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Biological and clinical perspectives of the actionable gene fusions and amplifications involving tyrosine kinase receptors in lung cancer

  • Maria Saigí
    Correspondence
    Corresponding authors Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain (M. Saigí). Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain. (M. Sanchez-C.)
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Enric Carcereny
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Teresa Morán
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Marc Cucurull
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Marta Domènech
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Ainhoa Hernandez
    Affiliations
    Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain

    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Anna Martinez-Cardús
    Affiliations
    Badalona·Applied Research Group in Oncology (B·ARGO). Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain
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  • Eva Pros
    Affiliations
    Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain
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  • Montse Sanchez-Cespedes
    Correspondence
    Corresponding authors Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain (M. Saigí). Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain. (M. Sanchez-C.)
    Affiliations
    Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain
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Open AccessPublished:June 18, 2022DOI:https://doi.org/10.1016/j.ctrv.2022.102430

      Highlights

      • Gene fusions and amplifications often activate receptor tyrosine kinases in lung cancer.
      • Next-generation sequencing is the gold standard for molecular diagnosis in lung cancer.
      • Genomic tests may not capture the root mechanism of resistance to targeted therapies.
      • Therapeutic options for the least prevalent fusions (e.g., NTRK, NRG1) are expanding.

      Abstract

      Identifying molecular oncogenic drivers is crucial for precision oncology. Genetic rearrangements, including gene fusions and gene amplification, involving and activating receptor tyrosine kinases (RTKs) are recurrent in solid tumors, particularly in non-small cell lung cancer. Advances in the tools to detect these alterations have deepened our understanding of the underlying biology and tumor characteristics and have prompted the development of novel inhibitors targeting activated RTKs. Nowadays, druggable oncogenic rearrangements are found in around 15% of lung adenocarcinomas. However, taken separately, each of these alterations has a low prevalence, which poses a challenge to their diagnosis. The identification and characterization of novel targetable oncogenic rearrangements in lung cancer continue to expand, as shown by the recent discovery of the CLIP1-LTK fusion found in 0.4% of lung adenocarcinomas. While tyrosine kinase inhibitors that block the activity of RTKs have represented a breakthrough in the therapeutic landscape by improving the prognosis of this disease, prolonged treatment inevitably leads to the development of acquired resistance. Here, we review the oncogenic fusions and gene amplifications involving RTK in lung cancer. We address the genetic and molecular structure of oncogenic RTKs and the methods to diagnose them, emphasizing the role of next-generation sequencing technologies. Furthermore, we discuss the therapeutic implications of the different tyrosine kinase inhibitors, including the current clinical trials and the mechanisms responsible for acquired resistance. Finally, we provide an overview of the use of liquid biopsies to monitor the course of the disease.

      Keywords

      Introduction

      Precision oncology has revolutionized the therapeutic landscape of advanced non-small cell lung cancer (NSCLC). Over the last two decades, the deep genomic profiling of lung cancers has enabled identifying many new genes involved in the development of this disease, including oncogenic drivers that have contributed to our understanding of lung carcinogenesis and have established an era of precision medicine in advanced-stage NSCLC [
      • Campbell J.D.
      • Alexandrov A.
      • Kim J.
      • Wala J.
      • Berger A.H.
      • Pedamallu C.S.
      • et al.
      Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas.
      ,
      • Schram A.M.
      • Chang M.T.
      • Jonsson P.
      • Drilon A.
      Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance.
      ].
      Tumors often have complex and unstable genomes that cause random mutations and genomic aberrations. While most of these alterations are unlikely to affect tumor development, some may involve essential genes that contribute to the oncogenic process. For instance, chromosomal rearrangements could result in gene fusions or gene amplifications that lead to the expression of oncoproteins. When the fusion or amplification involves a receptor tyrosine kinase (RTK), the tyrosine kinase domain (TKD) is activated—often constitutively and ligand-independent—and downstream effectors of the receptor receive constant signaling, causing uncontrolled cell growth and invasiveness [
      • Campbell J.D.
      • Alexandrov A.
      • Kim J.
      • Wala J.
      • Berger A.H.
      • Pedamallu C.S.
      • et al.
      Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas.
      ,
      • Schram A.M.
      • Chang M.T.
      • Jonsson P.
      • Drilon A.
      Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance.
      ]. Then, the tumor cell becomes dependent on this oncogenic RTK to maintain its malignant properties. This dependency, also called “oncogene addiction,” can be therapeutically approached with drugs that inhibit the activity of the oncoprotein. Currently, most RTKs inhibitors are designed to prevent either the binding of the ligand—often by using monoclonal or bispecific antibodies, and antibody-drug conjugates—or the binding of the ATP to the catalytic domain—mostly with small molecules. Among the latter, tyrosine kinase inhibitors (TKIs), which inhibit the catalytic domain of the target enzyme by preventing phosphorylation and subsequent signaling, are the most widely used in clinics [

      Jordan EJ, Kim HR, Arcila ME, Barron D, Chakravarty D, Gao J, et al. Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov 2017;7(6):596–609. doi: 10.1158/2159-8290.CD-16-1337.

      ]. On the other hand, other personalized therapeutic strategies include immune targeted approaches such as checkpoint blockade, personalized vaccines and/or chimeric antigen receptor T-cells [
      • Tsimberidou A.M.
      • Fountzilas E.
      • Nikanjam M.
      • Kurzrock R.
      Review of precision cancer medicine: Evolution of the treatment paradigm.
      ]. Slightly more than a dozen RTKs are genetically activated in NSCLC. Although gene fusions and gene amplifications are the most common mechanisms of RTKs’ activation, point mutations can also occur, as in MET, ERBB2, and EGFR (the latter is the most frequently RTK activated in lung cancer) [
      • Schram A.M.
      • Chang M.T.
      • Jonsson P.
      • Drilon A.
      Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance.
      ,

      Jordan EJ, Kim HR, Arcila ME, Barron D, Chakravarty D, Gao J, et al. Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov 2017;7(6):596–609. doi: 10.1158/2159-8290.CD-16-1337.

      ]. For still unknown reasons, activated RTKs prevail in lung cancer adenocarcinomas (LuADs) of relatively young females, since they are related to lower tobacco-exposure (light- or never-smokers)These patients are often diagnosed with advanced-stage cancer and have high incidences of pleural effusion and sclerotic bone and brain metastases as common patterns of tumor spread [
      • Sacher A.G.
      • Dahlberg S.E.
      • Heng J.
      • Mach S.
      • Jänne P.A.
      • Oxnard G.R.
      Association Between Younger Age and Targetable Genomic Alterations and Prognosis in Non-Small-Cell Lung Cancer.
      ,
      • Digumarthy S.R.
      • Mendoza D.P.
      • Lin J.J.
      • Rooney M.
      • Do A.
      • Chin E.
      • et al.
      Imaging features and patterns of metastasis in non-small cell lung cancer with RET rearrangements.
      ,

      Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3 tyrosine kinase fusions define a unique molecular subtype of non-small cell lung cancer. Clin Cancer Res 2014; 20 (15), 4107–14. doi: 10.1158/1078-0432.CCR-14-0284.

      ]. The recently discovered fusion CLIP1–LTK in LuADs, leading to the oncogenic activation of the RTK LTK [
      • Izumi H.
      • Matsumoto S.
      • Liu J.
      • Tanaka K.
      • Mori S.
      • Hayashi K.
      • et al.
      The CLIP1-LTK fusion is an oncogenicdriver in non-small-cell lung cancer.
      ], will likely share similar characteristics. Some exceptions are the activation of FGFRs, more common in lung squamous cell carcinomas (LuSCCs), and NTRKs, which occur in NSCLCs across gender, age, smoking history, and histopathology [

      Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3 tyrosine kinase fusions define a unique molecular subtype of non-small cell lung cancer. Clin Cancer Res 2014; 20 (15), 4107–14. doi: 10.1158/1078-0432.CCR-14-0284.

      ,

      Schram AM et al. Efficacy and safety of zenocutuzumab in advanced pancreas cancer and other solid tumors harboring NRG1 fusions. J Clin Oncol 2021. 39 (15): 3003-3003. DOI: 10.1200/JCO.2021.39.15_suppl.

      ]. Most of the alterations that activate RTKs are mutually exclusive between them and between alterations of molecules involved in signal transduction. This feature suggests that they are all functionally connected and confer similar growth advantages. This characteristic is highlighted in Fig. 1, in which an oncoplot has been drawn to show the associations between genetic alterations that affect RTK and signal transduction molecules among lung Tumor Cancer Genome Atlas.
      Figure thumbnail gr1
      Fig. 1Oncoplots showing the distribution of the oncogenic mutations/fusions/amplifications for each indicated gene in the LuAD and LuSCC cohorts of lung PanCancer atlas (data extracted from cbioportal, https://www.cbioportal.org/). LuAD: Lung adenocarcinoma; LuSCC: Lung squamous cell carcinomas, TCGA: Tumor Cancer Genome Atlas.
      Currently, at least six of the different oncogenic alterations affecting RTKs in NSCLC, including activating mutations in EGFR and MET (METex14), as well as fusions in ALK, ROS1, NTRK, and RET, are eligible to be treated with compounds approved by the US Food and Drug Administration (FDA) as standard-of-care therapy. Additional compounds are under development for tumors carrying MET amplification (METamp) or activating mutations in ERBB2 [

      Jordan EJ, Kim HR, Arcila ME, Barron D, Chakravarty D, Gao J, et al. Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov 2017;7(6):596–609. doi: 10.1158/2159-8290.CD-16-1337.

      ]. Other fusions (e.g., NRG1, and FGFRs) areinfrequently detected in routine clinical practice owing to their rarity. Emerging drugs such as zenocutuzumab, a bi-specific antibody, have reported activity in NRG1 tumors, regardless of histologic type [

      Wang R, Wang L, Li Y, Hu H, Shen L, Shen X, et al. FGFR1/3 tyrosine kinase fusions define a unique molecular subtype of non-small cell lung cancer. Clin Cancer Res 2014; 20 (15), 4107–14. doi: 10.1158/1078-0432.CCR-14-0284.

      ,
      • Izumi H.
      • Matsumoto S.
      • Liu J.
      • Tanaka K.
      • Mori S.
      • Hayashi K.
      • et al.
      The CLIP1-LTK fusion is an oncogenicdriver in non-small-cell lung cancer.
      ,

      Schram AM et al. Efficacy and safety of zenocutuzumab in advanced pancreas cancer and other solid tumors harboring NRG1 fusions. J Clin Oncol 2021. 39 (15): 3003-3003. DOI: 10.1200/JCO.2021.39.15_suppl.

