Cancer Treatment Reviews 109 (2022) 102430 Contents lists available at ScienceDirect Cancer Treatment Reviews journal homepage: www.elsevier.com/locate/ctrv Tumour Review Biological and clinical perspectives of the actionable gene fusions and amplifications involving tyrosine kinase receptors in lung cancer Maria Saigí a, b, *, Enric Carcereny a, b, Teresa Morán a, b, Marc Cucurull a, b, Marta Domènech a, b, Ainhoa Hernandez a, b, Anna Martinez-Cardús b, Eva Pros c, Montse Sanchez-Cespedes c, * a Department of Medical Oncology, Catalan Institute of Oncology (ICO), Badalona, Barcelona, Spain b Badalona⋅Applied Research Group in Oncology (B⋅ARGO). Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Badalona, Barcelona, Spain c Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Identifying molecular oncogenic drivers is crucial for precision oncology. Genetic rearrangements, including Lung cancer gene fusions and gene amplification, involving and activating receptor tyrosine kinases (RTKs) are recurrent in Gene fusions solid tumors, particularly in non-small cell lung cancer. Advances in the tools to detect these alterations have Gene amplification deepened our understanding of the underlying biology and tumor characteristics and have prompted the Tyrosine kinase receptors Tyrosine kinase inhibitors 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 target­ able 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 tech­ nologies. 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. Introduction essential genes that contribute to the oncogenic process. For instance, chromosomal rearrangements could result in gene fusions or gene am­ Precision oncology has revolutionized the therapeutic landscape of plifications that lead to the expression of oncoproteins. When the fusion advanced non-small cell lung cancer (NSCLC). Over the last two de­ or amplification involves a receptor tyrosine kinase (RTK), the tyrosine cades, the deep genomic profiling of lung cancers has enabled identi­ kinase domain (TKD) is activated—often constitutively and ligand- fying many new genes involved in the development of this disease, independent—and downstream effectors of the receptor receive con­ including oncogenic drivers that have contributed to our understanding stant signaling, causing uncontrolled cell growth and invasiveness [1,2]. of lung carcinogenesis and have established an era of precision medicine Then, the tumor cell becomes dependent on this oncogenic RTK to in advanced-stage NSCLC [1,2]. maintain its malignant properties. This dependency, also called “onco­ Tumors often have complex and unstable genomes that cause gene addiction,” can be therapeutically approached with drugs that random mutations and genomic aberrations. While most of these alter­ inhibit the activity of the oncoprotein. Currently, most RTKs inhibitors ations are unlikely to affect tumor development, some may involve are designed to prevent either the binding of the ligand—often by using * 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.) E-mail addresses: msaigi@iconcologia.net (M. Saigí), ecarcereny@iconcologia.net (E. Carcereny), mmoran@iconcologia.net (T. Morán), mcucurull@iconcologia. net (M. Cucurull), mdomenech@iconcologia.net (M. Domènech), ahernandezg@iconcologia.net (A. Hernandez), amartinezc@igtp.cat (A. Martinez-Cardús), epros@ carrerasresearch.org (E. Pros), mscespedes@carrerasresearch.org (M. Sanchez-Cespedes). https://doi.org/10.1016/j.ctrv.2022.102430 Received 21 March 2022; Received in revised form 20 May 2022; Accepted 15 June 2022 Available online 18 June 2022 0305-7372/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 monoclonal or bispecific antibodies, and antibody-drug conjugates—or to be treated with compounds approved by the US Food and Drug the binding of the ATP to the catalytic domain—mostly with small Administration (FDA) as standard-of-care therapy. Additional com­ molecules. Among the latter, tyrosine kinase inhibitors (TKIs), which pounds are under development for tumors carrying MET amplification inhibit the catalytic domain of the target enzyme by preventing phos­ (METamp) or activating mutations in ERBB2 [3]. Other fusions (e.g., phorylation and subsequent signaling, are the most widely used in NRG1, and FGFRs) areinfrequently detected in routine clinical practice clinics [3]. On the other hand, other personalized therapeutic strategies owing to their rarity. Emerging drugs such as zenocutuzumab, a bi- include immune targeted approaches such as checkpoint blockade, specific antibody, have reported activity in NRG1 tumors, regardless personalized vaccines and/or chimeric antigen receptor T-cells [4]. of histologic type [7–9]. A timeline depicting the identification of Slightly more than a dozen RTKs are genetically activated in NSCLC. selected oncogenic alterations (gene amplifications and fusions) in RTKs Although gene fusions and gene amplifications are the most common of NSCLC and the different treatments approved for each of the activated mechanisms of RTKs’ activation, point mutations can also occur, as in RTKs is represented in Fig. 2. MET, ERBB2, and EGFR (the latter is the most frequently RTK activated This review will discuss the most relevant gene fusions and gene in lung cancer) [2–3]. For still unknown reasons, activated RTKs prevail amplifications activating RTK in NSCLC, with special emphasis on the in lung cancer adenocarcinomas (LuADs) of relatively young females, genomic structure, diagnostic approaches, and available therapeutics of since they are related to lower tobacco-exposure (light- or never- each case. smokers)These patients are often diagnosed with advanced-stage can­ cer and have high incidences of pleural effusion and sclerotic bone and Oncogenic gene fusions involving RTK in NSCLC and their brain metastases as common patterns of tumor spread [5–7]. The clinical implications recently discovered fusion CLIP1–LTK in LuADs, leading to the onco­ genic activation of the RTK LTK [8], will likely share similar charac­ Gene fusions affecting the anaplastic lymphoma kinase receptor, ALK teristics. Some exceptions are the activation of FGFRs, more common in lung squamous cell carcinomas (LuSCCs), and NTRKs, which occur in ALK encodes a transmembrane RTK expressed in the nervous system NSCLCs across gender, age, smoking history, and histopathology [7,9]. during embryogenesis. ALK was first identified to fuse with NPM1 in Most of the alterations that activate RTKs are mutually exclusive be­ anaplastic large-cell lymphoma in 1994, but it was not until 2007 that tween them and between alterations of molecules involved in signal ALK fusions were reported in NSCLC [10]. At present, this alteration is transduction. This feature suggests that they are all functionally con­ considered to affect 2–7% of LuADs, mostly women and never-smokers. nected and confer similar growth advantages. This characteristic is Oncogenic ALK rearrangements fuse the intact kinase domain of ALK to highlighted in Fig. 1, in which an oncoplot has been drawn to show the the N–terminal regions of its partner, resulting in overexpression and associations between genetic alterations that affect RTK and signal constitutive, ligand-independent activation of ALK. Overall, more than transduction molecules among lung Tumor Cancer Genome Atlas. 20 fusion partners have been identified, with EML4 as the most preva­ Currently, at least six of the different oncogenic alterations affecting lent (Fig. 3) [11]. RTKs in NSCLC, including activating mutations in EGFR and MET Because ALK fusions involve large chromosomal inversions and (METex14), as well as fusions in ALK, ROS1, NTRK, and RET, are eligible translocations, fluorescent in situ hybridization (FISH) using break-apart Fig. 1. Oncoplots 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. 2 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 Fig. 2. Timeline 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. probes was the first method developed for detecting all ALK rear­ brigatinib over crizotinib in previously untreated LuAD patients with rangements and the first to receive approval by the FDA [12–13]. Since ALK-rearranged tumors [23]. Since both trials showed similar outcomes ALK fusions trigger an increase in the mRNA and protein levels, and toxicity profiles, alectinib and brigatinib represent a reasonable immunohistochemistry (IHC) can also be used. Owing to its high option for first-line treatment. Finally, lorlatinib, a selective and third- sensitivity and cost-effectiveness, IHC with the Ventana ALK D5F3 generation TKI that inhibits ALK and ROS1, has activity against most antibody gained FDA approval [14]. RNA-based next-generation known ALK drug-resistant mutations [24]. Based on the results of the sequencing (NGS) is also a valid technique to reveal ALK fusions as long CROWN trial, lorlatinib also gained FDA and EMA (European Medicines as the quality of RNA is optimal [15] (Table 1). Agency) approval as a first-line treatment [25]. Since no trial has Currently, five TKIs (i.e., crizotinib, ceritinib, alectinib, brigatinib, compared the efficacy of all ALK-TKIs in first-line, lorlatinib also seems a and lorlatinib) have been approved by the FDA to treat NSCLCs reasonable option, particularly when considering its activity in the harboring ALK translocations (Table 2). Crizotinib is a first-generation brain. More recently, the small-molecule ensartinib (X-396) demon­ TKI that inhibits ALK but also targets ROS1 and MET and was the first strated greater efficacy than crizotinib in both systemic and intracranial to show efficacy in previously treated patients [16–17]. Almost one- disease, thus representing another first-line therapeutic option for pa­ third of crizotinib-treated patients acquire resistance by mutations tients with ALK-positive NSCLC [26] (Table 2). affecting the kinase domain, with p.Leu1196Met and p.Gly1269Ala as Despite the therapeutic efficacy of these TKIs, the emergence of ac­ the most prevalent [18]. Ceritinib, alectinib, and brigatinib are second- quired resistance may limit their long-term benefits. The solvent-front p. generation ALK-TKIs that bind to the receptor with higher affinity than Gly1202Arg substitution impairs the correct binding of the drug to ALK crizotinib. These TKIs show activity against multiple mutations acquired and constitutes a common resistance mechanism to ALK-TKIs [27]. Be­ during the treatment with crizotinib and have an improved penetrance sides, ALK-independent (or off-target) resistance mechanisms such as into the central nervous system [18–20]. The results of the phase III METamp, detected in almost 15% of cases [28], loss of NF2, or histo­ ALEX trial evidenced better clinical outcomes for alectinib than for pathologic transformation into a neuroendocrine subtype have also been crizotinib in terms of median progression-free survival (mPFS) (34.8 vs reported [29]. 