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The significance of epidermal growth factor receptor uncommon mutations in non-small cell lung cancer: A systematic review and critical appraisal

Published:February 21, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101994

      Highlights

      • Uncommon mutations account for 10% of all EGFR mutations.
      • No specific studies have prospectively evaluated uncommon sensitizing mutations.
      • NGS gene panels have improved EGFR mutations detection accuracy.
      • Molecular Tumor Board is mandatory to optimize the treatment strategy for NSCLC.

      Abstract

      Uncommon epidermal growth factor receptor (EGFR) mutations collectively account for 10% of EGFR mutations, harboring heterogeneous molecular alterations within exons 18–21 with clinically variable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced Non-Small Cell Lung Cancer (NSCLC) patients. In addition, with the introduction of different NGS gene approach an improvement of EGFR mutations detection was reported. Today, no specific studies have prospectively evaluated uncommon sensitizing mutations in detail and no firm standard of care has been established in the first-line setting. The aim of this comprehensive review is to critically consider the clinical role of uncommon EGFR mutations highlighting the results of several in vitro and in vivo studies, which singly evaluated the sensitivity of uncommon mutations to currently European of Medicines Agency (EMA)-approved EGFR TKIs in cell lines, xenograft models and humans, in order to obtain a practical guide for refining the clinical decision-making process.

      Keywords

      Introduction

      Despite the increase of therapeutic options available in clinical setting, non-small cell lung cancer (NSCLC) still remains the leading cause of cancer-related death in both sexes worldwide, representing over 85% of all lung cancers [
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer statistics, 2019.
      ]. The clinical knowledge of the epidermal growth factor receptor (EGFR) molecular status, emerging over a decade ago, has led to a dramatic shift in the treatment paradigm of metastatic NSCLC. The prevalence of EGFR mutations ranges from 10% to 15% in Caucasian patients [
      • D'Angelo S.P.
      • Pietanza M.C.
      • Johnson M.L.
      • et al.
      Incidence of EGFR exon 19 deletions and L858R in tumor specimens from men and cigarette smokers with lung adenocarcinomas.
      ] and up to 50% in East-Asian patients [
      • Frol'kis I.V.
      Cellular mechanisms of the activating effect of vasopressin on smooth vascular muscles.
      ], mainly but not only with adenocarcinoma histology, female gender and quite peculiar of no smoker or former smoker patients [
      • Midha A.
      • Dearden S.
      • McCormack R.
      EGFR mutation incidence in non-small-cell lung cancer of adenocarcinoma histology: a systematic review and global map by ethnicity (mutMapII).
      ]. To date, a personalized therapeutic approach based on the detection of activating mutations in the kinase domain of EGFR correlated directly with sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in advanced NSCLC patients, represents the standard of care in the diagnostic setting of NSCLC patients [
      • Russo A.
      • Franchina T.
      • Ricciardi G.R.
      • et al.
      A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): Old successes and future perspectives.
      ]. So far either first- (erlotinib, gefitinib) or second- (afatinib, dacomitinib) or third-generation (osimertinib) EGFR TKIs have received the Food and Drug Administration (FDA) approval as standard first-line treatment options in EGFR mutated patients, leading to improved response and progression-free survival rates in several phase 3 trials [
      • Mok T.S.
      • Wu Y.L.
      • Thongprasert S.
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      ,
      • Rosell R.
      • Carcereny E.
      • Gervais R.
      • et al.
      Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
      ,
      • Sequist L.V.
      • Yang J.C.
      • Yamamoto N.
      • et al.
      Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.
      ,
      • Wu Y.L.
      • Cheng Y.
      • Zhou X.
      • et al.
      Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
      ,
      • Soria J.C.
      • Ohe Y.
      • Vansteenkiste J.
      • et al.
      Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer.
      ]. Most EGFR mutations are strong predictors of response to TKIs with the most “common” type of activating or sensitizing EGFR mutation being the in-frame deletion of exon 19 around the LRE motif (amino acid residues 747–750; ~45% of EGFR mutations), followed by p.L858R point mutation of exon 21 (~40% of EGFR mutations). The remaining 10% of EGFR mutations appeared to harbor heterogeneous molecular alterations within exons 18–21 (so-called “uncommon” mutations) with clinically variable responses to targeted drugs and shorter survival rates when compared to classical mutations [
      • Evans M.
      • O'Sullivan B.
      • Smith M.
      • et al.
      Large-scale EGFR mutation testing in clinical practice: analysis of a series of 18,920 non-small cell lung cancer cases.
      ], even if most Phase III trials excluded patients with these mutations from the analysis to define the clinical outcome (LUX-Lung 2, 3 and 6 being notable exceptions). Although, the introduction of Next Generation Sequencing (NGS) in the clinical setting has broadened the spectrum of detected mutations and medical oncologists may generally agree with the clinical usage of EGFR TKIs in this rare setting, no prospective large trials have prospectively evaluated uncommon sensitizing mutations in detail and no firm standard of care has been established. Specifically, retrospective studies of erlotinib and gefitinib seemed to show inconsistent responses [
      • De Pas T.
      • Toffalorio F.
      • Manzotti M.
      • et al.
      Activity of epidermal growth factor receptor-tyrosine kinase inhibitors in patients with non-small cell lung cancer harboring rare epidermal growth factor receptor mutations.
      ] while a combined post-hoc analysis of phase II-III trials revealed the clinical activity of afatinib in certain type of uncommon EGFR mutations [
      • Yang J.C.
      • Sequist L.V.
      • Geater S.L.
      • et al.
      Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6.
      ]. Even if in the FLAURA study upfront osimertinib has recently provided a statistically and clinically significant meaningful improvement in overall survival (OS) when compared only to first-generation TKIs (gefitinib and erlotinib) in EGFR-positive patients with classical mutations [
      • Cho J.H.
      • Sun J.
      • Lee S.
      • et al.
      An open-label, multicenter, phase II single arm trial of osimertinib in NSCLC patients with uncommon EGFR mutation(KCSG-LU15-09).
      ], small case series of osimertinib has led to equivocal benefit in NSCLC patients harboring different uncommon mutations [
      • Nasu S.
      • Shiroyama T.
      • Morita S.
      • et al.
      Osimertinib treatment was unsuccessful for lung adenocarcinoma with G719S, S768I, and T790M mutations.
      ]. Furthermore, very little is known about potential differences in TKI sensitivity among different EGFR exon19 deletions, even if recent evidences have proved equal sensitivity to first-line EGFR-TKIs [
      • Rossi S.
      • Toschi L.
      • Finocchiaro G.
      • et al.
      Impact of exon 19 deletion subtypes in EGFR-mutant metastatic non-small-cell lung cancer treated with first-line tyrosine kinase inhibitors.
      ].
      Therefore, this systematic review aims to critically evaluate the clinical role of uncommon EGFR mutations in TKI actionability highlighting the results of several in vitro and in vivo studies, which singly evaluated the sensitivity of uncommon mutations to currently European of Medicines Agency (EMA)-approved EGFR TKIs in cell lines, xenograft models and humans. In addition, current evidences towards a better selection of patients who are more likely to benefit from one TKI to another are discussed in order to obtain a practical guide for refining the clinical decision-making process.