      ]. A timeline depicting the identification of selected oncogenic alterations (gene amplifications and fusions) in RTKs of NSCLC and the different treatments approved for each of the activated RTKs is represented in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Timeline depicting the identification of selected oncogenic drivers in various malignancies, including NSCLC (in bold) and targeted therapies activity and approval for each of them. FDA: US Food and Drug Administration; NSCLC: Non-small cell lung cancer; TKI: Tyrosine kinase inhibitor.
      This review will discuss the most relevant gene fusions and gene amplifications activating RTK in NSCLC, with special emphasis on the genomic structure, diagnostic approaches, and available therapeutics of each case.

      Oncogenic gene fusions involving RTK in NSCLC and their clinical implications

      Gene fusions affecting the anaplastic lymphoma kinase receptor, ALK

      ALK encodes a transmembrane RTK expressed in the nervous system during embryogenesis. ALK was first identified to fuse with NPM1 in anaplastic large‐cell lymphoma in 1994, but it was not until 2007 that ALK fusions were reported in NSCLC [
      • Soda M.
      • Choi Y.L.
      • Enomoto M.
      • Takada S.
      • Yamashita Y.
      • Ishikawa S.
      • et al.
      Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.
      ]. At present, this alteration is considered to affect 2–7% of LuADs, mostly women and never-smokers. Oncogenic ALK rearrangements fuse the intact kinase domain of ALK to the N–terminal regions of its partner, resulting in overexpression and constitutive, ligand-independent activation of ALK. Overall, more than 20 fusion partners have been identified, with EML4 as the most prevalent (Fig. 3) [

      Choi YL, Takeuchi K, Soda M, Inamura K, Togashi Y, Hatano S, et al. Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res 2008;68(13):4971-6. doi: 10.1158/0008-5472.CAN-07-6158.

      ].
      Figure thumbnail gr3
      Fig. 3Molecular structure of oncogenic fusions in NSCLC involving RTK and the most common fusion partners in each case. The upper panel describes the fusion at the DNA level and the lower panel shows the wild-type and fusion proteins. The different domains of the RTKs are indicated; (*) indicates the absence of transmembrane domain which can occur in some cases. NSCLC: Non-small cell lung cancer; RTK: Receptor tyrosine kinase.TKD: Tyrosine kinase domain; MAM: MAM domain (meprin/A5/mu); LB: Ligand binding domain; LRR8: Leucine rich repeat; Ig2: Immunoglobulin domain; FN3:Fibronectin type III domain; LRRNT: Leucine rich repeat N-terminal domain; I-SET:  Immunoglobulin I-set domain; IgI, IgII; IgII: Immunoglobulin domains
      Because ALK fusions involve large chromosomal inversions and translocations, fluorescent in situ hybridization (FISH) using break-apart probes was the first method developed for detecting all ALK rearrangements and the first to receive approval by the FDA [
      • Conde E.
      • Suárez-Gauthier A.
      • Benito A.
      • Garrido P.
      • García-Campelo R.
      • Biscuola M.
      • et al.
      Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.
      ,
      • Morán T.
      • Quiroga V.
      • Gil Mde L.
      • Vilà L.
      • Pardo N.
      • Carcereny E.
      • et al.
      Targeting EML4-ALK driven non-small cell lung cancer (NSCLC).
      ]. Since ALK fusions trigger an increase in the mRNA and protein levels, immunohistochemistry (IHC) can also be used. Owing to its high sensitivity and cost-effectiveness, IHC with the Ventana ALK D5F3 antibody gained FDA approval [
      • Conde E.
      • Hernandez S.
      • Prieto M.
      • Martinez R.
      • Lopez-Rios F.
      Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.
      ]. RNA-based next-generation sequencing (NGS) is also a valid technique to reveal ALK fusions as long as the quality of RNA is optimal [
      • Lindeman N.I.
      • Cagle P.T.
      • Aisner D.L.
      • Arcila M.E.
      • Beasley M.B.
      • Bernicker E.H.
      • et al.
      Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the college of american pathologists, the international association for the study of lung cancer, and the association for molecular pathology.
      ] (Table 1).
      Table 1List of oncogenes altered by gene fusions and amplification in NSCLC.
      Driver fusion/ampHistopathologic predominance patternIncidenceCancer type for FDA approval techniqueIHCFISHNGS
      ALK fusionLuAD solid, mucinous cribiform, and signet ring cells2–7%NSCLCVentana ALK D5F3 IHC CDx assay*Vysis ALK break Apart FISH Probe Kit (Abbott Molecular)*NGS FoundationOne CDx*
      ROS1 fusionLuAD solid, mucinous cribiform, and signet ring cells2%NSCLCROS1 D4D6 IHC assay* Cell Signaling TechnologyVysis ROS1 break-apart FISH Probe KitNGS FoundationOne CDx*
      NTRK (1,2,3) fusionLuAD3.5%

      <1%

      <1%
      Solid tumorsLoxo/Ventana Pan-TRK IHC CDx*NANGS FoundationOne CDx*
      RET fusionLuAD solid, mucinous cribiform, and signet ring cells, LuSCC1–2%NSCLC, thyroidNANAOncomine Dx Target Test*
      FGFR3 fusionLuSCC/LuAD<1%NANANADNA/RNA-based NGS
      NRG1 fusionLuAD<1%NANANADNA/RNA-based NGS
      LTK fusionLuAD<1%NANANADNA/RNA-based NGS
      FGFR1 ampLuSCC15–25%NSCLC-SCCNANADNA-based NGS
      MET ampLuAD, LuSCC, PSC1–5%NANANADNA-based NGS
      ERBB2 ampLuAD2–5%Breast, gastric cancerHercepTest (Dako)*HER2 FISH pharmDx Kit (Dako)*DNA-based NGS
      KIT/PDGFRA ampLuAD, LuSCC1–2%NANANADNA-based NGS
      * FDA approved and validated in clinical trials.
      CD: Companion diagnostic; FDA: US Food and Drug Administration; FISH: Fluorescence in situ hybridization; IHC: Immunohistochemistry; LuAD: Lung adenocarcinoma; LuSCC: Lung squamous cell carcinoma; NA: None approved; NGS: Next-generation sequencing; NSCLC: Non-small cell lung cancer; PSC: Pulmonary sarcomatoid carcinoma.
      Currently, five TKIs (i.e., crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib) have been approved by the FDA to treat NSCLCs harboring ALK translocations (Table 2). Crizotinib is a first-generation TKI that inhibits ALK but also targets ROS1 and MET and was the first to show efficacy in previously treated patients [
      • Camidge D.R.
      • Bang Y.-J.
      • Kwak E.L.
      • Iafrate A.J.
      • Varella-Garcia M.
      • Fox S.B.
      • et al.
      Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study.
      ,
      • Solomon B.J.
      • Mok T.
      • Kim D.-W.
      • Wu Y.-L.
      • Nakagawa K.
      • Mekhail T.
      • et al.
      First-line crizotinib versus chemotherapy in ALK-positive lung cancer.
      ]. Almost one-third of crizotinib-treated patients acquire resistance by mutations affecting the kinase domain, with p.Leu1196Met and p.Gly1269Ala as the most prevalent [
      • Fontana D.
      • Ceccon M.
      • Gambacorti-Passerini C.
      • Mologni L.
      Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.
      ]. Ceritinib, alectinib, and brigatinib are second-generation ALK-TKIs that bind to the receptor with higher affinity than crizotinib. These TKIs show activity against multiple mutations acquired during the treatment with crizotinib and have an improved penetrance into the central nervous system [
      • Fontana D.
      • Ceccon M.
      • Gambacorti-Passerini C.
      • Mologni L.
      Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK.
      ,
      • Soria J.-C.
      • Tan D.S.W.
      • Chiari R.
      • Wu Y.-L.
      • Paz-Ares L.
      • Wolf J.
      • et al.
      First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study.
      ,
      • Shaw A.T.
      • Kim T.M.
      • Crinò L.
      • Gridelli C.
      • Kiura K.
      • Liu G.
      • et al.
      Ceritinib versus chemotherapy in patients with ALK-rearranged non-small-cell lung cancer previously given chemotherapy and crizotinib (ASCEND-5): a randomised, controlled, open-label, phase 3 trial.
      ]. The results of the phase III ALEX trial evidenced better clinical outcomes for alectinib than for crizotinib in terms of median progression-free survival (mPFS) (34.8 vs 10.9 months; hazard ratio [HR]: 0.43) -investigator assessed- and median overall survival (mOS) (not reached [NR] vs 57.4 months; HR: 0.67). On the basis of this study, alectinib became the most common first–line treatment for patients with ALK–rearranged tumors [
      • Peters S.
      • Camidge D.R.
      • Shaw A.T.
      • Gadgeel S.
      • Ahn J.S.
      • Kim D.-W.
      • et al.
      Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer.
      ,
      • Mok T.
      • Camidge D.R.
      • Gadgeel S.M.
      • Rosell R.
      • Dziadziuszko R.
      • Kim D.-W.
      • et al.
      Updated overall survival and final progression-free survival data for patients with treatment-naive advanced ALK-positive non-small-cell lung cancer in the ALEX study.
      ] (Table 2). Similarly, the ALTA1L trial demonstrated a superior mPFS for brigatinib over crizotinib in previously untreated LuAD patients with ALK-rearranged tumors [
      • Camidge D.R.
      • Kim H.R.
      • Ahn M.-J.
      • Yang J.C.H.
      • Han J.-Y.
      • Hochmair M.J.
      • et al.
      Brigatinib Versus Crizotinib in Advanced ALK Inhibitor–Naive ALK-Positive Non–Small Cell Lung Cancer: Second Interim Analysis of the Phase III ALTA-1L Trial.
      ]. Since both trials showed similar outcomes and toxicity profiles, alectinib and brigatinib represent a reasonable option for first-line treatment. Finally, lorlatinib, a selective and third-generation TKI that inhibits ALK and ROS1, has activity against most known ALK drug-resistant mutations [
      • Shaw A.T.
      • Solomon B.J.
      • Besse B.
      • Bauer T.M.
      • Lin C.-C.
      • Soo R.A.
      • et al.
      ALK Resistance mutations and efficacy of lorlatinib in advanced anaplastic lymphoma kinase-positive non-small-cell lung cancer.
      ]. Based on the results of the CROWN trial, lorlatinib also gained FDA and EMA (European Medicines Agency) approval as a first-line treatment [
      • Shaw A.T.
      • Bauer T.M.
      • de Marinis F.
      • Felip E.
      • Goto Y.
      • Liu G.
      • et al.
      First-line lorlatinib or crizotinib in advanced ALK-positive lung cancer.
      ]. Since no trial has compared the efficacy of all ALK-TKIs in first-line, lorlatinib also seems a reasonable option, particularly when considering its activity in the brain. More recently, the small-molecule ensartinib (X-396) demonstrated greater efficacy than crizotinib in both systemic and intracranial disease, thus representing another first-line therapeutic option for patients with ALK-positive NSCLC [
      • Horn L.
      • Wang Z.
      • Wu G.
      • Poddubskaya E.
      • Mok T.
      • Reck M.
      • et al.
      Ensartinib vs crizotinib for patients with anaplastic lymphoma kinase−positive non–small cell lung cancer: a randomized clinical trial.
      ] (Table 2).
      Table 2List of most relevant clinical trials and clinical efficacy of targeted therapies for NSCLC with tumors with driven oncogenic fusions and amplifications.
      BiomarkerTargeted Drug (type)TrialStudy typeORR (95% CI)mPFS months HR (95% CI)mOS months HR (95% CI)FDA approvalRef.
      ALKCrizotinibProfile 1001,