10.9 months; hazard ratio [HR]: 0.43) -investigator assessed- and me­ dian overall survival (mOS) (not reached [NR] vs 57.4 months; HR: Gene fusions affecting the c-ros oncogene 1 receptor, ROS1 0.67). On the basis of this study, alectinib became the most common first–line treatment for patients with ALK–rearranged tumors [21,22] ROS1 is a receptor with tyrosine kinase activity that shares structural (Table 2). Similarly, the ALTA1L trial demonstrated a superior mPFS for homology, within the TKD, with ALK and can also undergo genomic 3 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 Fig. 3. Molecular 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 trans­ membrane 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 Table 1 List of oncogenes altered by gene fusions and amplification in NSCLC. Driver Histopathologic predominance Incidence Cancer type for FDA IHC FISH NGS fusion/amp pattern approval technique ALK fusion LuAD solid, mucinous cribiform, 2–7% NSCLC Ventana ALK D5F3 IHC Vysis ALK break Apart FISH NGS and signet ring cells CDx assay* Probe Kit (Abbott Molecular) FoundationOne * CDx* ROS1 fusion LuAD solid, mucinous cribiform, 2% NSCLC ROS1 D4D6 IHC assay* Vysis ROS1 break-apart FISH NGS and signet ring cells Cell Signaling Technology Probe Kit FoundationOne CDx* NTRK LuAD 3.5% Solid tumors Loxo/Ventana Pan-TRK NA NGS (1,2,3) <1% IHC CDx* FoundationOne fusion <1% CDx* RET fusion LuAD solid, mucinous cribiform, 1–2% NSCLC, thyroid NA NA Oncomine Dx and signet ring cells, LuSCC Target Test* FGFR3 fusion LuSCC/LuAD <1% NA NA NA DNA/RNA-based NGS NRG1 fusion LuAD <1% NA NA NA DNA/RNA-based NGS LTK fusion LuAD <1% NA NA NA DNA/RNA-based NGS FGFR1 amp LuSCC 15–25% NSCLC-SCC NA NA DNA-based NGS MET amp LuAD, LuSCC, PSC 1–5% NA NA NA DNA-based NGS ERBB2 amp LuAD 2–5% Breast, gastric cancer HercepTest (Dako)* HER2 FISH pharmDx Kit DNA-based NGS (Dako)* KIT/PDGFRA LuAD, LuSCC 1–2% NA NA NA DNA-based NGS amp * 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 adenocarci­ noma; LuSCC: Lung squamous cell carcinoma; NA: None approved; NGS: Next-generation sequencing; NSCLC: Non-small cell lung cancer; PSC: Pulmonary sarcomatoid carcinoma. 4 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 Table 2 List of most relevant clinical trials and clinical efficacy of targeted therapies for NSCLC with tumors with driven oncogenic fusions and amplifications. Biomarker Targeted Drug Trial Study type ORR (95% CI) mPFS months HR mOS months HR FDA approval Ref. (type) (95% CI) (95% CI) ALK Crizotinib Profile 1001, Phase I (ALK 60.8% (52.3–68.9) 9.7 (7.7–12.8) 12; OS rate: 74.8% Accelerated 16 ≥1st L cohort n = (66.4–81.5) approval (2011) 143) Profile 1007, ≥2nd L Phase III (n = 65% vs 20% 7.7 vs 3; HR: 0.49 20.3 vs 22.8 HR: Approved (2013) 17 (vs CT) 173) (0.37–0.64) 1.02 (0.68–1.54) Profile 1014, 1st L (vs Phase III (n = 75% vs 45% 10.9 vs 7; HR: 0.45 NR vs 47.5; HR: 18 CT) 343) (0.35–0.60) 0.76 (0.55–1.05) Ceritinib ASCEND 4, 1st L (vs Phase III (n = 72.5% (65.5–78.7) vs 16.6 vs 8.1; HR: NA Approved (2014) 19 CT) 376) 26.7% (20.5–33.7) 0.55 (0.42–0.73) ASCEND 5, ≥2nd L (vs Phase III (n = 39.1% (30.2–48.7) vs 5.4 vs 1.6; HR: 0⋅49 NA 20 CT) 231) 6.9% (3.0–13.1%) (0.36–0.67) Alectinib ALEX (vs crizotinib) Phase III (n = 82.9% (75.0–88.5) vs 34.8 vs 10.9; HR NR vs 57.4; HR: Approved (2017) 22 1st L 303) 75.5% (67.0–82.0) 0.43 (0.32–0.58) 0.67 (0.46–0.98) Brigatinib ALTA 1L (vs Phase III (n = 71% (62–78) vs 60% 24 vs 11; HR 0.49 NA Approved (2020) 23 crizotinib), 1st L 275) (54–66) (0.35–0.68) Lorlatinib NCT01970865, ALK- Phase I/II (n 48% (42–55) NA NA Accelerated 24 TKI pre-tt = 275) approval (2018) CROWN (vs Phase III (n = 76% (68–83) vs 58% NR vs 9.3; HR 0.28 NA Approved (2021) 25 crizotinib), 1st L 296) (49–66) (0.19–0.41) Ensartinib or EXALT-3 (vs Phase III (n = 75% vs 67% 25.8 vs 12. 7; HR NA 26 X-396 crizotinib), 1st L 290) 0.51 (0.35–0.72) (multi-TKI) ROS1 Crizotinib Profile 1001, ≥1st L Phase I (ROS- 72% (58–84%) 19.3 (15.2–39.1) 51.4 (29.3-NR) Approved (2016) 33 1 cohort n = 50) EURCROSS, Europe, Phase II (n = 70% (51–85) 10.1 (20-NR) NA 34 TKI-naïve 34) Lorlatinib NCT01970865, TKI- Phase II (n = 62% (TKI-naïve)/ NA NA Approved (2019) 35 naïve and pre-tt 69) 35% (pre-tt) Entrectinib ALKA 372–001, Phase I/II (n 77.4% 24.6 (11.4–34.8) NA Approved (2019) 36 (multi-TKI) STARTK-1, TKI-naïve = 53) Ceritinib NCT 01964157, TKI- Phase II (n = 62% 9.3 (0–22) all /19.3 24 (5–43) 37 (multi-TKI) naïve and pre-tt 32) (1–37) tt-naïve Repotrectinib or TRIDENT-1, TKI-naïve Phase II (n = 86% NA NA 38 TPX-0005 7) (multi-TKI) Biomarker Targeted Drug (type) Trial Study type ORR (95% CI) mPFS months mOS months FDA approval Ref. HR (95% CI) HR (95% CI) NTRK Larotrectinib LOXO-TRK-14001, Phase I & II 79% (72–88) 28.3 (22.1-NR) 44.4 (36.5- Approved for solid 45, (Selective-TKI) SCOUT & NAVIGATE (n=159) NR) agnostic tumors 46 (2018) Entrectinib ALKA-372–001, Phase I & II 57% (43.2–70.8) 11 (9–14.9) 21 (14.9-NA) Approved for solid 47 (multi-TKI) STARTRK-1 & (n=54) agnostic tumors STARTRK-2 (2019) Selitrectinib or LOXO- NCT03215511 (TKI Phase I (n=20) 36% NA NA Orphan drug 49 195 (selective-TKI) pre-tt) designation Repotrectinib or TPX- TRIDENT-1 (TKI pre- Phase II (n=6) 50% NA NA 50 0005 (multi-TKI) tt) RET Cabozantinib NCT01639508, pre-tt Phase II (n=25) 28% 5.5 (3.8–9.4) 9.9 (8.1-NR) 56 (multi-TKI) Vandetanib LURET, pre-tt Phase II (n=19) 53% 6.5 (2.8–9.5) 13.5 57 (multi-TKI) (9.8–29.1) Lenvatinib NCT01877083, pre-tt Phase II (n=25) 16% 7.3 (3.6–10.2) NA 58 (multi-TKI) Selpercatinibor LOXO- LIBRETTO-001,1st L or Phase I/II 68% (pre-tt, 18.4 (13.8–24) NA Accelerated 59 292 pre-tt (n=105) n=105)/85% (tt- /NR (9.2-NR) approval (2020) (selective-TKI) naïve, n=34) Pralsetinib or BLU- ARROW, 1st L or pre-tt Phase I/II 58% (pre-tt n=48)/ NA NA Accelerated 60 667 (selective-TKI) (n=114 NSCLC) 71% (tt-naïve, n=7) approval (2020) MET amp Crizotinib Profile 1001, ≥1st L Phase I (MET GCN≥4: 44%; GCN 6.7/1.9/1.8 NA 81 cohort=37) 2-<4: 14%; GCN<2: 33% Capmatinib GEOMETRY, ≥1st L Phase II, 40%/29% 4.2/4.1 9.6/10.6 82 GCN≥10 (n=14/55) 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. 