      Methods

      We searched for different study types including NSCLC cell lines, xenograft models or in human trials, retrospective or prospective studies, case reports, preclinical researches, and systematic reviews (Fig. 1 – PRISMA plot) regarding uncommon EGFR mutation treated with a first-, second- or third-generation TKI.
      Figure thumbnail gr1
      Fig. 1PRISMA flow diagram showing the selection algorithm of retrieved papers according to the inclusion/exclusion criteria.
      A systematic review was conducted up to December 2019 through Medline (Pubmed) and Cochrane-Library, without language restrictions. Some unpublished studies were searched online and results were obtained from the American Society of Clinical Oncology (ASCO) and the European Society of Medical Oncology (ESMO) conference abstracts, as well as unpublished data or results from ongoing studies available on the National Institute of Health (NIH) website (www.clinicaltrials.gov) were also considered as a source of grey literature. We used a free term strategy, producing the following search string: (“[NSCLC]” AND “[EGFR]” AND “[EGFR TKI]” AND “[EGFR GENE VARIANTS]” AND “[UNCOMMON]” OR “[RARE MUTATIONS]” OR “[MINOR MUTATIONS]”) and references from relevant articles was carried out. Working in duplicate, two different reviewers (V.G. and A.G.) independently reviewed the articles by title and abstract and the full text of potentially significant articles. A third reviewer (U.M.) resolved disagreements between the other reviewers.
      About inclusion criteria, data relative to EGFR mutations in lung cancers were extracted from the Catalogue of Somatic Mutations in Cancer (COSMIC) database, release version 90 (last access 10-8-2019). When considering in vitro and xenograft models studies, the half maximal inhibitory concentration (IC50), which represents the concentration of TKI that is required for 50% inhibition of EGFR viable cells in vitro and is comparable to the half maximal effective concentration in vivo, was evaluated to discuss the different drug sensitivity patterns among the distinct TKIs [
      • Sebaugh J.L.
      Guidelines for accurate EC50/IC50 estimation.
      ]. IC50 values were given and eventually converted in terms of µmol. Mutations were considered to be resistant if the tested TKI showed IC50 values >0.1 µmol whereas considered to be sensitive with IC50 values ≤0.1 µmol [

      Stevens E. Medicinal chemistry: the modern drug discovery process.

      ]. As regards in human studies, progression-free survival (PFS), measured from the first day of TKI treatment until the first objective or clinical sign of disease progression or death, was matched to IC50 values and evaluated to compare the efficacy of specific TKIs among the different subsets of EGFR mutations.

      Results

      Exon 18

      Since EGFR mutations in exon 18 are not fully characterized and have been reported only by a limited number of studies [
      • Wu J.Y.
      • Yu C.J.
      • Chang Y.C.
      • Yang C.H.
      • Shih J.Y.
      • Yang P.C.
      Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer.
      ,
      • Beau-Faller M.
      • Prim N.
      • Ruppert A.M.
      • et al.
      Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network.
      ], it is therefore difficult to draw conclusions as to their true prevalence, molecular spectrum, clinical-pathological features and drug efficacy. Exon 18 mutations collectively account for 3–4% of all EGFR mutations [
      • Kobayashi Y.
      • Togashi Y.
      • Yatabe Y.
      • et al.
      EGFR Exon 18 mutations in lung cancer: molecular predictors of augmented sensitivity to Afatinib or Neratinib as compared with first- or third-generation TKIs.
      ], more commonly including point mutations that encompass a glycine change to serine, alanine or cysteine (p.G719S/A/C in the 97% of cases) within the codon 719, and less frequently involving the codon 709 [
      • Massarelli E.
      • Johnson F.M.
      • Erickson H.S.
      • Wistuba I.I.
      • Papadimitrakopoulou V.
      Uncommon epidermal growth factor receptor mutations in non-small cell lung cancer and their mechanisms of EGFR tyrosine kinase inhibitors sensitivity and resistance.
      ]. Although displaying variable responses to TKIs and being associated with poor prognosis when compared to classic activating mutations [
      • Leduc C.
      • Merlio J.P.
      • Besse B.
      • et al.
      Clinical and molecular characteristics of non-small-cell lung cancer (NSCLC) harboring EGFR mutation: results of the nationwide French Cooperative Thoracic Intergroup (IFCT) program.
      ], patients with p.G719A/C/S point mutations do not appear to be resistant to EGFR TKIs but do exhibit different sensitivity profile. The p.G719S mutation appeared to be less sensitive to gefitinib than erlotinib in preclinical studies [
      • Kancha R.K.
      • von Bubnoff N.
      • Peschel C.
      • Duyster J.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      ], but response and PFS rates with first-generation TKIs did not significantly differ in the clinical setting [
      • Otsuka T.
      • Mori M.
      • Yano Y.
      • et al.
      Effectiveness of tyrosine kinase inhibitors in Japanese patients with non-small cell lung cancer harboring minor epidermal growth factor receptor mutations: results from a Multicenter Retrospective Study (HANSHIN Oncology Group 0212).
      ]. Even if not yet evaluated in clinical studies, a consistently augmented sensitivity of p.G719S to afatinib was rather demonstrated in vitro and xenograft studies while osimertinib showed only intermediate sensitivity [
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      ,
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Masuzawa K.
      • Yasuda H.
      • Hamamoto J.
      • et al.
      Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations.
      ]. Likewise, p.G719C appeared to be sensitive to both first-generation TKIs from preclinical data [
      • Furuyama K.
      • Harada T.
      • Iwama E.
      • et al.
      Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.
      ,
      • Tam I.Y.
      • Leung E.L.
      • Tin V.P.
      • et al.
      Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.
      ] though with improved progression-free and overall survival rates in favor of erlotinib [
      • Klughammer B.
      • Brugger W.
      • Cappuzzo F.
      • et al.
      Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations.
      ], and confirmed to be very sensitive to afatinib with IC50 values in the low micromolar range and, only in xenograft models studies, to osimertinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. In contrast, p.G719A proved to be uniformly resistant to erlotinib and gefitinib in preclinical and clinical studies while showing marked sensitivity in vitro and in xenograft studies to afatinib and, less pronouncedly, to osimertinib [
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      ,
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Conversely, even if more often presenting as complex mutations with sensitizing p.G719S/A/C or p.L858R mutations, the vast majority of point mutations involving codon 709 (p.E709K/A/G/V) were revealed to be homogeneously resistant to first-generation TKIs [
      • Tam I.Y.
      • Leung E.L.
      • Tin V.P.
      • et al.
      Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.
      ,
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ] while demonstrating intense sensitivity to last-generation TKIs taken into account, mostly to afatinib. Intriguingly, p.E709K proved to be more sensitive to afatinib with at least two times lower IC50 values when compared to osimertinib, whereas p.E709A turned out to be very sensitive to afatinib in the low micromolar range and quite insensitive to osimertinib in xenograft studies [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ], confirming the suggested differential activity of TKIs toward specific EGFR activating and resistance mutations.
      Regarding exon 18 deletions, p.E709_T710delinsD has been considered to be the most common, even though it can be missed when using diagnostic commercial kits [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. According to in vitro and xenograft models studies, this deletion appeared to show higher sensitivity to afatinib, even when compared to osimertinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. However, clinical data with afatinib are scant [
      • Kobayashi Y.
      • Togashi Y.
      • Yatabe Y.
      • et al.
      EGFR Exon 18 mutations in lung cancer: molecular predictors of augmented sensitivity to Afatinib or Neratinib as compared with first- or third-generation TKIs.
      ] and only modest objective response rates to first-generation TKIs have been observed in selected case reports (Fig. 2) [
      • Ackerman A.
      • Goldstein M.A.
      • Kobayashi S.
      • Costa D.B.
      EGFR delE709_T710insD: a rare but potentially EGFR inhibitor responsive mutation in non-small-cell lung cancer.
      ].
      Figure thumbnail gr2
      Fig. 2Overview and assessment of EGFR TKIs activity in cell lines (IV), xenograft models (XM) and patients (IH) harboring exon 18 mutations [aexpressed in term of micromolar (µM) concentration, bexpressed in term of median months of progression-free survival (PFS)]. The drug sensitivity was color-coded according to the scheme indicated at the top right and was categorized as sensitive, resistant, controversial or not available based on literature data.