      ≥1st L
      Phase I (ALK cohort n = 143)

      60.8% (52.3–68.9)9.7 (7.7–12.8)





      12; OS rate: 74.8% (66.4–81.5)Accelerated approval (2011)16
      Profile 1007, ≥2nd L (vs CT)Phase III (n = 173)65% vs 20%7.7 vs 3; HR: 0.49 (0.37–0.64)20.3 vs 22.8 HR: 1.02 (0.68–1.54)Approved (2013)17
      Profile 1014, 1st L (vs CT)Phase III (n = 343)75% vs 45%10.9 vs 7; HR: 0.45 (0.35–0.60)NR vs 47.5; HR: 0.76 (0.55–1.05)18
      CeritinibASCEND 4, 1st L (vs CT)Phase III (n = 376)72.5% (65.5–78.7) vs 26.7% (20.5–33.7)16.6 vs 8.1; HR: 0.55 (0.42–0.73)NAApproved (2014)19
      ASCEND 5, ≥2nd L (vs CT)Phase III (n = 231)

      39.1% (30.2–48.7) vs 6.9% (3.0–13.1%)5.4 vs 1.6; HR: 0·49 (0.36–0.67)

      NA20
      AlectinibALEX (vs crizotinib) 1st LPhase III (n = 303)82.9% (75.0–88.5) vs 75.5% (67.0–82.0)34.8 vs 10.9; HR 0.43 (0.32–0.58)NR vs 57.4; HR: 0.67 (0.46–0.98)Approved (2017)22
      BrigatinibALTA 1L (vs crizotinib), 1st LPhase III (n = 275)71% (62–78) vs 60% (54–66)24 vs 11; HR 0.49 (0.35–0.68)NAApproved (2020)23
      LorlatinibNCT01970865, ALK-TKI pre-ttPhase I/II (n = 275)48% (42–55)NANAAccelerated approval (2018)24
      CROWN (vs crizotinib), 1st LPhase III (n = 296)76% (68–83) vs 58% (49–66)NR vs 9.3; HR 0.28 (0.19–0.41)NAApproved (2021)25
      Ensartinib or

      X-396

      (multi-TKI)
      EXALT-3 (vs crizotinib), 1st LPhase III (n = 290)75% vs 67%25.8 vs 12. 7; HR 0.51 (0.35–0.72)NA26
      ROS1CrizotinibProfile 1001, ≥1st LPhase I (ROS-1 cohort n = 50)72% (58–84%)19.3 (15.2–39.1)51.4 (29.3-NR)Approved (2016)33
      EURCROSS, Europe, TKI-naïvePhase II (n = 34)70% (51–85)10.1 (20-NR)NA34
      LorlatinibNCT01970865, TKI-naïve and pre-ttPhase II (n = 69)62% (TKI-naïve)/35% (pre-tt)NANAApproved (2019)35
      Entrectinib

      (multi-TKI)
      ALKA 372–001, STARTK-1, TKI-naïvePhase I/II (n = 53)77.4%24.6 (11.4–34.8)NAApproved (2019)36
      Ceritinib

      (multi-TKI)
      NCT 01964157, TKI-naïve and pre-ttPhase II (n = 32)62%9.3 (0–22) all /19.3 (1–37) tt-naïve24 (5–43)37
      Repotrectinib or TPX-0005

      (multi-TKI)
      TRIDENT-1, TKI-naïvePhase II (n = 7)86%NANA38
      BiomarkerTargeted Drug (type)TrialStudy typeORR (95% CI)mPFS months HR (95% CI)mOS months HR (95% CI)FDA approvalRef.
      NTRKLarotrectinib

      (Selective-TKI)
      LOXO-TRK-14001, SCOUT & NAVIGATEPhase I & II (n=159)79% (72–88)28.3 (22.1-NR)44.4 (36.5-NR)Approved for solid agnostic tumors (2018)45, 46
      Entrectinib

      (multi-TKI)
      ALKA-372–001, STARTRK-1 & STARTRK-2Phase I & II (n=54)57% (43.2–70.8)11 (9–14.9)21 (14.9-NA)Approved for solid agnostic tumors (2019)47
      Selitrectinib or LOXO-195 (selective-TKI)NCT03215511 (TKI pre-tt)Phase I (n=20)36%NANAOrphan drug designation49
      Repotrectinib or TPX-0005 (multi-TKI)TRIDENT-1 (TKI pre-tt)Phase II (n=6)50%NANA50
      RETCabozantinib

      (multi-TKI)
      NCT01639508, pre-ttPhase II (n=25)28%5.5 (3.8–9.4)9.9 (8.1-NR)56
      Vandetanib

      (multi-TKI)
      LURET, pre-ttPhase II (n=19)53%6.5 (2.8–9.5)13.5 (9.8–29.1)57
      Lenvatinib

      (multi-TKI)
      NCT01877083, pre-ttPhase II (n=25)16%7.3 (3.6–10.2)NA58
      Selpercatinibor LOXO-292

      (selective-TKI)
      LIBRETTO-001,1st L or pre-ttPhase I/II (n=105)68% (pre-tt, n=105)/85% (tt-naïve, n=34)18.4 (13.8–24) /NR (9.2-NR)NAAccelerated approval (2020)59
      Pralsetinib or BLU-667 (selective-TKI)ARROW, 1st L or pre-ttPhase I/II (n=114 NSCLC)58% (pre-tt n=48)/71% (tt-naïve, n=7)NANAAccelerated approval (2020)60
      MET ampCrizotinibProfile 1001, ≥1st LPhase I (MET cohort=37)GCN≥4: 44%; GCN 2-<4: 14%; GCN<2: 33%6.7/1.9/1.8NA81
      CapmatinibGEOMETRY, ≥1st LPhase II, GCN≥10 (n=14/55)40%/29%4.2/4.19.6/10.682
      95% CI: 95% confidence interval; FDA: US Food and Drug Administration; GCN: Gain copy number; HR: Hazard ratio; L: Line (of treatment); mOS: Median overall survival; mPFS: Median progression-free survival; NA: Not assessed; NR: Not reached; NSCLC: Non-small cell lung cancer; ORR: Overall response rate; pre-tt: Pre-treated; TKI: Tyrosine kinase inhibitor; tt-naïve: Treatment naïve.
      Despite the therapeutic efficacy of these TKIs, the emergence of acquired resistance may limit their long-term benefits. The solvent-front p.Gly1202Arg substitution impairs the correct binding of the drug to ALK and constitutes a common resistance mechanism to ALK-TKIs [

      Gainor JF, Dardaei L, Yoda S, Friboulet L, Leshchiner I, Katayama R, et al. Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK-rearranged lung cancer. Cancer Discov 2016;6(10):1118–33. doi: 10.1158/2159-8290.CD-16-0596.

      ]. Besides, ALK-independent (or off-target) resistance mechanisms such as METamp, detected in almost 15% of cases [

      Dagogo-Jack I, Yoda S, Lennerz JK, Langenbucher A, Lin JJ, Rooney MM et al. MET alterations are a recurring and actionable resistance mechanism in ALK-positive lung cancer. Clin Cancer Res 2020;26 (11):2535-45. doi: 10.1158/1078-0432.CCR-19-3906.

      ], loss of NF2, or histopathologic transformation into a neuroendocrine subtype have also been reported [

      Coleman N, Wotherspoon A, Yousaf N, Popat S.. Transformation to neuroendocrine carcinoma as a resistance mechanism to lorlatinib. Lung Cancer 2019;134:117-20. doi: 10.1016/j.lungcan.2019.05.025.

      ].

      Gene fusions affecting the c-ros oncogene 1 receptor, ROS1

      ROS1 is a receptor with tyrosine kinase activity that shares structural homology, within the TKD, with ALK and can also undergo genomic rearrangements to create fusion proteins [
      • Bergethon K.
      • Shaw A.T.
      • Ignatius Ou S.-H.
      • Katayama R.
      • Lovly C.M.
      • McDonald N.T.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      ]. For ALK and other RTK fusions, the fusion partner provides a dimerization domain that induces constitutive oligomerization and, thus, activation of the kinase. However, since most ROS1 fusion partners lack this dimerization domain, the mechanism for ROS1 oncogenic activation remains unknown [

      Neel DS, Allegakoen DV, Olivas V, Mayekar MK, Hemmati G, Chatterjee N, et al. Differential subcellular localization regulates oncogenic signaling by ROS1 kinase fusion proteins. Cancer Res 2019; 79(3): 546–56 oi: 10.1158/0008-5472.CAN-18-1492.