5 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 rearrangements to create fusion proteins [30]. For ALK and other RTK resistance to these TKIs can eventually arise by the emergence of sec­ fusions, the fusion partner provides a dimerization domain that induces ondary mutations at the kinase domain of NTRK [48]. Next-generation constitutive oligomerization and, thus, activation of the kinase. How­ agents, such as selitrectinib (LOXO-195) and repotrectinib, were ever, since most ROS1 fusion partners lack this dimerization domain, the designed to address on-target resistance mechanisms and have shown mechanism for ROS1 oncogenic activation remains unknown [31]. In promising activities in clinical trials (Table 2) [49–51]. Given the potent the ROS1 fusions, the kinase domain of ROS1 is paired with a wide range clinical activity of the TRK inhibitors, the current clinical guidelines of partners, the most common being CD74 (Fig. 3) [30]. Chromosomal recommend testing all NSCLCs for NTRK fusions. rearrangements of the ROS1 proto-oncogene occur in about 1–2% of LuADs and more often in never-smokers [30]. The diagnosis of ROS1 Gene fusions affecting the REarranged during transfection receptor, RET fusions can be performed by IHC—the most cost-effective method—and confirmed by FISH, but NGS can also be used (Table 1) [32]. The RET proto-oncogene encodes a transmembrane RTK involved in Crizotinib was the first TKI to demonstrate activity in ROS1-rear­ numerous developmental pathways and multiple malignancies, ranged tumors, showing promising overall response rates (ORR) (mPFS including multiple endocrine neoplasia 2, papillary thyroid cancer, and of 19.3 months and mOS of 51.4 months (95% CI 29.3- NR) in a subset of NSCLC. Unlike other RTKs, RET does not directly bind to its ligands but mostly pretreated patients with advanced-stage NSCLC [33]. At present, requires glycosyl-phosphatidylinositol-anchored (GFRalpha1-4) co- crizotinib remains the recommended first-line therapy for these patients receptors. Indeed, ligands of the glial cell line-derived neurotrophic [34]. Lorlatinib has also demonstrated efficacy in both TKI-naïve and factor family bind to one of the four co-receptors, which subsequently TKI-pretreated patients with ROS1-rearranged tumors [35]. Entrectinib, allow RET dimerization, autophosphorylation, and activation, leading which also targets TRKA/B/C and ALK, has shown activity against these to downstream activation signaling [52]. RET fusions can be found in tumors as well, inducing meaningful intracranial responses in patients 1–3% of LuADs and are more common in young, female, never-smoker with brain metastases [36]. While these three drugs have gained FDA patients (Table 1). More than ten RET fusion partners have been approval, other agents such as ceritinib, brigatinib, repotrectinib (TPX- described, being KIF5B the most frequent (Fig. 3) [52,53]. Unlike ALK 0005), and DS-6051b are currently being evaluated in phase I and II and ROS1 fusions, RET fusions cannot be properly detected by IHC trials [37–39] (Table 2). because of the low sensitivity and highly variable specificity of the The genetic mechanisms that confer resistance to crizotinib in tu­ available antibodies. On the other hand, FISH is a sensitive but rather mors with ROS1 fusions are equivalent to those in ALK-rearranged unspecific approach to test for RET fusions in NSCLC, owing to the NSCLC. The p.Gly2032Arg mutation, which is structurally analogous to frequent presence of RET rearrangements not resulting in oncogenic RET the p.Gly1202Arg mutation in ALK, is the most common, although other [54]. Therefore, NGS is the most appropriate option for diagnosing RET mutations (e.g., p.Asp2033Asn and p.Ser1986Phe) have also been re­ fusions [55] (Table 1). ported [40]. Since not all on-target mutations are equally sensitive to the Several multitarget kinase inhibitors, such as cabozantinib, vande­ new generation of inhibitors, tumor stratification according to the type tanib, and lenvatinib—approved for treating patients with advanced of mutation will be required. medullar thyroid cancer—have limited activity in RET-rearranged NSCLC and important adverse effects due to their off-target activity Gene fusions affecting the neurotrophic tropomyosin tyrosine kinase [56–58]. More recently, two highly potent and selective RET-TKI, sel­ receptor, NTRK percatinib and pralsetinib, have gained FDA approval for lung and thyroid cancers. In the LIBRETTO-001 trial on advanced NSCLC, sel­ The NTRK-family of genes comprises three members (NTRK1, percatinib (LOXO-292) was given to pretreated (ORR of 65%) and