      Exon 19

      As previously discussed, the most frequently identified EGFR mutation category are exon 19 in-frame deletions, so-termed “common” mutations which account for approximately 45% of all EGFR alterations and are collectively known to be highly sensitive to all generations of EGFR-targeted TKIs. Actually, this subgroup of mutations do represent at least 30 different deletions with or without amino acid insertion/substitution, eventually displaying different sensitivity profile in both the preclinical and clinical setting [
      • Tian Y.
      • Zhao J.
      • Ren P.
      • et al.
      Different subtypes of EGFR exon19 mutation can affect prognosis of patients with non-small cell lung adenocarcinoma.
      ].
      The most frequently observed EGFR exon 19 deletion, which is nested around the conserved LRE string leading to elimination of 5 amino acids between residues 746–750 (delE746-A750) [
      • Murray S.
      • Dahabreh I.J.
      • Linardou H.
      • Manoloukos M.
      • Bafaloukos D.
      • Kosmidis P.
      Somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor and tyrosine kinase inhibitor response to TKIs in non-small cell lung cancer: an analytical database.
      ], showed improved objective response and PFS rates to first-generation EGFR TKIs when compared to uncommon exon 19 deletions occurring at different amino acid positions, so-called non-LRE deletions [
      • Chung K.P.
      • Wu S.G.
      • Wu J.Y.
      • et al.
      Clinical outcomes in non-small cell lung cancers harboring different exon 19 deletions in EGFR.
      ]. However, commercially available kits for EGFR testing used in daily practice detect only specific deletions in exon 19 and may miss uncommon deletions. Among these, an interesting finding from Kohsaka et al. study was that both afatinib and osimertinib reduced the viability of cells expressing uncommon exon 19 deletions in xenograft assays with a remarkably augmented sensitivity compared to gefitinib and erlotinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Nevertheless, clinical data directly comparing the efficacy of TKIs among minor deletions of exon 19 are lacking.
      Clinical retrospective data showed that deletions occurred throughout almost the entire exon 19 amino acid string from E746 to D761 involving 16 amino acids with over half of the subtypes being complex with an accompanying insertion [
      • Su J.
      • Zhong W.
      • Zhang X.
      • et al.
      Molecular characteristics and clinical outcomes of.
      ]. Indeed, a number of other exon 19 deletion mutations have been less frequently observed between amino acids 745 and 753, and associated with sensitivity to TKIs [
      • Sharma S.V.
      • Bell D.W.
      • Settleman J.
      • Haber D.A.
      Epidermal growth factor receptor mutations in lung cancer.
      ]. Namely, these mutations seemed to exhibit structural similarities with common exon 19 deletions and often consist of complex insertion-deletions (indels) leading to replacement of the deleted amino acids with a non-native residue (such as the p.L747-A750delinsP where a proline residue results to be introduced in substitution) [
      • Sharma S.V.
      • Bell D.W.
      • Settleman J.
      • Haber D.A.
      Epidermal growth factor receptor mutations in lung cancer.
      ]. Although being associated with in vitro and in vivo sensitivity to first-generation EGFR TKIs and afatinib with lower clinical responses comparing to classical mutation [
      • Lin Y.T.
      • Liu Y.N.
      • Wu S.G.
      • Yang J.C.
      • Shih J.Y.
      Epidermal growth factor receptor tyrosine kinase inhibitor-sensitive exon 19 insertion and exon 20 insertion in patients with advanced non-small-cell lung cancer.
      ], several differences in sensitivity and response to EGFR TKIs among patients with these complex mutations have been reported [
      • Frega S.
      • Lorenzi M.
      • Fassan M.
      • et al.
      Clinical features and treatment outcome of non-small cell lung cancer (NSCLC) patients with uncommon or complex epidermal growth factor receptor (EGFR) mutations.
      ]. Accordingly, p.L747_A750delinsP have shown poorer PFS rates when compared to the classical sensitizing p.E746_A750, and even to the less frequently detected p.L747_P753delinsS which actually demonstrated to be broadly sensitive to all generation TKIs in the preclinical setting [
      • Stewart T.
      • Truini A.
      • DeVeaux M.
      • Zelterman D.
      • Walther Z.
      • Wurtz A.
      Differential outcomes in patients with uncommon EGFR exon 19 mutations.
      ]. No firm conclusions can be drawn since patients harboring complex deletions starting with leucine in position 747 plus insertions had significantly shorter PFS to first-generation TKIs [
      • Kaneda T.
      • Hata A.
      • Tomioka H.
      • et al.
      Possible differential EGFR-TKI efficacy among exon 19 deletional locations in EGFR-mutant non-small cell lung cancer.
      ] while showing no significant differences in PFS and OS to gefitinib in other retrospective series [
      • Sutiman N.
      • Tan S.W.
      • Tan E.H.
      • et al.
      EGFR mutation subtypes influence survival outcomes following first-line Gefitinib therapy in advanced Asian NSCLC patients.
      ]. To date, preclinical findings has recently showed that the p.L747-A750 > P complex mutation is completely inhibited by low doses of afatinib whereas being less sensitive to erlotinib or osimertinib, underlining important differences among specific exon 19 complex deletions that have been also confirmed in the clinical setting [
      • Truini A.
      • Starrett J.H.
      • Stewart T.
      • et al.
      The EGFR Exon 19 mutant L747–A750>P exhibits distinct sensitivity to tyrosine kinase inhibitors in lung adenocarcinoma.
      ]. Moreover, even if certain indel mutations showed a slightly increased sensitivity to first-generation TKIs in some in vitro studies [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ], xenograft studies suggested that these genotypes were particularly responsive to afatinib and osimertinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ], even if few clinical data appeared to favor the only use of afatinib [
      • He M.
      • Capelletti M.
      • Nafa K.
      • et al.
      EGFR exon 19 insertions: a new family of sensitizing EGFR mutations in lung adenocarcinoma.
      ] while retrospective series have recently proved equal sensitivity to first-line EGFR TKIs among certain exon 19 deletion subtypes with further comparable activity of gefitinib and afatinib in this subset of patients (Fig. 3) [
      • Rossi S.
      • Toschi L.
      • Finocchiaro G.
      • et al.
      Impact of exon 19 deletion subtypes in EGFR-mutant metastatic non-small-cell lung cancer treated with first-line tyrosine kinase inhibitors.
      ].
      Figure thumbnail gr3
      Fig. 3Overview and assessment of EGFR TKIs activity in cell lines (IV), xenograft models (XM) and patients (IH) harboring exon 19 mutations [aexpressed in term of micromolar (µM) concentration, b expressed in term of median months of progression-free survival (PFS)]. The drug sensitivity was color-coded according to the scheme indicated at the top right and was categorized as sensitive, controversial or not available based on literature data.