      ]. In the ROS1 fusions, the kinase domain of ROS1 is paired with a wide range of partners, the most common being CD74 (Fig. 3) [
      • Bergethon K.
      • Shaw A.T.
      • Ignatius Ou S.-H.
      • Katayama R.
      • Lovly C.M.
      • McDonald N.T.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      ]. Chromosomal rearrangements of the ROS1 proto-oncogene occur in about 1–2% of LuADs and more often in never-smokers [
      • Bergethon K.
      • Shaw A.T.
      • Ignatius Ou S.-H.
      • Katayama R.
      • Lovly C.M.
      • McDonald N.T.
      • et al.
      ROS1 rearrangements define a unique molecular class of lung cancers.
      ]. The diagnosis of ROS1 fusions can be performed by IHC—the most cost-effective method—and confirmed by FISH, but NGS can also be used (Table 1) [
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • Zhang C.
      • Lee C.
      • Jänne P.A.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      ].
      Crizotinib was the first TKI to demonstrate activity in ROS1-rearranged tumors, showing promising overall response rates (ORR) (mPFS of 19.3 months and mOS of 51.4 months (95% CI 29.3- NR) in a subset of mostly pretreated patients with advanced-stage NSCLC [
      • Shaw A.T.
      • Riely G.J.
      • Bang Y.-J.
      • Kim D.-W.
      • Camidge D.R.
      • Solomon B.J.
      • et al.
      Crizotinib in ROS1- rearranged advanced non-small-cell lung cancer (NSCLC): updated results, including overall survival, from PROFILE 1001.
      ]. At present, crizotinib remains the recommended first-line therapy for these patients [
      • Michels S.
      • Massutí B.
      • Schildhaus H.-U.
      • Franklin J.
      • Sebastian M.
      • Felip E.
      • et al.
      Safety and efficacy of crizotinib in patients with advanced or metastatic ROS1-rearranged lung cancer (EUCROSS): A European phase II clinical trial.
      ]. Lorlatinib has also demonstrated efficacy in both TKI-naïve and TKI-pretreated patients with ROS1-rearranged tumors [
      • Peters S.
      • Shaw A.T.
      • Besse B.
      • Felip E.
      • Solomon B.J.
      • Soo R.A.
      • et al.
      Impact of lorlatinib on patient-reported outcomes in patients with advanced ALK-positive or ROS1-positive non-small cell lung cancer.
      ]. Entrectinib, which also targets TRKA/B/C and ALK, has shown activity against these tumors as well, inducing meaningful intracranial responses in patients with brain metastases [
      • Drilon A.
      • Siena S.
      • Dziadziuszko R.
      • Barlesi F.
      • Krebs M.G.
      • Shaw A.T.
      • et al.
      Entrectinib in ROS1 fusion-positive non-small-cell lung cancer: integrated analysis of three phase 1–2 trials.
      ]. While these three drugs have gained FDA approval, other agents such as ceritinib, brigatinib, repotrectinib (TPX-0005), and DS-6051b are currently being evaluated in phase I and II trials [
      • Lim S.M.
      • Kim H.R.
      • Lee J.-S.
      • Lee K.H.
      • Lee Y.-G.
      • Min Y.J.
      • et al.
      Open-label, multicenter, phase II study of ceritinib in patients with non-small-cell lung cancer harboring ROS1 rearrangement.
      ,
      • Cho B.C.
      • Drilon A.E.
      • Doebele R.C.
      • Kim D.-W.
      • Lin J.J.
      • Lee J.
      • et al.
      Safety and preliminary clinical activity of repotrectinib in patients with advanced ROS1 fusion-positive non-small cell lung cancer (TRIDENT-1 study).
      ,
      • Fujiwara Y.
      • Takeda M.
      • Yamamoto N.
      • Nakagawa K.
      • Nosaki K.
      • Toyozawa R.
      • et al.
      Safety and pharmacokinetics of DS-6051b in Japanese patients with non-small cell lung cancer harboring ROS1 fusions: a phase I study.
      ] (Table 2).
      The genetic mechanisms that confer resistance to crizotinib in tumors with ROS1 fusions are equivalent to those in ALK-rearranged NSCLC. The p.Gly2032Arg mutation, which is structurally analogous to the p.Gly1202Arg mutation in ALK, is the most common, although other mutations (e.g., p.Asp2033Asn and p.Ser1986Phe) have also been reported [
      • Davare M.A.
      • Vellore N.A.
      • Wagner J.P.
      • Eide C.A.
      • Goodman J.R.
      • Drilon A.
      • et al.
      Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors.
      ]. Since not all on-target mutations are equally sensitive to the new generation of inhibitors, tumor stratification according to the type of mutation will be required.

      Gene fusions affecting the neurotrophic tropomyosin tyrosine kinase receptor, NTRK

      The NTRK-family of genes comprises three members (NTRK1, NTRK2, and NTRK3) that encode tropomyosin receptor kinases (TRK) A, B, and C, respectively. These kinase receptors are physiologically involved in neuronal development and differentiation. The aberrant activation of NTRK, which results in the constitutive activation of downstream pathways, appears mainly through gene fusions and several partners have been described (Fig. 3) [
      • Cocco E.
      • Scaltriti M.
      • Drilon A.
      NTRK fusion-positive cancers and TRK inhibitor therapy.
      ]. These alterations have a prevalence of 0.2–0.4% across several solid tumor types in adult cancers, regardless of gender and smoking habit, with higher prevalence in pediatric population (1.34%). Among adult patients, NTRK fusions are more prevalent in salivary gland cancers (2.43%), soft tissue sarcoma (1.27%) and thyroid cancers (1.28%),albeit their frequency in LuADs is low (<1%) [

      Westphalen, C.B., Krebs, M.G., Le Tourneau, C. et al. Genomic context of NTRK1/2/3 fusion-positive tumours from a large real-world population. npj Precis. Onc.5, 69 (2021). doi.org/10.1038/s41698-021-00206-y.

      ]. Notably, the rare fusion ETV6–NTRK3 is detectable in nearly all patients with secretory carcinoma of the breast and with mammary analogue secretory carcinoma of the salivary glands and is, thus, considered pathognomonic in these two histologically identical tumor types [
      • Tognon C.
      • Knezevich S.R.
      • Huntsman D.
      • Roskelley C.D.
      • Melnyk N.
      • Mathers J.A.
      • et al.
      Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma.
      ]. IHC screening confirmed by FISH or by NGS are appropriate methods for diagnosing these fusions [
      • Hechtman J.F.
      • Benayed R.
      • Hyman D.M.
      • Drilon A.
      • Zehir A.
      • Frosina D.
      • et al.
      Pan-Trk immunohistochemistry is an efficient and reliable screen for the detection of NTRK fusions.
      ] (Table 1).
      The first generation of TKIs that blocked the activity of RTKs were larotrectinib (a selective TRK inhibitor) and entrectinib (a multikinase inhibitor also targeting ROS1 and ALK), and both conferred durable responses in patients with metastatic disease (ORR of larotrectinib: 75% and ORR of entrectinib: 61.2%, with a mPFS of 13.8 months (95% CI, 10.1–19.9), regardless of tumor histology, age, or fusion partner [
      • Drilon A.
      • Laetsch T.W.
      • Kummar S.
      • DuBois S.G.
      • Lassen U.N.
      • Demetri G.D.
      • et al.
      Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children.
      ,
      • Drilon A.
      • Tan D.S.W.
      • Lassen U.N.
      • Leyvraz S.
      • Liu Y.
      • Patel J.D.
      • et al.
      Efficacy and Safety of Larotrectinib in Patients With Tropomyosin Receptor Kinase Fusion-Positive Lung Cancers.
      ,
      • Demetri G.D.
      • De Braud F.
      • Drilon A.
      • Siena S.
      • Patel M.R.
      • Cho B.C.
      • et al.
      Updated Integrated Analysis of the Efficacy and Safety of Entrectinib in Patients With NTRK Fusion-Positive Solid Tumors.
      ]. The activity of larotrectinib have also been reported in 15 patients with NSCLC harboring NTRK fusions, showing a ORR of 73%, mPFS of 35.4 months (95% CI, 5.3–35.4), and mOS of 40.7 months (95% CI, 17.2 to not estimable) [
      • Drilon A.
      • Tan D.S.W.
      • Lassen U.N.
      • Leyvraz S.
      • Liu Y.
      • Patel J.D.
      • et al.
      Efficacy and Safety of Larotrectinib in Patients With Tropomyosin Receptor Kinase Fusion-Positive Lung Cancers.
      ]. These two TKIs gained tumor-agnostic regulatory accelerated approvals by the FDA to treat cancers harboring NTRK-fusions (Table 2) [
      • Drilon A.
      • Laetsch T.W.
      • Kummar S.
      • DuBois S.G.
      • Lassen U.N.
      • Demetri G.D.
      • et al.
      Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children.
      ,
      • Demetri G.D.
      • De Braud F.
      • Drilon A.
      • Siena S.
      • Patel M.R.
      • Cho B.C.
      • et al.
      Updated Integrated Analysis of the Efficacy and Safety of Entrectinib in Patients With NTRK Fusion-Positive Solid Tumors.
      ]. However, like others RTKs, resistance to these TKIs can eventually arise by the emergence of secondary mutations at the kinase domain of NTRK [

      Fuse MJ, Okada K, Oh-Hara T, Ogura H, Fujita N, Katayama R. Mechanisms of resistance to NTRK inhibitors and therapeutic strategies in NTRK1-rearranged cancers. Mol Cancer Ther 2017;16(10):2130–43. doi: 10.1158/1535-7163.MCT-16-0909.

      ]. Next-generation agents, such as selitrectinib (LOXO-195) and repotrectinib, were designed to address on-target resistance mechanisms and have shown promising activities in clinical trials (Table 2) [

      Hyman D, Kummar S, Farago A, et al. CT127 - Phase I and expanded access experience of LOXO-195 (BAY 2731954), a selective next-generation TRK inhibitor (TRKi). American Association for Cancer Research Annual Meeting 2019; 29 March–3 April 2019: Atlanta, Georgia, USA. doi: 10.1158/1538-7445.AM2019-CT127.

      ,
      • Drilon A.
      • Cho B.C.
      • Kim D.
      • Lee J.
      • Lin J.J.
      • Zhu V.
      • et al.
      4 536 - Safety and preliminary clinical activity of repotrectinib in patients with advanced ROS1/TRK fusion-positive solid tumors (TRIDENT-1 study).
      ,

      Drilon A, Nagasubramanian R, Blake JF, Ku N, Tuch BB, Ebata K, et al. A next-generation TRK kinase inhibitor overcomes acquired resistance to prior TRK kinase inhibition in patients with TRK fusion-positive solid tumors. Cancer Discov 2017;7(9):963–72. doi: 10.1158/2159-8290.CD-17-0507.

      ]. Given the potent clinical activity of the TRK inhibitors, the current clinical guidelines recommend testing all NSCLCs for NTRK fusions.