NTRK2, and NTRK3) that encode tropomyosin receptor kinases (TRK) A, treatment-naïve patients (ORR of 85%) [59] (Table 2). Pralsetinib’s B, and C, respectively. These kinase receptors are physiologically (BLU-667) efficacy was established in the phase I/II ARROW trial with involved in neuronal development and differentiation. The aberrant similar results [60]. Both drugs were well tolerated with an acceptable activation of NTRK, which results in the constitutive activation of safety profile and relevant intracranial activity regardless of the RET- downstream pathways, appears mainly through gene fusions and several fusion partner (ORR of selpercatinib: 91% and ORR of pralsetinib: 78%). partners have been described (Fig. 3) [41]. These alterations have a However, p.Gly810Arg/Ser/Cys mutations have been described as ac­ prevalence of 0.2–0.4% across several solid tumor types in adult cancers, quired resistance mechanisms to selective RET inhibitors [61]. Second- regardless of gender and smoking habit, with higher prevalence in pe­ generation RET-TKIs (e.g., TPX-0046 [NCT04161391] and BOS-172738 diatric population (1.34%). Among adult patients, NTRK fusions are [NCT03780517]) are currently being tested in clinical trials [62]. more prevalent in salivary gland cancers (2.43%), soft tissue sarcoma (1.27%) and thyroid cancers (1.28%),albeit their frequency in LuADs is Gene fusions affecting the fibroblast growth factor receptors, FGFRs low (<1%) [42]. Notably, the rare fusion ETV6–NTRK3 is detectable in nearly all patients with secretory carcinoma of the breast and with FGFRs are a family of RTKs expressed on the cell membrane that play mammary analogue secretory carcinoma of the salivary glands and is, crucial roles in developmental and adult cells. The human family of thus, considered pathognomonic in these two histologically identical FGFRs consists of four members: FGFR1, FGFR2, FGFR3, and FGFR4 tumor types [43]. IHC screening confirmed by FISH or by NGS are [63]. Despite being encoded by different genes, the four members share appropriate methods for diagnosing these fusions [44] (Table 1). high homology. The aberrant activation of FGFRs, caused by activating The first generation of TKIs that blocked the activity of RTKs were gene fusions, amplifications, or mutations involving the TKD, is a larotrectinib (a selective TRK inhibitor) and entrectinib (a multikinase frequent oncogenic mechanism in different types of cancer, such as inhibitor also targeting ROS1 and ALK), and both conferred durable glioblastomas, cholangiocarcinomas, breast cancer, and bladder cancer, responses in patients with metastatic disease (ORR of larotrectinib: 75% among others [63]. Although fusions affecting FGFRs are uncommon, and ORR of entrectinib: 61.2%, with a mPFS of 13.8 months (95% CI, they have been described in lung cancer, mostly in LuSCCs. The most 10.1–19.9), regardless of tumor histology, age, or fusion partner widely recognized is the fusion between FGFR3 and TACC3 [9] (Fig. 3). [45–47]. The activity of larotrectinib have also been reported in 15 Fusions at FGFR1, FGFR2, and FGFR4 are even rarer and still poorly patients with NSCLC harboring NTRK fusions, showing a ORR of 73%, characterized. Alterations at FGFR1 in lung cancer are mostly gene mPFS of 35.4 months (95% CI, 5.3–35.4), and mOS of 40.7 months (95% amplification and are found in up to 25% of LuSCCs, approximately CI, 17.2 to not estimable) [46]. These two TKIs gained tumor-agnostic (Table 1) [64]. regulatory accelerated approvals by the FDA to treat cancers In contrast to ALK, ROS1, and RET, FGFR-fusions are more frequent harboring NTRK-fusions (Table 2) [45,47]. However, like others RTKs, in LuSCC from smokers and in poorly differentiated tumors. NGS is the 6 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 standard technique to diagnose FGFR-fusions. Several drugs targeting [74]. The most frequent gene amplification in lung cancer is FGFR1, FGFRs are currently under development and their efficacy is being described in up to approximately 25% of LuSCC, as above mentioned evaluated in several basket trials regardless of histopathologic subtype. (Table 1) [64]. The early-phase clinical trial and first–in–human study (NCT01703481) tested the TKI erdafitinib (JNJ–42756493), a pan-FGFR inhibitor for Gene amplification affecting the mesenchymal-epithelial transition factor patients with detectable FGFR1-3 alterations, but yielded only a few receptor, MET responses with an acceptable toxicity profile [65]. Similar efficacy has been reported for the ATP-competitive broad-spectrum kinase inhibitor The MET proto-oncogene encodes the RTK for hepatocyte growth ARQ–087 in NSCLC patients with tumors harboring FGFR genetic acti­ factor and regulates a genetic program associated with cell proliferation vation [66]. Finally, infigratinib, an FGFR1–3 selective inhibitor, has and invasion of the extracellular matrix. MET mainly exists in epithelial proven effective in FGFR altered tumors, particularly in chol­ cells and plays an important role in embryogenesis, tumor growth, and angiocarcinomas [67] (Table 2). metastasis [75]. In NSCLC, primary METamp has been reported in around 1–5% of tumors (Table 1), but, importantly, it also constitutes a Other gene fusions affecting RTKs in NSCLC mechanism for acquired resistance to EGFR-TKIs, as reported in 5–20% of the cases [76], but also in other oncogenic RTK such as ALK