      Exon 20

      Besides classical sensitizing mutations, exon 20 insertions represent the third most frequently detected EGFR mutation accounting for approximately 1.5–2.5% of all NSCLCs and 6% of all EGFR-mutated patients [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ,
      • Sequist L.V.
      • Bell D.W.
      • Lynch T.J.
      • Haber D.A.
      Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer.
      ]. Among uncommon mutations, exon 20 insertions are the most prevalent and heterogeneous group of aberrations. They consist of in-frame insertions or duplications across a span of ~15 amino acids that encompass residues from 761 to 775 with the vast majority being resistant to EGFR TKIs [
      • Vyse S.
      • Huang P.H.
      Targeting.
      ] and experiencing longer PFS with platinum-based chemotherapy in retrospective studies [
      • Oxnard G.R.
      • Lo P.C.
      • Nishino M.
      • et al.
      Natural history and molecular characteristics of lung cancers harboring EGFR exon 20 insertions.
      ]. To date those exon 20 insertions, traditionally considered to be the most prevalent and to confer resistance to all generation EGFR TKIs [
      • Yasuda H.
      • Kobayashi S.
      • Costa D.B.
      EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications.
      ], include: p.A763_Y764insFQEA, p.A767_V769dupASV, p.V769_D770insASV and p.D770_N771insSVD.
      Preclinical evidences suggested that insertions between codons 769 to 775 could lead to drug resistance whereas more proximal codons might have an activation mechanism and structure that more closely resemble those of classical sensitizing EGFR mutations eventually predicting TKI sensitivity [
      • Arcila M.E.
      • Chaft J.E.
      • Nafa K.
      • et al.
      Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas.
      ]. Accordingly, in cell lines p.A763_Y764insFQEA showed sensitivity at lower concentrations than 0.1 µmol/L to erlotinib, in the very low micromolar range to afatinib and osimertinib whereas retaining slight sensitivity only to gefitinib [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. Thus, p.A763_Y764insFQEA appeared to display sensitivity to all approved EGFR TKIs in the preclinical setting and to be associated with improved clinical outcomes following treatment with only second-line erlotinib (median PFS of 17.4 months, median OS of 24 months) [
      • Klughammer B.
      • Brugger W.
      • Cappuzzo F.
      • et al.
      Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations.
      ], claiming for more robust clinical first-line data to establish the best treatment choice even when compared to chemotherapy-based regimens. p.A767_V769dupASV was associated with resistance to first-generation EGFR TKIs in both preclinical [
      • Masuzawa K.
      • Yasuda H.
      • Hamamoto J.
      • et al.
      Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations.
      ] and clinical setting [
      • Yasuda H.
      • Park E.
      • Yun C.H.
      • et al.
      Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.
      ,
      • Wu J.Y.
      • Wu S.G.
      • Yang C.H.
      • et al.
      Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response.
      ] while showing a wide therapeutic window for afatinib and osimertinib in cell lines studies [
      • Yasuda H.
      • Park E.
      • Yun C.H.
      • et al.
      Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.
      ] as well as a lack of response to afatinib in a xenograft assay [
      • Vyse S.
      • Huang P.H.
      Targeting.
      ]. Likewise, despite the need for more solid in vivo data, in vitro findings confirmed that p.V769_D770insASV was not even sensitive to high doses of first-generation TKIs (>3 µmol/L) while demonstrating promising activity especially for afatinib (<0.1 µmol/L) comparing to osimertinib (0.333 µmol/L) [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]; strikingly, osimertinib showed an interesting tumor growth inhibition to a greater degree than afatinib in certain in vivo xenograft models [
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      ], though a similar slight activity for both afatinib and osimertinib (=0.1 µmol/L) was observed in other xenograft models assays expressing the p.V769_D770insASV mutation [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Both preclinical and clinical data seemed to confirm p.D770_N771insSVD as a resistant mutation to first-generation TKIs, even if associated with short PFS and longer overall survival rates have been shown in two patients in the SATURN study [
      • Klughammer B.
      • Brugger W.
      • Cappuzzo F.
      • et al.
      Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations.
      ]; notably, in spite of the absence of clinical evidences in literature, afatinib and osimertinib showed only slight activity [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. Nevertheless, currently missing clinical data are warranted to prospectively investigate the efficacy of all generation TKIs in patients harboring uncommon exon 20 insertions, even if some phase II trials evaluating the role of osimertinib are ongoing (NCT03191149; NCT13414814).
      Regarding exon 20 point mutations, the single p.S768I mutation represents approximately 1% of all EGFR mutations, even if often existing as compound mutations [
      • Zhu X.
      • Bai Q.
      • Lu Y.
      • et al.
      Response to tyrosine kinase inhibitors in lung adenocarcinoma with the rare epidermal growth factor receptor mutation S768I: a retrospective analysis and literature review.
      ]. Although the actual frequency of the complex mutation remains unclear with the single mutation experiencing inferior outcomes compared to tumors with compound p.S768I mutations [
      • O'Kane G.M.
      • Bradbury P.A.
      • Feld R.
      • et al.
      Uncommon EGFR mutations in advanced non-small cell lung cancer.
      ], the majority of available clinical findings reported responses to TKIs in tumors with complex p.S768I mutations. As a matter of fact, the combined post-hoc analysis of LUX-lung 2, 3 and 6 trials reported excellent objective responses and longest survival rates (median PFS of 14.7 months) for patients with p.S768I mutations treated with afatinib, even if seven of eight patients presented with complex mutations [
      • Yang J.C.
      • Sequist L.V.
      • Geater S.L.
      • et al.
      Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6.
      ]. As for first-generation TKIs, preclinical findings seemed to resemble the relative resistance of this point mutation to erlotinib and gefitinib [
      • Kancha R.K.
      • von Bubnoff N.
      • Peschel C.
      • Duyster J.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      ,
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ], consequently resulting in inferior clinical reported outcomes in most retrospective series [
      • Pallan L.
      • Taniere P.
      • Koh P.
      Rare EGFR exon 20 S768I mutation predicts resistance to targeted therapy: a report of two cases.
      ,
      • Leventakos K.
      • Kipp B.R.
      • Rumilla K.M.
      • Winters J.L.
      • Yi E.S.
      • Mansfield A.S.
      S768I mutation in EGFR in patients with lung cancer.
      ]. Furthermore, in vitro and xenograft studies were consistent with clinical reports observed in patients harboring the p.S768I mutation, demonstrating the superiority of afatinib (IC50 = 0.0007 µmol/L) when compared to first-generation TKIs and, to a lesser extent, to osimertinib which retained a satisfactory sensitivity profile (IC50 = 0.049 µmol/L) [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. From the clinical standpoint, in a prospective multicenter phase II trial osimertinib has been only recently associated with a median PFS of 12.3 months in patients with just the p.S768I mutation [
      • Cho J.H.
      • Lim S.H.
      • An H.J.
      • et al.
      Osimertinib for patients with non-small-cell lung cancer harboring uncommon EGFR mutations: a multicenter, open-label, Phase II Trial (KCSG-LU15-09).
      ], although further results of other ongoing phase II prospective clinical trials are awaited (NCT03434418).
      In addition to the drug-induced selection of a threonine-methionine amino acidic substitution at position 790 (T790M) which is thought to be the most common mechanism of resistance to first-generation TKIs [
      • Ko B.
      • Paucar D.
      • Halmos B.
      T790M: revealing the secrets of a gatekeeper.
      ], the acquired exon 20p.C797S point mutation represents the most common resistance mechanism to third-generation TKIs [
      • Thress K.S.
      • Paweletz C.P.
      • Felip E.
      • et al.
      Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M.
      ]. Based on preclinical models, the single p.C797S mutation appeared to confer resistance to most of irreversible TKIs, affecting the cysteine residue at position 797 used by these drugs to covalently bind with the receptor [
      • Yu Z.
      • Boggon T.J.
      • Kobayashi S.
      • et al.
      Resistance to an irreversible epidermal growth factor receptor (EGFR) inhibitor in EGFR-mutant lung cancer reveals novel treatment strategies.
      ,
      • Avizienyte E.
      • Ward R.A.
      • Garner A.P.
      Comparison of the EGFR resistance mutation profiles generated by EGFR-targeted tyrosine kinase inhibitors and the impact of drug combinations.
      ]. In vitro studies seemed to be consistent with the available xenograft models assays, confirming to be associated with complete resistance to osimertinib (IC50 = 5 µmol/L) while showing only slight sensitivity to first-generation TKIs and afatinib (IC50 = 0.5 µmol/L) [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. As suggested by previous in vitro evidences showing that first-generation EGFR TKIs seemed to exert their efficacy independent of the cysteine at position 797 [
      • Niederst M.J.
      • Hu H.
      • Mulvey H.E.
      • et al.
      The allelic context of the C797S mutation acquired upon treatment with third-generation EGFR inhibitors impacts sensitivity to subsequent treatment strategies.
      ], it is noteworthy that a combination of first- and third-generation EGFR TKIs resulted to be more clinically effective only when p.C797S and p.T790M mutations have been detected in trans [
      • Arulananda S.
      • Do H.
      • Musafer A.
      • Mitchell P.
      • Dobrovic A.
      • John T.
      Combination Osimertinib and Gefitinib in C797S and T790M EGFR-mutated non-small cell lung cancer.
      ,
      • Wang Z.
      • Yang J.J.
      • Huang J.
      • et al.
      Lung adenocarcinoma harboring EGFR T790M and in trans C797S responds to combination therapy of first- and third-generation EGFR TKIs and shifts allelic configuration at resistance.
      ], even if clear clinical outcomes are currently not available. Intriguingly, the definition of trans configuration was related to the location of mutations that, however, could exist on different DNA strands occurring either within different alleles of the same clone or in different tumor cell clones, ultimately leading to different clinical scenarios [
      • Leone A.
      C797S and T790M EGFR mutations in non-small cell lung cancer. In trans or in separate clones?.
      ]. On the other hand, if mutations were detected in cis, the EGFR activity appeared to be suppressed neither by the EGFR TKIs alone nor in combination (Fig. 4) [
      • Del Re M.
      • Crucitta S.
      • Gianfilippo G.
      • et al.
      Understanding the mechanisms of resistance in.
      ].
      Figure thumbnail gr4
      Fig. 4Overview and assessment of EGFR TKIs activity in cell lines (IV), xenograft models (XM) and patients (IH) harboring exon 20 mutations [aexpressed in term of micromolar (µM) concentration, bexpressed in term of median months of progression-free survival (PFS)]. The drug sensitivity was color-coded according to the scheme indicated at the top right and was categorized as sensitive, resistant, controversial or not available based on literature data.