      Gene fusions affecting the REarranged during transfection receptor, RET

      The RET proto-oncogene encodes a transmembrane RTK involved in numerous developmental pathways and multiple malignancies, including multiple endocrine neoplasia 2, papillary thyroid cancer, and NSCLC. Unlike other RTKs, RET does not directly bind to its ligands but requires glycosyl-phosphatidylinositol-anchored (GFRalpha1-4) co-receptors. Indeed, ligands of the glial cell line-derived neurotrophic factor family bind to one of the four co-receptors, which subsequently allow RET dimerization, autophosphorylation, and activation, leading to downstream activation signaling [
      • Breit S.N.
      • Brown D.A.
      • Tsai V.W.
      The GDF15-GFRAL Pathway in health and metabolic disease: friend or foe?.
      ]. RET fusions can be found in 1–3% of LuADs and are more common in young, female, never-smoker patients (Table 1). More than ten RET fusion partners have been described, being KIF5B the most frequent (Fig. 3) [
      • Breit S.N.
      • Brown D.A.
      • Tsai V.W.
      The GDF15-GFRAL Pathway in health and metabolic disease: friend or foe?.
      ,
      • Gautschi O.
      • Milia J.
      • Filleron T.
      • Wolf J.
      • Carbone D.P.
      • Owen D.
      • et al.
      Targeting RET in patients with RET-rearranged lung cancers: results from the global, multicenter RET registry.
      ]. Unlike ALK and ROS1 fusions, RET fusions cannot be properly detected by IHC because of the low sensitivity and highly variable specificity of the available antibodies. On the other hand, FISH is a sensitive but rather unspecific approach to test for RET fusions in NSCLC, owing to the frequent presence of RET rearrangements not resulting in oncogenic RET [
      • Radonic T.
      • Geurts-Giele W.R.R.
      • Samsom K.G.
      • Roemen G.M.J.M.
      • von der Thüsen J.H.
      • Thunnissen E.
      • et al.
      RET Fluorescence in situ hybridization analysis is a sensitive but highly unspecific screening method for RET fusions in lung cancer.
      ]. Therefore, NGS is the most appropriate option for diagnosing RET fusions [
      • Ju Y.S.
      • Lee W.-C.
      • Shin J.-Y.
      • Lee S.
      • Bleazard T.
      • Won J.-K.
      • et al.
      A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing.
      ] (Table 1).
      Several multitarget kinase inhibitors, such as cabozantinib, vandetanib, and lenvatinib—approved for treating patients with advanced medullar thyroid cancer—have limited activity in RET-rearranged NSCLC and important adverse effects due to their off-target activity [
      • Drilon A.
      • Rekhtman N.
      • Arcila M.
      • Wang L.u.
      • Ni A.
      • Albano M.
      • et al.
      Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial.
      ,
      • Yoh K.
      • Seto T.
      • Satouchi M.
      • Nishio M.
      • Yamamoto N.
      • Murakami H.
      • et al.
      Vandetanib in patients with previously treated RET-rearranged advanced non-small-cell lung cancer (LURET): an open-label, multicentre phase 2 trial.
      ,
      • Hida T.
      • Velcheti V.
      • Reckamp K.L.
      • Nokihara H.
      • Sachdev P.
      • Kubota T.
      • et al.
      A phase 2 study of lenvatinib in patients with RET fusion-positive lung adenocarcinoma.
      ]. More recently, two highly potent and selective RET-TKI, selpercatinib and pralsetinib, have gained FDA approval for lung and thyroid cancers. In the LIBRETTO-001 trial on advanced NSCLC, selpercatinib (LOXO-292) was given to pretreated (ORR of 65%) and treatment-naïve patients (ORR of 85%) [
      • Drilon A.
      • Oxnard G.R.
      • Tan D.S.W.
      • Loong H.H.F.
      • Johnson M.
      • Gainor J.
      • et al.
      Efficacy of selpercatinib in RET fusion-positive non-small-cell lung cancer.
      ] (Table 2). Pralsetinib’s (BLU-667) efficacy was established in the phase I/II ARROW trial with similar results [
      • Gainor J.F.
      • Curigliano G.
      • Kim D.-W.
      • Lee D.H.
      • Besse B.
      • Baik C.S.
      • et al.
      Pralsetinib for RET fusion-positive non-small-cell lung cancer (ARROW): a multi-cohort, open-label, phase 1/2 study.
      ]. Both drugs were well tolerated with an acceptable safety profile and relevant intracranial activity regardless of the RET-fusion partner (ORR of selpercatinib: 91% and ORR of pralsetinib: 78%). However, p.Gly810Arg/Ser/Cys mutations have been described as acquired resistance mechanisms to selective RET inhibitors [
      • Solomon B.J.
      • Tan L.
      • Lin J.J.
      • Wong S.Q.
      • Hollizeck S.
      • Ebata K.
      • et al.
      RET solvent front mutations mediate acquired resistance to selective RET inhibition in RET-driven malignancies.
      ]. Second-generation RET-TKIs (e.g., TPX-0046 [NCT04161391] and BOS-172738 [NCT03780517]) are currently being tested in clinical trials [
      • Thein K.Z.
      • Velcheti V.
      • Mooers B.H.M.
      • Wu J.
      • Subbiah V.
      Precision therapy for RET-altered cancers with RET inhibitors.
      ].

      Gene fusions affecting the fibroblast growth factor receptors, FGFRs

      FGFRs are a family of RTKs expressed on the cell membrane that play crucial roles in developmental and adult cells. The human family of FGFRs consists of four members: FGFR1, FGFR2, FGFR3, and FGFR4 [

      Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, et al. Identification of targetable FGFR gene fusions in diverse cancers. Cancer Discov 2013;3(6):636-47. doi: 10.1158/2159-8290.CD-13-0050. Epub 2013 Apr 4.

      ]. Despite being encoded by different genes, the four members share high homology. The aberrant activation of FGFRs, caused by activating gene fusions, amplifications, or mutations involving the TKD, is a frequent oncogenic mechanism in different types of cancer, such as glioblastomas, cholangiocarcinomas, breast cancer, and bladder cancer, among others [

      Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, et al. Identification of targetable FGFR gene fusions in diverse cancers. Cancer Discov 2013;3(6):636-47. doi: 10.1158/2159-8290.CD-13-0050. Epub 2013 Apr 4.

      ]. Although fusions affecting FGFRs are uncommon, they have been described in lung cancer, mostly in LuSCCs. The most widely recognized is the fusion between FGFR3 and TACC3 [

      Schram AM et al. Efficacy and safety of zenocutuzumab in advanced pancreas cancer and other solid tumors harboring NRG1 fusions. J Clin Oncol 2021. 39 (15): 3003-3003. DOI: 10.1200/JCO.2021.39.15_suppl.

      ] (Fig. 3). Fusions at FGFR1, FGFR2, and FGFR4 are even rarer and still poorly characterized. Alterations at FGFR1 in lung cancer are mostly gene amplification and are found in up to 25% of LuSCCs, approximately (Table 1) [
      • Qin A.
      • Johnson A.
      • Ross J.S.
      • Miller V.A.
      • Ali S.M.
      • Schrock A.B.
      • et al.
      Detection of known and novel FGFR fusions in non-small cell lung cancer by comprehensive genomic profiling.
      ].
      In contrast to ALK, ROS1, and RET, FGFR-fusions are more frequent in LuSCC from smokers and in poorly differentiated tumors. NGS is the standard technique to diagnose FGFR-fusions. Several drugs targeting FGFRs are currently under development and their efficacy is being evaluated in several basket trials regardless of histopathologic subtype. The early-phase clinical trial and first–in–human study (NCT01703481) tested the TKI erdafitinib (JNJ–42756493), a pan-FGFR inhibitor for patients with detectable FGFR1-3 alterations, but yielded only a few responses with an acceptable toxicity profile [
      • Tabernero J.
      • Bahleda R.
      • Dienstmann R.
      • Infante J.R.
      • Mita A.
      • Italiano A.
      • et al.
      Phase I dose-escalation study of JNJ–42756493, an oral pan-fibroblast growth factor receptor inhibitor, in patients with advanced solid tumors.
      ]. Similar efficacy has been reported for the ATP-competitive broad-spectrum kinase inhibitor ARQ–087 in NSCLC patients with tumors harboring FGFR genetic activation [
      • Papadopoulos K.P.
      • El-Rayes B.F.
      • Tolcher A.W.
      • Patnaik A.
      • Rasco D.W.
      • Harvey R.D.
      • et al.
      A Phase 1 study of ARQ 087, an oral pan-FGFR inhibitor in patients with advanced solid tumours.
      ]. Finally, infigratinib, an FGFR1–3 selective inhibitor, has proven effective in FGFR altered tumors, particularly in cholangiocarcinomas [

      Milind M. Javle MM, Kelley RK, Springfeld C, Abou-Alfa GC, Macarulla T, Tanasanvimon et al. A phase II study of infigratinib in previously treated advanced/metastatic cholangiocarcinoma with FGFR gene fusions/alterations. DOI: 10.1200/JCO.2021.39.3_suppl.TPS356.

      ] (Table 2).

      Other gene fusions affecting RTKs in NSCLC

      A variety of other rare fusions involving receptors with tyrosine kinase activity have been identified in NSCLC, including MET and NRG1 [
      • Pan Y.
      • Zhang Y.
      • Ye T.
      • Zhao Y.
      • Gao Z.
      • Yuan H.
      • et al.
      Detection of Novel NRG1, EGFR, and MET fusions in lung adenocarcinomas in the chinese population.
      ,

      Fernandez-Cuesta L, Plenker D, Osada H, Sun R, Menon R, Leenders F, et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014;4(4):415–22. doi: 10.1158/2159-8290.CD-13-0633.

      ]. NRG1 is the ligand of the HER family and, despite not being an RTK itself, triggers the activation of RTKs. Although the prevalence of these fusions is very low, drugs targeting these proteins or the pathways regulated by them are under evaluation, and data from small-cohort studies and case reports have been obtained [
      • Gay N.D.
      • Wang Y.
      • Beadling C.
      • Warrick A.
      • Neff T.
      • Corless C.L.
      • et al.
      Durable response to afatinib in lung adenocarcinoma harboring NRG1 gene fusions.
      ,

      Drilon A, Somwar R, Mangatt BP, Edgren H, Desmeules P, Ruusulehto A, et al. Response to ERBB3-directed targeted therapy in NRG1-rearranged cancers. Cancer Discov 2018;8(6):686–95. doi: 10.1158/2159-8290.CD-17-1004.

      ].
      Fusions involving MET were initially discovered in an osteogenic sarcoma cell line (TPR–MET) and are very rare events in lung cancer (about 0.5%). A wide variety of fusion partners have been identified, including HLA–DRB1, KIF5B, PTPRZ1, STARD3NL, and ST7, although the biology of these alterations and their therapeutic implications have not been evaluated yet [

      Fernandez-Cuesta L, Plenker D, Osada H, Sun R, Menon R, Leenders F, et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014;4(4):415–22. doi: 10.1158/2159-8290.CD-13-0633.