tumors A variety of other rare fusions involving receptors with tyrosine ki­ treated with ALK-TKIs [28]. Currently, METamp is defined by focal gains nase activity have been identified in NSCLC, including MET and NRG1 in MET copy number relative to the centromere of chromosome 7 (ratio [68–69]. NRG1 is the ligand of the HER family and, despite not being an MET/CEP7), although there is no well-established consensus on the RTK itself, triggers the activation of RTKs. Although the prevalence of most appropriate copy number cut-off. However, a higher ratio of MET/ these fusions is very low, drugs targeting these proteins or the pathways CEP7 copy number seems to predict a better response to MET inhibitors regulated by them are under evaluation, and data from small-cohort [77]. METamp can be measured using FISH or NGS [76,77], but IHC of studies and case reports have been obtained [70–71]. total MET protein is not considered a good surrogate marker for MET­ Fusions involving MET were initially discovered in an osteogenic amp tumors since strong MET immunostaining has been observed in sarcoma cell line (TPR–MET) and are very rare events in lung cancer many tumors without gene amplification, mostly in LuADs [73] (about 0.5%). A wide variety of fusion partners have been identified, (Table 1). including HLA–DRB1, KIF5B, PTPRZ1, STARD3NL, and ST7, although Notably, MET activation can also occur by point mutations that the biology of these alterations and their therapeutic implications have affect, mostly, consensus splice sites, leading to the elimination of exon not been evaluated yet [69]. 14 (METex14) [78]. METex14 mutations are observed in 2–4% of Finally, the fusion CLIP1-LTK has recently been discovered in a NSCLCs and in both LuADs and LuSCCs, with a higher incidence in Japanese cohort of LuADs as an oncogenic driver, affecting around 0.4% pulmonary sarcomatoid carcinomas [79,80]. These mutations have also of NSCLCs (Table 1). This fusion leads to the activation of the LTK been reported in other malignancies, such as gastric (7%) or colorectal (leukocyte receptor tyrosine kinase), an RTK and a member of the ros/ (0–9%) cancers [78]. insulin receptor family of tyrosine kinases whose function is still not well Crizotinib has shown activity against MET activated lung tumors understood. Tumors with the CLIP1-LTK fusion responded to lorlatinib, [81]. More recently, the FDA approved two specific MET-TKIs, capma­ although clinical validation of this new oncogenic driver and clinical tinib and tepotinib, to treat NSCLC patients with METex14 mutant tu­ development of novel therapeutic agents are warranted [8]. mors [76]. Note that for the EMA approval, patients harboring METex14 Neuroregulins (NRGs) are the ligands of the EGFR family of proteins should have progressed to prior immunotherapy and/or platinum-based and are encoded by four genes (NRG1, NRG2, NRG3, and NRG4). NRG1 chemotherapy. Capmatinib has also shown activity in METamp tumors, is the best characterized and is associated with the invasive mucinous particularly in those with a high gain copy number (≥10) (Table 2) [82]. subtype of LuAD, typically diagnosed in women and never-smokers Furthermore, the dual EGFR and MET inhibition has proven effective in (Table 1). Rearrangements of NRG1 in solid tumors lead to aberrant those cases in which METamp was the acquired resistance mechanism to activation of HER2/HER3 and, subsequently, of the PI3K-AKT-mTOR EGFR-TKI [83]. and RAS/MAPK cascade. Albeit several fusion partners have been described, CD74 remains the most common [68–69] (Fig. 3). Afatinib, Gene amplification affecting the epidermal growth factor receptors HER an irreversible inhibitor of EGFR, represents a therapeutic option for patients with tumors with NRG1 fusions and several case reports have The HER family, also called EGFR family, or ERBB family when been published in this sense [70]. In addition, blocking HER3 also referring to the gene, comprises four transmembrane receptor tyrosine showed promising activity in these patients [71]. kinases: EGFR (or HER1), HER2, HER3, and HER4. These receptors signal through homo- and hetero-dimerization and promote cell prolif­ Oncogenic gene amplifications involving RTK in NSCLC and their clinical eration, motility, and invasion [84]. EGFR amplification (EGFRamp), in implications association with EGFR protein overexpression, has been reported in both LuAD and LuSCC patients and, in LuADs, can co-exist with EGFR mu­ Besides gene fusions, gene amplification represents another mecha­ tations [85]. EGFRamp has also been described as a resistance mecha­ nism that activates oncogenes during cancer development. Gene nism to third-generation EGFR-TKIs [85]. Despite the many available amplification can be defined as an expansion in the number of copies of EGFR-TKIs, none of them has shown remarkable activity against a gene or a chromosomal region that occurs during the DNA replication EGFRamp tumors. Monoclonal antibodies targeting EGFR, such as process [72]. The amplified DNA can be organized as extrachromosomal cetuximab, have also provided disappointing results in NSCLC, in elements, as repeated units at a single locus, or scattered throughout the contrast to those in colorectal and head and neck cancer [86]. Within the genome. Similar to gene fusions, oncogenic activation by gene amplifi­ HER family, ERBB2, encoding HER2, shows oncogenic alterations in cation