      Exon 21

      The most commonly identified EGFR mutation occurring within exon 21 includes the point mutation which substitutes arginine for Leucine at codon 858 (p.L858R). However, a heterogeneous group of other non-classical EGFR point mutations may occur besides L858R and have been collectively associated with decreased sensitivity and poor clinical outcomes to first-generation TKIs in both preclinical and clinical studies [
      • Pilotto S.
      • Rossi A.
      • Vavalà T.
      • et al.
      Outcomes of first-generation EGFR-TKIs against non-small-cell lung cancer harboring uncommon EGFR mutations: a post hoc analysis of the BE-POSITIVE study.
      ,
      • Banno E.
      • Togashi Y.
      • Nakamura Y.
      • et al.
      Sensitivities to various epidermal growth factor receptor-tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: what is the optimal epidermal growth factor receptor-tyrosine kinase inhibitor?.
      ].
      The p.L861Q mutation represents the second most frequent mutation occurring within the exon 21, accounting for approximately 1–2% of all EGFR mutations and not rarely presenting as complex mutation. Despite preclinical data suggested a decreased sensitivity to gefitinib and erlotinib in both in vitro [
      • Kancha R.K.
      • von Bubnoff N.
      • Peschel C.
      • Duyster J.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      ] and xenograft studies [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ], retrospective data showed that some patients harboring the p.L861Q mutation exhibited a modest response to first-generation EGFR TKI treatment [
      • Otsuka T.
      • Mori M.
      • Yano Y.
      • et al.
      Effectiveness of tyrosine kinase inhibitors in Japanese patients with non-small cell lung cancer harboring minor epidermal growth factor receptor mutations: results from a Multicenter Retrospective Study (HANSHIN Oncology Group 0212).
      ]. On the other hand, available clinical data with afatinib appeared to corroborate preclinical findings of broad activity, considering that the in vitro sensitivity of this mutation in the low micromolar range showed no significant differences in IC50 values comparing to osimertinib while being, more relevantly, consistent with improved PFS as well as OS rates [
      • Yang J.C.
      • Sequist L.V.
      • Geater S.L.
      • et al.
      Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6.
      ]. Moreover, clinical data investigating the role of osimertinib in such patients have recently showed high response rates along with interesting PFS rates of 15.2 months [
      • Cho J.H.
      • Lim S.H.
      • An H.J.
      • et al.
      Osimertinib for patients with non-small-cell lung cancer harboring uncommon EGFR mutations: a multicenter, open-label, Phase II Trial (KCSG-LU15-09).
      ], confirming its in vitro efficacy [
      • Kobayashi Y.
      • Mitsudomi T.
      Not all epidermal growth factor receptor mutations in lung cancer are created equal: perspectives for individualized treatment strategy.
      ]. Other more rarely detected gene variants may occur within the exon 21 and include point mutations that generally appeared to be much more sensitive to afatinib and osimertinib (Fig. 5), even if clinical data are missing and first-generation TKIs were revealed to be slightly active in the prclinical setting (for instance, in p.A864T cell lines [
      • Kancha R.K.
      • von Bubnoff N.
      • Peschel C.
      • Duyster J.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      ] and in p.L861R xenograft models assays) [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ].
      Figure thumbnail gr5
      Fig. 5Overview and assessment of EGFR TKIs activity in cell lines (IV), xenograft models (XM) and patients (IH) harboring exon 21 mutations [aexpressed in term of micromolar (µM) concentration, bexpressed in term of median months of progression-free survival (PFS)]. The drug sensitivity was color-coded according to the scheme indicated at the top right and was categorized as sensitive, resistant, controversial or not available based on literature data.