      ].
      Finally, the fusion CLIP1-LTK has recently been discovered in a Japanese cohort of LuADs as an oncogenic driver, affecting around 0.4% of NSCLCs (Table 1). This fusion leads to the activation of the LTK (leukocyte receptor tyrosine kinase), an RTK and a member of the ros/insulin receptor family of tyrosine kinases whose function is still not well understood. Tumors with the CLIP1-LTK fusion responded to lorlatinib, although clinical validation of this new oncogenic driver and clinical development of novel therapeutic agents are warranted [
      • Izumi H.
      • Matsumoto S.
      • Liu J.
      • Tanaka K.
      • Mori S.
      • Hayashi K.
      • et al.
      The CLIP1-LTK fusion is an oncogenicdriver in non-small-cell lung cancer.
      ].
      Neuroregulins (NRGs) are the ligands of the EGFR family of proteins and are encoded by four genes (NRG1, NRG2, NRG3, and NRG4). NRG1 is the best characterized and is associated with the invasive mucinous subtype of LuAD, typically diagnosed in women and never-smokers (Table 1). Rearrangements of NRG1 in solid tumors lead to aberrant activation of HER2/HER3 and, subsequently, of the PI3K-AKT-mTOR and RAS/MAPK cascade. Albeit several fusion partners have been described, CD74 remains the most common [
      • Pan Y.
      • Zhang Y.
      • Ye T.
      • Zhao Y.
      • Gao Z.
      • Yuan H.
      • et al.
      Detection of Novel NRG1, EGFR, and MET fusions in lung adenocarcinomas in the chinese population.
      ,

      Fernandez-Cuesta L, Plenker D, Osada H, Sun R, Menon R, Leenders F, et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014;4(4):415–22. doi: 10.1158/2159-8290.CD-13-0633.

      ] (Fig. 3). Afatinib, an irreversible inhibitor of EGFR, represents a therapeutic option for patients with tumors with NRG1 fusions and several case reports have been published in this sense [
      • Gay N.D.
      • Wang Y.
      • Beadling C.
      • Warrick A.
      • Neff T.
      • Corless C.L.
      • et al.
      Durable response to afatinib in lung adenocarcinoma harboring NRG1 gene fusions.
      ]. In addition, blocking HER3 also showed promising activity in these patients [

      Drilon A, Somwar R, Mangatt BP, Edgren H, Desmeules P, Ruusulehto A, et al. Response to ERBB3-directed targeted therapy in NRG1-rearranged cancers. Cancer Discov 2018;8(6):686–95. doi: 10.1158/2159-8290.CD-17-1004.

      ].

      Oncogenic gene amplifications involving RTK in NSCLC and their clinical implications

      Besides gene fusions, gene amplification represents another mechanism that activates oncogenes during cancer development. Gene amplification can be defined as an expansion in the number of copies of a gene or a chromosomal region that occurs during the DNA replication process [

      Albertson DG. Gene amplification in cancer. Trends Genet 2006;22(8):447-55. doi: 10.1016/j.tig.2006.06.007.

      ]. The amplified DNA can be organized as extrachromosomal elements, as repeated units at a single locus, or scattered throughout the genome. Similar to gene fusions, oncogenic activation by gene amplification leads to a supraphysiological increase in the expression of the oncogene [
      • Pros E.
      • Lantuejoul S.
      • Sanchez-Verde L.
      • Castillo S.D.
      • Bonastre E.
      • Suarez-Gauthier A.
      • et al.
      Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.
      ]. In the case of RTKs, this allows the receptor to become constitutively active. Gene amplification in cancer cells can ensue by expansion during the process of tumor development, by de novo occurrence, or by clonal selection, as an adaptative strategy to bypass the pressure exerted during therapeutics. The latter constitutes a common mechanism for the acquired resistance to different TKI targeting RTKs [
      • Rotow J.
      • Bivona T.G.
      Understanding and targeting resistance mechanisms in NSCLC.
      ]. The most frequent gene amplification in lung cancer is FGFR1, described in up to approximately 25% of LuSCC, as above mentioned (Table 1) [
      • Qin A.
      • Johnson A.
      • Ross J.S.
      • Miller V.A.
      • Ali S.M.
      • Schrock A.B.
      • et al.
      Detection of known and novel FGFR fusions in non-small cell lung cancer by comprehensive genomic profiling.
      ].

      Gene amplification affecting the mesenchymal-epithelial transition factor receptor, MET

      The MET proto-oncogene encodes the RTK for hepatocyte growth factor and regulates a genetic program associated with cell proliferation and invasion of the extracellular matrix. MET mainly exists in epithelial cells and plays an important role in embryogenesis, tumor growth, and metastasis [
      • Comoglio P.M.
      • Trusolino L.
      • Boccaccio C.
      Known and novel roles of the MET oncogene in cancer: a coherent approach to targeted therapy.
      ]. In NSCLC, primary METamp has been reported in around 1–5% of tumors (Table 1), but, importantly, it also constitutes a mechanism for acquired resistance to EGFR-TKIs, as reported in 5–20% of the cases [
      • Guo R.
      • Luo J.
      • Chang J.
      • Rekhtman N.
      • Arcila M.
      • Drilon A.
      MET-dependent solid tumours - molecular diagnosis and targeted therapy.
      ], but also in other oncogenic RTK such as ALK tumors treated with ALK-TKIs [

      Dagogo-Jack I, Yoda S, Lennerz JK, Langenbucher A, Lin JJ, Rooney MM et al. MET alterations are a recurring and actionable resistance mechanism in ALK-positive lung cancer. Clin Cancer Res 2020;26 (11):2535-45. doi: 10.1158/1078-0432.CCR-19-3906.

      ]. Currently, METamp is defined by focal gains in MET copy number relative to the centromere of chromosome 7 (ratio MET/CEP7), although there is no well-established consensus on the most appropriate copy number cut-off. However, a higher ratio of MET/CEP7 copy number seems to predict a better response to MET inhibitors [
      • Noonan S.A.
      • Berry L.
      • Lu X.
      • Gao D.
      • Barón A.E.
      • Chesnut P.
      • et al.
      Identifying the appropriate FISH criteria for defining MET copy number-driven lung adenocarcinoma through oncogene overlap analysis.
      ]. METamp can be measured using FISH or NGS [
      • Guo R.
      • Luo J.
      • Chang J.
      • Rekhtman N.
      • Arcila M.
      • Drilon A.
      MET-dependent solid tumours - molecular diagnosis and targeted therapy.
      ,
      • Noonan S.A.
      • Berry L.
      • Lu X.
      • Gao D.
      • Barón A.E.
      • Chesnut P.
      • et al.
      Identifying the appropriate FISH criteria for defining MET copy number-driven lung adenocarcinoma through oncogene overlap analysis.
      ], but IHC of total MET protein is not considered a good surrogate marker for METamp tumors since strong MET immunostaining has been observed in many tumors without gene amplification, mostly in LuADs [
      • Pros E.
      • Lantuejoul S.
      • Sanchez-Verde L.
      • Castillo S.D.
      • Bonastre E.
      • Suarez-Gauthier A.
      • et al.
      Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.
      ] (Table 1).
      Notably, MET activation can also occur by point mutations that affect, mostly, consensus splice sites, leading to the elimination of exon 14 (METex14) [

      Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, et al. Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov 2015; 5(8):850–9. doi: 10.1158/2159-8290.CD-15-0285.

      ]. METex14 mutations are observed in 2–4% of NSCLCs and in both LuADs and LuSCCs, with a higher incidence in pulmonary sarcomatoid carcinomas [
      • Schrock A.B.
      • Frampton G.M.
      • Suh J.
      • Chalmers Z.R.
      • Rosenzweig M.
      • Erlich R.L.
      • et al.
      Characterization of 298 Patients with lung cancer harboring MET exon 14 skipping alterations.
      ,
      • Saigi M.
      • McLeer-Florin A.
      • Pros E.
      • Nadal E.
      • Brambilla E.
      • Sanchez-Cespedes M.
      Genetic screening and molecular characterization of MET alterations in non-small cell lung cancer.
      ]. These mutations have also been reported in other malignancies, such as gastric (7%) or colorectal (0–9%) cancers [

      Frampton GM, Ali SM, Rosenzweig M, Chmielecki J, Lu X, Bauer TM, et al. Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov 2015; 5(8):850–9. doi: 10.1158/2159-8290.CD-15-0285.

      ].
      Crizotinib has shown activity against MET activated lung tumors [
      • Camidge D.R.
      • Otterson G.A.
      • Clark J.W.
      • Ignatius Ou S.-H.
      • Weiss J.
      • Ades S.
      • et al.
      Crizotinib in Patients With MET-Amplified NSCLC.
      ]. More recently, the FDA approved two specific MET-TKIs, capmatinib and tepotinib, to treat NSCLC patients with METex14 mutant tumors [
      • Guo R.
      • Luo J.
      • Chang J.
      • Rekhtman N.
      • Arcila M.
      • Drilon A.
      MET-dependent solid tumours - molecular diagnosis and targeted therapy.
      ]. Note that for the EMA approval, patients harboring METex14 should have progressed to prior immunotherapy and/or platinum-based chemotherapy. Capmatinib has also shown activity in METamp tumors, particularly in those with a high gain copy number (≥10) (Table 2) [
      • Wolf J.
      • Seto T.
      • Han J.-Y.
      • Reguart N.
      • Garon E.B.
      • Groen H.J.M.
      • et al.
      Capmatinib in MET exon 14-mutated or MET-amplified non-small-cell lung cancer.
      ]. Furthermore, the dual EGFR and MET inhibition has proven effective in those cases in which METamp was the acquired resistance mechanism to EGFR-TKI [
      • Sequist L.V.
      • Han J.-Y.
      • Ahn M.-J.
      • Cho B.C.
      • Yu H.
      • Kim S.-W.
      • et al.
      Osimertinib plus savolitinib in patients with EGFR mutation-positive, MET-amplified, non-small-cell lung cancer after progression on EGFR tyrosine kinase inhibitors: interim results from a multicentre, open-label, phase 1b study.
      ].