leads to a supraphysiological increase in the expression of the NSCLCs through either point mutations or gene amplification and rep­ oncogene [73]. In the case of RTKs, this allows the receptor to become resents a novel targetable RTK. These alterations are most prevalent in constitutively active. Gene amplification in cancer cells can ensue by LuADs from never-smoker women. Most ERBB2 mutations are localized expansion during the process of tumor development, by de novo occur­ in exon 20, within the TKD, and the most frequent mutation is the p. rence, or by clonal selection, as an adaptative strategy to bypass the Tyr772_Ala775dup (c.2313_2324dup), found in 1–2% of LuADs [87]. pressure exerted during therapeutics. The latter constitutes a common On the other hand, ERBB2 amplification (ERBB2amp) has been described mechanism for the acquired resistance to different TKI targeting RTKs in around 3% of NSCLCs, either de novo or as a mechanism of acquired 7 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 resistance to EGFR-TKIs [87]. In contrast to breast and gastric cancer, Table 3 where ERBB2 overexpression is relatively common and has met with a List of pros and cons of diagnostic techniques for the detection of gene fusions notable success of anti-HER2 therapies, targeting ERBB2 in NSCLC re­ and gene amplification in clinical cancer management. mains challenging. Although ERBB2amp shows strong HER2 protein Method Pros Cons levels [73], initial trials of the anti-HER2 monoclonal antibody trastu­ FISH Well-established approach The cut-off values should be zumab, alone or in combination with chemotherapy or pertuzumab, No need for complex and standardized for each gene displayed only modest activity in ERBB2amp NSCLCs and yielded expensive equipment Not able to identify the fusion negative results in HER2-overexpressing patients [88]. In the recent Useful as a validation partner involved approach after positive IHC or Limited to detect phase II DESTINY-Lung01 trial, the antibody-drug conjugate trastuzu­ NGS intrachromosomic translocations mab deruxtecan showed durable anticancer activity in patients with previously treated HER2-mutant NSCLC, regardless of HER2 expression IHC Well-established approach IHC score should be standardized and amplification status [89]. No need for complex and Not able to identify the fusion expensive equipment partner involved Useful for preselecting tumors Specificity might relay on the Gene amplification affecting the growth factor receptors KIT and PDGFRA for confirmatory FISH testing antibody Allows to describe The c-KIT proto-oncogene encodes a transmembrane receptor tyro­ morphologic characteristics sine kinase, KIT, expressed in several normal human tissues. Its ligand is and tumor heterogeneity Excellent sensitivity for the stem cell factor that mediates KIT dimerization and activation. certain antibodies Activating mutations at KIT have been documented in various neo­ More cost-effective than FISH plasms, particularly in gastrointestinal stromal tumors, among others NGS Large amount of genetic Depends on the quality and the [90]. These mutations confer sensitivity to the TKI imatinib [91]. On the information is provided at amount of sample other hand, the members of the platelet-derived growth factor receptor once Needs dedicated bioinformatics In fusions, breakpoints are personnel to maintain a clinical (PDGFR) family are protein-tyrosine kinases encoded by two genes: characterized at single NGS service PDGFRA and PDGFRB [92]. The functional receptors consist of the nucleotide resolution Requires complex and expensive PDGFRα/α and PDGFRβ/β homodimers and the PDGFRα/β heterodimer. Detection of unknown equipment This RTK family plays an essential role in embryonic development and translocation partners Multiple samples can be wound healing in adults [92]. KIT and PDGFRA are closely located in the pooled and sequenced same arm of chromosome 4q12 and have been shown to co-amplify in together about 2% of NSCLCs, both in LuADs and LuSCCs [73]. KIT/PDGFRA Suitable turn-around time amplification has also been described as a resistance mechanism to ALK- RT-PCR based High specificity with robust Results depend on the quality of TKIs in LuADs with ALK fusion [93]. To this day, these alterations have techniques and detailed information RNA No need for complex and Needs to be designed according not been explored as therapeutic targets in lung cancer. expensive equipment to known fusion breakpoints In fusions, breakpoints can be Unable to detect unknown Lung cancer molecular testing in clinics: State of the art characterized at single partners nucleotide resolution Unconclusive for the detection of gene amplification 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 squa­ FISH: Fluorescent in situ hybridization, IHC: Immunohistochemistry, NGS: Next- mous cell carcinoma patients diagnosed with lung cancer since these generation sequencing, RT-PCR: Reverse transcriptase polymerase chain reaction. alterations are present in about 30% of the cases [2,3]. Different diag­ nostic methods, including IHC, FISH, reverse transcriptase PCR, and DNA/RNA-based NGS, can be used to detect gene fusions and amplifi­ NSCLC patients. ctDNA has proven suitable to detect gene mutations and cations. A list of benefits and limitations of these approaches and the fusions involving RTKs with reasonable sensitivity and specificity across different