      Complex mutations

      Complex EGFR mutations, commonly defined as compound or double or multiple mutations in the EGFR tyrosine kinase domain, have been frequently detected by NGS but their clinical value remain ambiguous with variable and not fully elucidated responses to TKIs [
      • Kobayashi S.
      • Canepa H.M.
      • Bailey A.S.
      • et al.
      Compound EGFR mutations and response to EGFR tyrosine kinase inhibitors.
      ].
      The prevalence of detection rate of compound EGFR mutations has increased from 4% to 14% over the past years [
      • Wu J.Y.
      • Yu C.J.
      • Chang Y.C.
      • Yang C.H.
      • Shih J.Y.
      • Yang P.C.
      Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer.
      ,
      • Kobayashi S.
      • Canepa H.M.
      • Bailey A.S.
      • et al.
      Compound EGFR mutations and response to EGFR tyrosine kinase inhibitors.
      ]. More recently, with the introduction of NGS analysis, the identification of complex mutations was reported in up to 25% of the EGFR-mutated population [
      • Kim E.Y.
      • Cho E.N.
      • Park H.S.
      • et al.
      Compound EGFR mutation is frequently detected with co-mutations of actionable genes and associated with poor clinical outcome in lung adenocarcinoma.
      ], suggesting that the frequency of these mutations is other than insignificant. In most cases these mutations consist of one classical sensitizing mutation together with a rare partner mutation of unknown clinical significance or an atypical uncommon mutation [
      • Kim E.Y.
      • Cho E.N.
      • Park H.S.
      • et al.
      Compound EGFR mutation is frequently detected with co-mutations of actionable genes and associated with poor clinical outcome in lung adenocarcinoma.
      ]. The sensitivity pattern of the accompanying mutation seemed to significantly affect the efficacy of the TKI in most compound mutations [
      • Tam I.Y.
      • Leung E.L.
      • Tin V.P.
      • et al.
      Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.
      ].
      Recent preclinical evidences seemed to suggest that second-generation TKIs such as afatinib may have a broader inhibitory profile against EGFR compound mutations than first- or even third-generation TKIs, even if, as expected, less effective than osimertinib in T790M mutations [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Indeed, preclinical evidences suggested the association of gefitinib and erlotinib with significantly diminished inhibitory activity against certain emerging complex mutations (e.g. p.L858R + p.E709A/G/K) which actually resulted to be strongly inhibited in the low micromolar range by afatinib and, less markedly, osimertinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Moreover, the in vitro characterization p.S768I + p.G719A was confirmed in xenograft models assays, resulting to be resistant to first-generation TKIs also in the rare clinical setting [
      • Wu J.Y.
      • Wu S.G.
      • Yang C.H.
      • et al.
      Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response.
      ] and highly sensitive to afatinib, while being only slightly inhibited by osimertinib [
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ]. Clinical retrospective data investigating the efficacy of gefitinib in NSCLC patients harboring complex mutations seemed to corroborate the preclinical findings, demonstrating a median PFS of 6.0 and 10.0 months in patients with p.L858R + p.S768I and p.L858R + p.E709K, respectively, while even shorter survival rates in patient presenting with p.G719S/A/C mutations in combination with other atypical EGFR gene alterations were observed [
      • Peng L.
      • Song Z.G.
      • Jiao S.C.
      Efficacy analysis of tyrosine kinase inhibitors on rare non-small cell lung cancer patients harboring complex EGFR mutations.
      ]. Among all the uncommon gene alterations, these compound mutations seemed to be associated with improved clinical outcomes, even if a high degree of heterogeneity plays a crucial role in it [
      • Kim E.Y.
      • Cho E.N.
      • Park H.S.
      • et al.
      Compound EGFR mutation is frequently detected with co-mutations of actionable genes and associated with poor clinical outcome in lung adenocarcinoma.
      ]. Other retrospective patient series suggested that complex mutations containing the classical sensitizing p.L858R or exon 19 deletions showed poorer PFS rates when treated with first-generation TKIs and compared to single mutations (median PFS of 5.3 vs 8.0 months, respectively) [
      • Wu J.Y.
      • Yu C.J.
      • Chang Y.C.
      • Yang C.H.
      • Shih J.Y.
      • Yang P.C.
      Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer.
      ], whereas in the study by Peng et al. no significant differences in PFS and OS rates between NSCLC patients with single mutation involving Leucine in position 858 and in association with other exon 18–21 co-mutations were observed with gefitinib (Table 1) [
      • Peng L.
      • Song Z.
      • Jiao S.
      Comparison of uncommon EGFR exon 21 L858R compound mutations with single mutation.
      ].
      Table 1Overview of EGFR TKIs activity in cell lines (IV), xenograft models (XM) and patients (IH) harboring complex mutations.
      EGFR exonNucleotide variantProtein changeGefitinibErlotinibAfatinibOsimertinib
      IV
      Expressed in term ofmicromolar (µM) concentration.
      XM
      Expressed in term ofmicromolar (µM) concentration.
      IH
      Expressed in term of median months of progression-free survival (PFS)].
      IV
      Expressed in term ofmicromolar (µM) concentration.
      XM
      Expressed in term ofmicromolar (µM) concentration.
      IH
      Expressed in term of median months of progression-free survival (PFS)].
      IV
      Expressed in term ofmicromolar (µM) concentration.
      XM
      Expressed in term ofmicromolar (µM) concentration.
      IH
      Expressed in term of median months of progression-free survival (PFS)].
      IV
      Expressed in term ofmicromolar (µM) concentration.
      XM
      Expressed in term ofmicromolar (µM) concentration.
      IH
      Expressed in term of median months of progression-free survival (PFS)].
      18