      Gene amplification affecting the epidermal growth factor receptors HER

      The HER family, also called EGFR family, or ERBB family when referring to the gene, comprises four transmembrane receptor tyrosine kinases: EGFR (or HER1), HER2, HER3, and HER4. These receptors signal through homo- and hetero-dimerization and promote cell proliferation, motility, and invasion [
      • Wang Z.
      ErbB Receptors and Cancer.
      ]. EGFR amplification (EGFRamp), in association with EGFR protein overexpression, has been reported in both LuAD and LuSCC patients and, in LuADs, can co-exist with EGFR mutations [

      Nukaga S, Yasuda H, Tsuchihara K, Hamamoto J, Masuzawa K, Kawada I, et al. Amplification of EGFR wild-type alleles in non-small cell lung cancer cells confers acquired resistance to mutation-selective EGFR tyrosine kinase inhibitors. Cancer Res 2017;77(8):2078-89. 10.1158/0008-5472.CAN-16-2359.

      ]. EGFRamp has also been described as a resistance mechanism to third-generation EGFR-TKIs [

      Nukaga S, Yasuda H, Tsuchihara K, Hamamoto J, Masuzawa K, Kawada I, et al. Amplification of EGFR wild-type alleles in non-small cell lung cancer cells confers acquired resistance to mutation-selective EGFR tyrosine kinase inhibitors. Cancer Res 2017;77(8):2078-89. 10.1158/0008-5472.CAN-16-2359.

      ]. Despite the many available EGFR-TKIs, none of them has shown remarkable activity against EGFRamp tumors. Monoclonal antibodies targeting EGFR, such as cetuximab, have also provided disappointing results in NSCLC, in contrast to those in colorectal and head and neck cancer [
      • Mazzarella L.
      • Guida A.
      • Curigliano G.
      Cetuximab for treating non-small cell lung cancer.
      ]. Within the HER family, ERBB2, encoding HER2, shows oncogenic alterations in NSCLCs through either point mutations or gene amplification and represents a novel targetable RTK. These alterations are most prevalent in LuADs from never-smoker women. Most ERBB2 mutations are localized in exon 20, within the TKD, and the most frequent mutation is the p.Tyr772_Ala775dup (c.2313_2324dup), found in 1–2% of LuADs [
      • Jebbink M.
      • de Langen A.J.
      • Boelens M.C.
      • Monkhorst K.
      • Smit E.F.
      The force of HER2 - A druggable target in NSCLC?.
      ]. On the other hand, ERBB2 amplification (ERBB2amp) has been described in around 3% of NSCLCs, either de novo or as a mechanism of acquired resistance to EGFR-TKIs [
      • Jebbink M.
      • de Langen A.J.
      • Boelens M.C.
      • Monkhorst K.
      • Smit E.F.
      The force of HER2 - A druggable target in NSCLC?.
      ]. In contrast to breast and gastric cancer, where ERBB2 overexpression is relatively common and has met with a notable success of anti-HER2 therapies, targeting ERBB2 in NSCLC remains challenging. Although ERBB2amp shows strong HER2 protein levels [
      • Pros E.
      • Lantuejoul S.
      • Sanchez-Verde L.
      • Castillo S.D.
      • Bonastre E.
      • Suarez-Gauthier A.
      • et al.
      Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.
      ], initial trials of the anti-HER2 monoclonal antibody trastuzumab, alone or in combination with chemotherapy or pertuzumab, displayed only modest activity in ERBB2amp NSCLCs and yielded negative results in HER2-overexpressing patients [
      • Gatzemeier U.
      • Groth G.
      • Butts C.
      • Van Zandwijk N.
      • Shepherd F.
      • Ardizzoni A.
      • et al.
      Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer.
      ]. In the recent phase II DESTINY-Lung01 trial, the antibody-drug conjugate trastuzumab deruxtecan showed durable anticancer activity in patients with previously treated HER2-mutant NSCLC, regardless of HER2 expression and amplification status [
      • Li B.T.
      • Smit E.F.
      • Goto Y.
      • Nakagawa K.
      • Udagawa H.
      • Mazières J.
      • et al.
      DESTINY-Lung01 trial investigators. trastuzumab deruxtecan in HER2-mutant non-small-cell lung cancer.
      ].

      Gene amplification affecting the growth factor receptors KIT and PDGFRA

      The c-KIT proto-oncogene encodes a transmembrane receptor tyrosine kinase, KIT, expressed in several normal human tissues. Its ligand is the stem cell factor that mediates KIT dimerization and activation. Activating mutations at KIT have been documented in various neoplasms, particularly in gastrointestinal stromal tumors, among others [
      • Sihto H.
      • Sarlomo-Rikala M.
      • Tynninen O.
      • Tanner M.
      • Andersson L.C.
      • Franssila K.
      • et al.
      KIT and platelet-derived growth factor receptor alpha tyrosine kinase gene mutations and KIT amplifications in human solid tumors.
      ]. These mutations confer sensitivity to the TKI imatinib [
      • Heinrich M.C.
      • Corless C.L.
      • Demetri G.D.
      • Blanke C.D.
      • von Mehren M.
      • Joensuu H.
      • et al.
      Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.
      ]. On the other hand, the members of the platelet-derived growth factor receptor (PDGFR) family are protein-tyrosine kinases encoded by two genes: PDGFRA and PDGFRB [
      • Betsholtz C.
      • Karlsson L.
      • Lindahl P.
      Developmental roles of platelet-derived growth factors.
      ]. The functional receptors consist of the PDGFRα/α and PDGFRβ/β homodimers and the PDGFRα/β heterodimer. This RTK family plays an essential role in embryonic development and wound healing in adults [
      • Betsholtz C.
      • Karlsson L.
      • Lindahl P.
      Developmental roles of platelet-derived growth factors.
      ]. KIT and PDGFRA are closely located in the same arm of chromosome 4q12 and have been shown to co-amplify in about 2% of NSCLCs, both in LuADs and LuSCCs [
      • Pros E.
      • Lantuejoul S.
      • Sanchez-Verde L.
      • Castillo S.D.
      • Bonastre E.
      • Suarez-Gauthier A.
      • et al.
      Determining the profiles and parameters for gene amplification testing of growth factor receptors in lung cancer.
      ]. KIT/PDGFRA amplification has also been described as a resistance mechanism to ALK-TKIs in LuADs with ALK fusion [
      • Katayama R.
      • Shaw A.T.
      • Khan T.M.
      • Mino-Kenudson M.
      • Solomon B.J.
      • Halmos B.
      • et al.
      Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers.
      ]. To this day, these alterations have not been explored as therapeutic targets in lung cancer.

      Lung cancer molecular testing in clinics: State of the art

      According to clinical guidelines, an upfront genomic profiling test should be a priority to detect targetable oncogenic alterations in advanced non-squamous cell carcinomas and light/non-smokers squamous cell carcinoma patients diagnosed with lung cancer since these alterations are present in about 30% of the cases [
      • Schram A.M.
      • Chang M.T.
      • Jonsson P.
      • Drilon A.
      Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance.
      ,

      Jordan EJ, Kim HR, Arcila ME, Barron D, Chakravarty D, Gao J, et al. Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov 2017;7(6):596–609. doi: 10.1158/2159-8290.CD-16-1337.

      ]. Different diagnostic methods, including IHC, FISH, reverse transcriptase PCR, and DNA/RNA-based NGS, can be used to detect gene fusions and amplifications. A list of benefits and limitations of these approaches and the different validated diagnostic methods for each gene fusion and amplification are listed in Table 3. Conventional methods, such as IHC or FISH, have been widely implemented and are approved methodologies to detect gene amplification and specific fusions, including ALK or ROS1 [
      • Conde E.
      • Suárez-Gauthier A.
      • Benito A.
      • Garrido P.
      • García-Campelo R.
      • Biscuola M.
      • et al.
      Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.
      ,
      • Morán T.
      • Quiroga V.
      • Gil Mde L.
      • Vilà L.
      • Pardo N.
      • Carcereny E.
      • et al.
      Targeting EML4-ALK driven non-small cell lung cancer (NSCLC).
      ,
      • Conde E.
      • Hernandez S.
      • Prieto M.
      • Martinez R.
      • Lopez-Rios F.
      Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.
      ,
      • Sholl L.M.
      • Sun H.
      • Butaney M.
      • Zhang C.
      • Lee C.
      • Jänne P.A.
      • et al.
      ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas.
      ,
      • Shaw A.T.
      • Riely G.J.
      • Bang Y.-J.
      • Kim D.-W.
      • Camidge D.R.
      • Solomon B.J.
      • et al.
      Crizotinib in ROS1- rearranged advanced non-small-cell lung cancer (NSCLC): updated results, including overall survival, from PROFILE 1001.
      ]. However, based on the increasingly frequent need for a comprehensive genomic evaluation, NGS panels are becoming the preferred approach. Because NGS provides a great deal of genetic information that needs to be understood and classified according to clinical evidence within an appropriate time frame, its assessment should be provided by an expert molecular tumor board [
      • Mosele F.
      • Remon J.
      • Mateo J.
      • Westphalen C.B.
      • Barlesi F.
      • Lolkema M.P.
      • et al.
      Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group.
      ]. Despite the advantages of the NGS technology, access to NGS panels and treatments varies broadly among the different health systems worldwide.
      Table 3List of pros and cons of diagnostic techniques for the detection of gene fusions and gene amplification in clinical cancer management.
      MethodProsCons
      FISHWell-established approach

      No need for complex and expensive equipment

      Useful as a validation approach after positive IHC or NGS
      The cut-off values should be standardized for each gene

      Not able to identify the fusion partner involved

      Limited to detect intrachromosomic translocations

      IHCWell-established approach

      No need for complex and expensive equipment

      Useful for preselecting tumors for confirmatory FISH testing

      Allows to describe morphologic characteristics and tumor heterogeneity

      Excellent sensitivity for certain antibodies

      More cost-effective than FISH
      IHC score should be standardized

      Not able to identify the fusion partner involved

      Specificity might relay on the antibody
      NGSLarge amount of genetic information is provided at once

      In fusions, breakpoints are characterized at single nucleotide resolution

      Detection of unknown translocation partners

      Multiple samples can be pooled and sequenced together

      Suitable turn-around time
      Depends on the quality and the amount of sample

      Needs dedicated bioinformatics personnel to maintain a clinical NGS service

      Requires complex and expensive equipment

      RT-PCR based techniquesHigh specificity with robust and detailed information

      No need for complex and expensive equipment

      In fusions, breakpoints can be characterized at single nucleotide resolution
      Results depend on the quality of RNA

