopsies are thought to offer a better capture of intra-tumour heterogeneity and represent an attractive alternative to tissue biopsy at the time of resistance to first-generation TKIs . Interestingly, the use of liquid biopsies reveals an unexpected heterogeneity of genomic molecular events leading to EGFR-TKI resistance. Forty-six per cent of patients presented more than one putative resistance mechanism according to ctDNA profiling after first-line treatment with a first-generation EGFR-TKI . Taking advantage of highly sensitive technology (ddPCR, NGS), ctDNA has the potential to accurately represent the population of intra-tumour clones and previous studies focusing on solid biopsies may have underestimated the co-existence of multiple resistance mechanism within the same patient. However, longitudinal validations are still needed to track emergence of resistance to third-generation EGFR inhibitors and to support large application in thoracic oncology routine practice. In the clinic, various trials are testing combination therapies to overcome or postpone resistance to third-generation EGFR inhibitors (Figure 1). Notably, MET inhibition appears promising as in vitro and in vivo evidence showed that it can delay onset of resistance in EGFR T790M-mutated lung cancer models. In the TATTON study, savolitinib (AZD6094, MET inhibitor) showed clinical activity irrespective of T790M status in EGFR-mutant tumours progressing after first-generation TKIs, with an acceptable toxicity profile . The METLUNG phase III study did not show any benefit of onartuzumab (an anti-MET antibody) associated with erlotinib in first-line treatment of EGFR-mutant NSCLC, but included patients with a wide range of MET expression without any c-met gene copy number evaluation . As described in this review, the RAS/MAPK pathway is commonly altered downstream in response to third-generation EGFR-TKIs and MEK inhibitors also delay acquired resistance in combination to third-generation EGFR inhibitors in preclinical models. Consequently, this combination is actually being tested in early clinical trials for T790M-mutated NSCLC (NCT02143466, NCT02580708) (Figure 1). Numerous combination strategies are being tested in early clinical trials, notably concomitant treatments of EGFR-targeted agents with immune checkpoint blockers (ICBs) are being studied in phase I/II trials. Occurrence of grade 3 and 4 interstitial lung disease has been reported in 38% (13/34) of patients exposed to the combination of osimertinib plus durvalumab , warranting further safety evaluation of concomitant use with ICB. Moreover, a recent meta-analysis revealed that EGFR-mutated patients do not derive substantial benefit from ICB monotherapy . Anti-angiogenic agents are of particular interest in the context of EGFR-mutated tumours, as a promising clinical benefit of bevacizumab adjunction to erlotinib in the first-line setting was suggested by the BELIEF phase II study . Whether the combination of anti-angiogenic antibodies with third-generation EGFR-TKIs could provide relevant clinical benefit is being tested in early clinical trials (NCT02803203; NCT02789345). To date, resistance mechanisms to third-generation EGFR-TKIs were mainly observed after prior exposure to first- or second-generation EGFR inhibitors. Whether comparable tumour adaptation will be observed in the case of first line exposure to novel EGFR inhibitors remains unknown. The ongoing FLAURA study comparing osimertinib to gefinitib or erlotinib in this setting will probably provide valuable insights (NCT02296125). Of note, data about the cohort of 60 patients treated upfront in the AURA phase I study of osimertinib has been recently reported . Interestingly, according to plasma sample analyses at the moment of disease progression, putative resistance mechanisms included alterations in genes involved in escape to osimertinib given after early-generation TKIs (MET, EGFR, KRAS amplifications, PIK3CA, KRAS and HER2 activating mutations) (Figure 1). In addition, the emergence of C797S, MEK1, HER2 and JAK3 mutations, whose respective pathway activation may engender resistance to targeted treatments , have also been reported . Importantly, no emergence of EGFR T790M mutation was detected, which is predictable considering the pharmacodynamics of osimertinib. Concerning triple-mutant EGFR NSCLC (activating-mutation/T790M/C797S), new avenues for treatment strategies have been hypothesized from preclinical studies (Figure 2). A new C797S mutant-selective inhibitor EAI045 has shown encouraging preclinical activity in combination with cetuximab, but only in the case of the EGFR L858R activating mutation [78, 79]. According to in vitro experiments, when C797S emerged in trans of the T790M allele, tumours remain sensitive to first- and third-generation EGFR-TKI combinations, whereas tumours remain broadly resistant if C797S emerged in the cis position of T790M allele . A cis allelic position of C797S mutation with the T790M allele was observed in 66% (8/12) of cases (with available information), supporting the clinical relevance of this EGFR-TKI combination strategy (Table 2). A recent publication reported encouraging preclinical activity of brigatinib in combination with cetuximab in triple mutants with EGFR exon19 mutations . Finally, if the EGFR T790M mutation is lost when resistance emerges, it is noteworthy that the EGFR C797S resistance mutation is sensitive to first-generation TKIs. Figure 2. View largeDownload slide Therapeutic perspectives in EGFR triple-mutant tumours. Under therapeutic pressure, sequential mutation acquisition drives resistance to EGFR-TKI. In red are displayed the combination strategies identified according to in vitro studies. Figure 2. View largeDownload slide Therapeutic perspectives in EGFR triple-mutant tumours. Under therapeutic pressure, sequential mutation acquisition drives resistance to EGFR-TKI. In red are displayed the combination strategies identified according to in vitro studies. Discussion Conclusion This review emphasizes the considerable inter- and intra-patient heterogeneity of resistance mechanisms developed by EGFR-mutated tumours in response to third-generation TKIs. In the near future, the use of longitudinal ctDNA monitoring as well as repeated biopsies will be key in capturing the complex clonal evolution occurring in tumours bearing an oncogene addiction. Deepening our understanding of non-genetic resistance mechanisms is crucial to develop novel therapeutic strategies to prolong the survival of patients. Disclosure J-CS has received honoraria from AstraZeneca, Astex, Clovis, GSK, Gammamabs, Lilly, MSD, Mission Therapeutics, Merus, Pfizer, Pharmamar, Pierre Fabre, Roche-Genentech, Sanofi, Servier, Symphogen and Takeda. All remaining authors have declared no conflicts of interest. Funding AstraZeneca has provided a sponsorship grant towards this independent publication. Key Message - Third generation EGFR-TKI inhibitors are among the most promising targeted therapies in oncology in reference to recently published trials showing impressive clinical benefit in EGFR T790M-positive lung cancer.- However, heterogeneous resistance mechanisms always emerged and there is currently a clear need for subsequent therapeutic strategies. - Recapituating and classifying the resistance mechanisms reported experimentally and clinically to date, this review emphazises the considerable intra and interpatient heterogeneity of this phenomenon. - Deepening our understanding of the complex clonal evolution occurring in oncogene-addicted tumors will be key to overcome the acquired resistance to novel TKI inhibitors. References 1 Torre LA, Bray F, Siegel RL et al. Global cancer statistics, 2012. Ca Cancer J Clin 2015; 65( 2): 87– 108. Google Scholar CrossRef Search ADS PubMed 2 Detterbeck FC, Boffa DJ, Tanoue LT. The new lung cancer staging system. 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Annals of Oncology – Oxford University Press
Published: Jan 1, 2018
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