      +

      18
      c.2126A > C

      +

      c.2155G > T
      p.E709A

      +

      p.G719C
      0.032



      0.512
      • Tam I.Y.
      • Leung E.L.
      • Tin V.P.
      • et al.
      Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      12.0
      • Klughammer B.
      • Brugger W.
      • Cappuzzo F.
      • et al.
      Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      18

      +

      18
      c.2126A > C

      +

      c.2155G > A
      p.E709A

      +

      p.G719S
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      8.0
      • Peng L.
      • Song Z.G.
      • Jiao S.C.
      Efficacy analysis of tyrosine kinase inhibitors on rare non-small cell lung cancer patients harboring complex EGFR mutations.
      -8.3
      • Klughammer B.
      • Brugger W.
      • Cappuzzo F.
      • et al.
      Examining treatment outcomes with erlotinib in patients with advanced non-small cell lung cancer whose tumors harbor uncommon EGFR mutations.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      18

      +

      21
      c.2155G > A

      +

      c.2582 T > A
      p.G719S

      +

      p.L861Q
      32.0
      • Otsuka T.
      • Mori M.
      • Yano Y.
      • et al.
      Effectiveness of tyrosine kinase inhibitors in Japanese patients with non-small cell lung cancer harboring minor epidermal growth factor receptor mutations: results from a Multicenter Retrospective Study (HANSHIN Oncology Group 0212).
      0.1–0.05
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      0.005
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      18

      +

      21
      c.2126A > C

      +

      c.2573 T > G
      p.E709A

      +

      p.L858R
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      18

      +

      21
      c.2126A > C

      +

      c.2573 T > G


      p.E709A

      +

      p.L858R

      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.01
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      18

      +

      21
      c.2126A > G +

      c.2573 T > G


      p.E709G

      +

      p.L858R

      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      18

      +

      21
      c.2125G > A + c.2573 T > Gp.E709K

      +

      p.L858R
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      10.0
      • Malapelle U.
      • Sirera R.
      • Jantus-Lewintre E.
      • et al.
      Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer.


      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      18
      c.2303G > T

      +

      c.2156G > C
      p.S768I

      +

      p.G719A
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      1.2
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      5.0–7.0
      • Peng L.
      • Song Z.G.
      • Jiao S.C.
      Efficacy analysis of tyrosine kinase inhibitors on rare non-small cell lung cancer patients harboring complex EGFR mutations.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      18
      c.2303G > T

      +

      c.2155G > T
      p.S768I

      +

      p.G719C




      0.01
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.






      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.






      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.






      0.001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.


      20

      +

      18
      c.2303G > T

      +

      c.2155G > T
      p.S768I

      +

      p.G719C
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.01
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      18
      c.2303G > T

      +

      c.2155G > A
      p.S768I

      +

      p.G719S
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      18
      c.2303G > T

      +

      c.2155G > A
      p.S768I

      +

      p.G719S
      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      20
      c.2369C > T

      +

      c.2390G > C
      p.T790M

      +

      p.C797S
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      20

      +

      21
      c.2303G > T

      +

      c.2573 T > G
      p.S768I

      +

      p.L858R
      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      6.0
      • Malapelle U.
      • Sirera R.
      • Jantus-Lewintre E.
      • et al.
      Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer.
      -21.7
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      0.05
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      21

      +

      18
      c.2582 T > A

      +

      c.2156G > C
      p.L861Q

      +

      p.G719A
      0.1
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.


      0.1–0.05
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.


      0.05
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      0.005
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      0.0005
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      0.005
      • Floc'h N.
      • Martin M.J.
      • Riess J.W.
      • et al.
      Antitumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 insertions.
      21

      +

      21
      c.2582 T > A

      +

      c.2573 T > G
      p.L861Q

      +

      p.L858R
      0.01
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0001
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      21

      +

      21
      c.2582 T > A

      +

      c.2573 T > G
      p.L861Q

      +

      p.L858R
      >0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.1
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.0005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      0.005
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      a Expressed in term of micromolar (µM) concentration.
      b Expressed in term of median months of progression-free survival (PFS)].