      Needs to be designed according to known fusion breakpoints

      Unable to detect unknown partners

      Unconclusive for the detection of gene amplification

      FISH: Fluorescent in situ hybridization, IHC: Immunohistochemistry, NGS: Next-generation sequencing, RT-PCR: Reverse transcriptase polymerase chain reaction.
      Tumor biopsies are the most common source of cancer cells for genotyping and categorizing tumors for clinical decisions. Good quality RNA and DNA can be obtained from them, and biopsies preserve the morphological features of the tumor. Histopathologic transformation as a resistance mechanism to TKIs can be determined. However, in lung cancer, tumor tissue extraction requires invasive procedures, becoming a caveat when multiple re-biopsies are needed to monitor the course of the disease. For this reason, alternative sources of tumor DNA are needed. A blood-based test using cell-free circulating tumor DNA (ctDNA), also called “liquid biopsy,” is a potential surrogate source of tumor DNA for diagnostic, prognostic, and therapeutic biomarkers in NSCLC patients. ctDNA has proven suitable to detect gene mutations and fusions involving RTKs with reasonable sensitivity and specificity across tumor types and is gaining interest in cancer monitoring [
      • Aggarwal C.
      • Rolfo C.D.
      • Oxnard G.R.
      • Gray J.E.
      • Sholl L.M.
      • Gandara D.R.
      Strategies for the successful implementation of plasma-based NSCLC genotyping in clinical practice.
      ]. ctDNA represents a non-invasive, rapid, and cost-effective strategy for obtaining DNA from tumor cells. However, the technique has not yet been fully translated into clinical practice, and the variability in the amount of ctDNA released by the tumors to the bloodstream may prevent the standardization of the procedure [
      • Aggarwal C.
      • Rolfo C.D.
      • Oxnard G.R.
      • Gray J.E.
      • Sholl L.M.
      • Gandara D.R.
      Strategies for the successful implementation of plasma-based NSCLC genotyping in clinical practice.
      ]. In addition, the quantity of tumor ctDNA in the whole DNA extract can be excessively low to develop high-throughput analyses and can render artefactual mutations (false positive) or, according to the sensitivity of the method, false negative results. Large-scale screening and standardization of experimental steps could resolve these problems.

      Therapeutic challenges and opportunities in NSCLC patients harboring oncogenic fusions

      Despite incorporating novel and highly selective TKIs for lung tumors harboring specific oncogenic mutations, advanced-stage lung cancer remains largely incurable. Treatments facilitate the emergence of resistant clones or the selection of pre-existing resistant sub-clones, and relapsing is unavoidable. Acquired resistance, defined as progression after initial benefit, is mediated by different biologic mechanisms that allow tumor adaptation. Therefore, re-biopsy is always encouraged to identify the mechanisms underlying the acquired resistance and evaluate whether it can be approached therapeutically. The alterations that drive resistance include acquired mutations in the target oncogene (on–target resistance) and alterations in genes coding for proteins acting in parallel or downstream signaling pathways that allow bypassing the action of the TKI (off-target resistance) [
      • Camidge D.R.
      • Pao W.
      • Sequist L.V.
      Acquired resistance to TKIs in solid tumours: learning from lung cancer.
      ]. Furthermore, although not very common, histopathologic transformation, mostly from LuAD to small-cell lung carcinoma or LuSCC, represents another mechanism of resistance to targeted TKI and has been described in LuADs with mutations in EGFR or with ALK and ROS1 fusions [
      • Pros E.
      • Saigi M.
      • Alameda D.
      • Gomez-Mariano G.
      • Martinez-Delgado B.
      • Alburquerque-Bejar J.J.
      • et al.
      Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.
      ,
      • Cha Y.J.
      • Cho B.C.
      • Kim H.R.
      • Lee H.-J.
      • Shim H.S.
      A Case of ALK-Rearranged Adenocarcinoma with Small Cell Carcinoma-Like Transformation and Resistance to Crizotinib.
      ,
      • Hsieh M.-S.
      • Lin M.-W.
      • Lee Y.-H.
      Lung adenocarcinoma with sarcomatoid transformation after tyrosine kinase inhibitor treatment and chemotherapy.
      ]. In some cases, genetic alterations may be associated with this transformation, for instance, in some LuADs that have become resistant to EGFR-TKIs through the TACC3-FGFR3 fusion [
      • Pros E.
      • Saigi M.
      • Alameda D.
      • Gomez-Mariano G.
      • Martinez-Delgado B.
      • Alburquerque-Bejar J.J.
      • et al.
      Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.
      ]. The pre-existence of RB1 inactivation, common in mutant-EGFR LuADs, has also been associated with a higher predisposition to develop acquired resistance by histopathological transformation [
      • Pros E.
      • Saigi M.
      • Alameda D.
      • Gomez-Mariano G.
      • Martinez-Delgado B.
      • Alburquerque-Bejar J.J.
      • et al.
      Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.
      ]. Once the tumor undergoes histologic transformation, the prognosis is detrimental, and the therapeutic approach is undertaken on a case-by-case basis.
      Disease progression after treatment with TKIs can also occur because of an inadequate exposure of the drug to the receptor. The low penetration of these drugs into certain organs, particularly into the brain, poses additional challenges. This is particularly important for patients with NSCLCs harboring oncogenic driver fusions, who have an increased risk of developing brain metastases (20 to 40% at the time of diagnosis, which can increase up to 80% over the course of their disease) [
      • Tan A.C.
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      Brain metastases in lung cancers with emerging targetable fusion drivers.
      ]. Since brain metastases constitute an independent prognostic factor for worse overall survival and poorer quality of life, novel generation TKIs with improved intracranial activity are being designed. These TKIs need to demonstrate higher efficacy in reducing brain metastases than previous generations, to prevent the onset of new metastases, and to delay local therapeutic strategies, such as whole-brain radiotherapy and its related toxicity [
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      Brain metastases in patients with EGFR-mutated or ALK-rearranged non-small-cell lung cancers.
      ].
      Of note, a small proportion of NSCLC patients with targetable fusions can present oligometastatic disease at diagnosis or undergo oligoprogression under TKI treatment [
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      ]. This means that, while maintaining a systemic overall response to TKI, a subset of tumor clones has become resistant and has progressed into specific locations. Therefore, adding local ablative strategies should be considered to increase the disease’s control rate with prognostic implications [
      • Franceschini D.
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      ].
      Novel and specific inhibitors are the preferred therapeutic options for NSCLC patients with actionable driver oncogenes. Consequently, most immunotherapy trials exclude these patients, and the benefits of these treatments remain unclear for them. The majority of the currently available evidence comes from subgroup analysis from real-world data and clinical trials or from small trials specifically designated to address this issue [
      • Addeo A.
      • Passaro A.
      • Malapelle U.
      • Luigi Banna G.
      • Subbiah V.
      • Friedlaender A.
      Immunotherapy in non-small cell lung cancer harbouring driver mutations.
      ]. Actionable driver fusions are commonly found in never-smoker patients, which tend to be associated with a low tumor mutation burden and a less inflamed tumor microenvironment. The multicenter study Immunotarget retrospectively collected the clinical outcomes of NSCLC patients harboring driver mutations who were treated with immunotherapy. Its results showed low efficacy in patients harboring oncogenic driver fusions (e.g., ALK or RET), regardless of the levels of PD-L1 [
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      ].
      Finally, the clinical management of lung cancer patients, as for many other cancers, would need to consider genetic predisposition as another variable. Most NSCLCs with actionable oncogenic drivers, including fusions and gene amplification, are found in never-smokers and, often, in young individuals. To date, the explanation is unclear, and the possibility that these cancers arise within hereditary cancer syndromes should be considered. In this regard, the Li-Fraumeni syndrome is the most common hereditary syndrome associated with lung cancer development, especially EGFR-mutant tumors [
      • Pros E.
      • Saigi M.
      • Alameda D.
      • Gomez-Mariano G.
      • Martinez-Delgado B.
      • Alburquerque-Bejar J.J.
      • et al.
      Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.
      ,
      • Mezquita L.
      • Jové M.
      • Nadal E.
      • Kfoury M.
      • Morán T.
      • Ricordel C.
      • et al.
      High prevalence of somatic oncogenic driver alterations in patients with NSCLC and Li-fraumeni syndrome.
      ]. The occasional association of lung cancer with other cancer syndromes, such as BRCA1/2, among others, should not be ruled out either [
      • Pros E.
      • Saigi M.
      • Alameda D.
      • Gomez-Mariano G.
      • Martinez-Delgado B.
      • Alburquerque-Bejar J.J.
      • et al.
      Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.
      ].

      Conclusions

      TKIs have prompted significant improvements in the outcomes of patients with lung tumors harboring tyrosine kinase driven oncogenic mutations or rearrangements (fusions or gene amplification). Despite the low frequency of each mutation or rearrangement involving RTKs, altogether, they affect about 15% of NSCLCs. Currently, identifying these patients remains crucial because they can achieve survival for up to several years when treated with the appropriate inhibitor. The magnitude of the clinical benefits achieved with targeted therapies and the increasing number of specific clinical trials has prompted the integration of molecular profiling technologies in clinical and pathological settings. The key for succeeding with targeted therapeutics is integrating an accurate diagnosis with potent and selective therapeutics together with an optimal penetration of new, low-toxicity drugs into the central nervous system. Although several promising targeted drugs have been recently approved or are under clinical evaluation, optimal strategies to overcome resistance, including combinations with immunotherapy or chemotherapy, have not been clearly established. The future of precision medicine will likely integrate comprehensive genomic tumor characterization, dynamic monitoring of liquid biopsy and/or tissue guided re-biopsies, and the enrollment of patients into innovative clinical trials.

      CRediT authorship contribution statement

      Maria Saigí: Conceptualization, Writing – original draft, Writing – review & editing, Supervision. Enric Carcereny: Writing – review & editing. Teresa Morán: Writing – original draft, Writing – review & editing. Marc Cucurull: Writing – review & editing. Marta Domènech: Writing – original draft, Writing – review & editing. Ainhoa Hernandez: Writing – review & editing. Anna Martinez-Cardús: Writing – review & editing, Supervision. Eva Pros: Conceptualization, Writing – original draft, Writing – review & editing, Supervision. Montse Sanchez-Cespedes: Conceptualization, Writing – original draft, Writing – review & editing, Supervision.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      This work was supported by Spanish grant from ICAPEM (ICAPEM becas 2021), to EP, and by Spanish grant form Asociación Española Contra el Cancer (AECC) (grant number GCB14142170MONT) to MSC. We thank M Rey providing language help and O Romero for data analysis support.

      Funding

      MS is supported by a Juan-Rodés contract from the Instituto de Salud Carlos III (JR20/00015).

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