      Discussion

      In this review, we summarized and critically discussed in vitro and in vivo data to provide a comprehensive overview of incidence, sensitivity pattern and outcomes of less frequently detected EGFR mutations in both preclinical and clinical setting of advanced non-small cell lung cancer. Despite these populations of EGFR mutations have been collectively named “uncommon” to be distinguished from the classical sensitizing mutations with well-described clinical significance, we may consider them as “untested” in the light of the underestimated incidence obtained even with the most commonly FDA-approved Real–Time polymerase chain reaction (PCR)-based assays used in clinical trials [
      • O'Kane G.M.
      • Bradbury P.A.
      • Feld R.
      • et al.
      Uncommon EGFR mutations in advanced non-small cell lung cancer.
      ,
      • Tortorici S.
      • Difalco P.
      • Caradonna L.
      • Tetè S.
      Traditional endodontic surgery versus modern technique: a 5-year controlled clinical trial.
      ].
      Testing of inadequate and low-quality tissue sample as well as the heterogeneity in detection techniques has fatally resulted in inaccuracies and biases in the reported incidence of less common EGFR mutations, even in pivotal and practice-changing phase III randomized clinical trials [
      • O'Kane G.M.
      • Bradbury P.A.
      • Feld R.
      • et al.
      Uncommon EGFR mutations in advanced non-small cell lung cancer.
      ]. Indeed, the wide use of PCR-based commercial assays such as Therascreen (Qiagen Manchester, UK) or Cobas (Roche, Basel, Switzerland), which only considered “hot spots” thought predictive of TKI response, have posed significant diagnostic issues of detection sensitivity, even in a recent prospective post-hoc analysis that was precisely specified to evaluate the clinical activity of a second-generation TKI in patients harboring EGFR minor mutations [
      • Yang J.C.
      • Sequist L.V.
      • Geater S.L.
      • et al.
      Clinical activity of afatinib in patients with advanced non-small-cell lung cancer harbouring uncommon EGFR mutations: a combined post-hoc analysis of LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6.
      ,
      • Malapelle U.
      • Sirera R.
      • Jantus-Lewintre E.
      • et al.
      Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer.
      ]. For instance, the exon 18 “G719X mutation”, which have been extensively reported in literature in several preclinical and clinical series, actually referred to point mutations, leading to substitutions of the glycine at position 719 to other amino acidic residues (S/A/C) and consequently resulting in different in vitro and in vivo TKI actionability [
      • Kimura S.
      • Tanaka K.
      • Harada T.
      • et al.
      Sensitivity of epidermal growth factor receptor with single or double uncommon mutations to afatinib confirmed by a visual assay.
      ,
      • Kohsaka S.
      • Nagano M.
      • Ueno T.
      • et al.
      A method of high-throughput functional evaluation of.
      ,
      • Masuzawa K.
      • Yasuda H.
      • Hamamoto J.
      • et al.
      Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations.
      ,
      • Furuyama K.
      • Harada T.
      • Iwama E.
      • et al.
      Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.
      ,
      • Tam I.Y.
      • Leung E.L.
      • Tin V.P.
      • et al.
      Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.
      ]. More recently, given the need for a more comprehensive gene profiling in NSCLC metastatic setting, different NGS gene panels have been implemented into clinical practice, increasing significantly the reference range in term of gene regions coverage and limit of detection while leading to the improvement of EGFR mutations detection [
      • Pepe F.
      • De Luca C.
      • Smeraglio R.
      • et al.
      Performance analysis of SiRe next-generation sequencing panel in diagnostic setting: focus on NSCLC routine samples.
      ]. However, despite being present in at least 10% of patients with EGFR mutation-positive NSCLC because of recent improvements in detection methods, less common mutations have been excluded in most phase III trials, like the FLAURA study, evaluating the clinical efficacy of TKIs in advanced EGFR-mutated NSCLC patients [
      • Mok T.S.
      • Wu Y.L.
      • Thongprasert S.
      • et al.
      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
      ,
      • Rosell R.
      • Carcereny E.
      • Gervais R.
      • et al.
      Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.
      ,
      • Sequist L.V.
      • Yang J.C.
      • Yamamoto N.
      • et al.
      Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.
      ,
      • Wu Y.L.
      • Cheng Y.
      • Zhou X.
      • et al.
      Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
      ,
      • Soria J.C.
      • Ohe Y.
      • Vansteenkiste J.
      • et al.
      Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer.
      ]. Furthermore, even if several randomized clinical trials (LUX-Lung 7, FLAURA, ARCHER-1050) have demonstrated that second- and third-generation TKIs have led to improved PFS rates when compared to first-generation TKIs in a front-line setting, until recently no head-to-head comparisons between the different generations of TKIs have been conducted in patients with less common EGFR mutations [
      • Kohsaka S.
      • Petronczki M.
      • Solca F.
      • Maemondo M.
      Tumor clonality and resistance mechanisms in EGFR mutation-positive non-small-cell lung cancer: implications for therapeutic sequencing.
      ].
      When systematically reviewing the present literature, we observed that in vitro and xenograft studies seemed to resemble the mixed and mostly retrospective data available regarding the activity of all the EMA-approved EGFR TKIs in tumors harboring less frequently detected activating mutations. Even if clinical data with second- and third-generation TKIs are scant, exon 18p.E709K/A/G/V mutations appeared to be suitable for afatinib whereas p.G719A/C/S point mutations showed varying drug sensitivities and clinical responses to EGFR TKIs, depending on the precise amino acid substitution; in particular, p.G719C appeared to be sensitive to any type of EGFR TKIs whereas p.G719A was strongly inhibited by afatinib, thereby underlining the strong importance of very specific techniques in the diagnostic setting. Activating indel alterations within exon 19 appeared to be resistant to first-generation TKIs while retaining a remarked sensitivity to both afatinib and osimertinib. Exon 20 insertions confirmed their wide variability, showing interesting preclinical activity in favor of afatinib and osimertinib in some specific cases that, however, still need to be validated in ongoing prospective clinical trial. The most prevalent exon 20 mutations, including insertions and point mutations, were confirmed to be resistant to first-generation TKIs while afatinib and osimertinib showed promising preclinical activity, especially for p.A763_Y764insFQEA. As concerns exon 21, the p.L861Q point mutation exhibited a modest response to first-generation EGFR TKI treatment while showing remarkable preclinical and clinical prospective data in favor of afatinib and osimertinib. Regarding compound mutations, the insufficient and anecdotal available data seemed to be controversial, suggesting that the sensitivity pattern of the accompanying mutation seemed to significantly influence the TKI efficacy. However, afatinib (and to a lesser extent osimertinib) retained a strong preclinical activity against certain emerging complex mutations such as p.L858R + p.E709A/G/K or p.S768I + p.G719A. The trans and cis configurations of compound mutations, especially for the p.C797S mutation, has resulted to be another relevant point of discussion leading to different patterns of sensitivity and resistance, respectively, since co-existing in cis mutations likely appeared to have an additive impact on the tertiary structure of EGFR underlying their role in terms of acquired resistance [
      • Kohsaka S.
      • Petronczki M.
      • Solca F.
      • Maemondo M.
      Tumor clonality and resistance mechanisms in EGFR mutation-positive non-small-cell lung cancer: implications for therapeutic sequencing.
      ]. In summary, considering the high molecular heterogeneity, the relative paucity of clinical data and the lack of formal statistical comparisons, such observations might be valuable options for clinical practice, strongly encouraging the implementation of a NGS-based approach to specifically characterize the EGFR mutational status in NSCLC patients [
      • Pepe F.
      • De Luca C.
      • Smeraglio R.
      • et al.
      Performance analysis of SiRe next-generation sequencing panel in diagnostic setting: focus on NSCLC routine samples.
      ,
      • Tortorici S.
      • Burruano F.
      • Buzzanca M.L.
      • Difalco P.
      • Cabibi D.
      • Maresi E.
      Cervico-facial actinomycosis: epidemiological and clinical coomments.
      ].
      In this fascinating and very complex landscape and in absence of head-to-head prospective randomized data, the role of such observations reported in this review, entrusted with the management of a multidisciplinary team (Molecular Tumor Board), may represent the key weapon to better define and optimize the treatment strategy for NSCLC patients harboring less frequently detected EGFR mutations.
      Funding
      The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

      CRediT authorship contribution statement

      Valerio Gristina: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing - original draft. Umberto Malapelle: Conceptualization, Investigation, Supervision, Visualization, Writing - review & editing. Antonio Galvano: Writing - review & editing, Formal analysis, Investigation, Methodology, Software, Writing - original draft. Pasquale Pisapia: Writing - original draft, Investigation, Software. Francesco Pepe: Writing - original draft, Investigation, Software, Writing - original draft. Christian Rolfo: Supervision, Validation, Visualization, Writing - review & editing. Silvia Tortorici: Supervision, Validation, Visualization, Writing - review & editing. Viviana Bazan: Methodology, Supervision, Validation, Writing - review & editing. Giancarlo Troncone: Conceptualization, Project administration, Resources, Supervision, Validation, Visualization, Writing - review & editing. Antonio Russo: Conceptualization, Project administration, Resources, Supervision, Validation, Visualization, Writing - review & editing.

      Declaration of Competing Interest

      Dr. Malapelle reports personal fees from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen, Merck, BMS for participation in a speaker bureau; personal fees from Boehringer Ingelheim, MSD, Amgen, Merck, BMS for acting in an advisory role, financial support from Boehringer Ingelheim and Amgen that was paid directly to his institution outside the submitted work. Prof. Rolfo reports personal fees from Merck Sharp and Dohme, Astrazeneca, for participation in a speaker bureau; personal fees from Mylan, Archer, Merck Serono, Inivata for acting in an advisory scientific role; and has research collaborations with Biomarkers and Guardant Health outside the submitted work. Prof. Troncone reports personal fees from Roche for participation in a speaker bureau; personal fees from MSD, Pfizer for acting in an advisory role. The remaining authors declare no potential conflicts of interest.

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