validated diagnostic methods for each gene fusion and ampli­ tumor types and is gaining interest in cancer monitoring [95]. ctDNA fication are listed in Table 3. Conventional methods, such as IHC or represents a non-invasive, rapid, and cost-effective strategy for obtain­ FISH, have been widely implemented and are approved methodologies ing DNA from tumor cells. However, the technique has not yet been fully to detect gene amplification and specific fusions, including ALK or ROS1 translated into clinical practice, and the variability in the amount of [12–14,32–33]. However, based on the increasingly frequent need for a ctDNA released by the tumors to the bloodstream may prevent the comprehensive genomic evaluation, NGS panels are becoming the standardization of the procedure [95]. In addition, the quantity of tumor preferred approach. Because NGS provides a great deal of genetic in­ ctDNA in the whole DNA extract can be excessively low to develop high- formation that needs to be understood and classified according to clin­ throughput analyses and can render artefactual mutations (false posi­ ical evidence within an appropriate time frame, its assessment should be tive) or, according to the sensitivity of the method, false negative results. provided by an expert molecular tumor board [94]. Despite the advan­ Large-scale screening and standardization of experimental steps could tages of the NGS technology, access to NGS panels and treatments varies resolve these problems. broadly among the different health systems worldwide. Tumor biopsies are the most common source of cancer cells for Therapeutic challenges and opportunities in NSCLC patients harboring genotyping and categorizing tumors for clinical decisions. Good quality oncogenic fusions RNA and DNA can be obtained from them, and biopsies preserve the morphological features of the tumor. Histopathologic transformation as Despite incorporating novel and highly selective TKIs for lung tu­ a resistance mechanism to TKIs can be determined. However, in lung mors harboring specific oncogenic mutations, advanced-stage lung cancer, tumor tissue extraction requires invasive procedures, becoming cancer remains largely incurable. Treatments facilitate the emergence of a caveat when multiple re-biopsies are needed to monitor the course of resistant clones or the selection of pre-existing resistant sub-clones, and the disease. For this reason, alternative sources of tumor DNA are relapsing is unavoidable. Acquired resistance, defined as progression needed. A blood-based test using cell-free circulating tumor DNA after initial benefit, is mediated by different biologic mechanisms that (ctDNA), also called “liquid biopsy,” is a potential surrogate source of allow tumor adaptation. Therefore, re-biopsy is always encouraged to tumor DNA for diagnostic, prognostic, and therapeutic biomarkers in identify the mechanisms underlying the acquired resistance and 8 M. Saigí et al. Cancer Treatment Reviews 109 (2022) 102430 evaluate whether it can be approached therapeutically. The alterations Conclusions that drive resistance include acquired mutations in the target oncogene (on–target resistance) and alterations in genes coding for proteins acting TKIs have prompted significant improvements in the outcomes of in parallel or downstream signaling pathways that allow bypassing the patients with lung tumors harboring tyrosine kinase driven oncogenic action of the TKI (off-target resistance) [96]. Furthermore, although not mutations or rearrangements (fusions or gene amplification). Despite very common, histopathologic transformation, mostly from LuAD to the low frequency of each mutation or rearrangement involving RTKs, small-cell lung carcinoma or LuSCC, represents another mechanism of altogether, they affect about 15% of NSCLCs. Currently, identifying resistance to targeted TKI and has been described in LuADs with mu­ these patients remains crucial because they can achieve survival for up tations in EGFR or with ALK and ROS1 fusions [97–99]. In some cases, to several years when treated with the appropriate inhibitor. The genetic alterations may be associated with this transformation, for magnitude of the clinical benefits achieved with targeted therapies and instance, in some LuADs that have become resistant to EGFR-TKIs the increasing number of specific clinical trials has prompted the inte­ through the TACC3-FGFR3 fusion [97]. The pre-existence of RB1 inac­ gration of molecular profiling technologies in clinical and pathological tivation, common in mutant-EGFR LuADs, has also been associated with settings. The key for succeeding with targeted therapeutics is integrating a higher predisposition to develop acquired resistance by histopatho­ an accurate diagnosis with potent and selective therapeutics together logical transformation [97]. Once the tumor undergoes histologic with an optimal penetration of new, low-toxicity drugs into the central transformation, the prognosis is detrimental, and the therapeutic nervous system. Although several promising targeted drugs have been approach is undertaken on a case-by-case basis. recently approved or are under clinical evaluation, optimal strategies to Disease progression after treatment with TKIs can also occur because overcome resistance, including combinations with immunotherapy or of an inadequate exposure of the drug to the receptor. The low pene­ chemotherapy, have not been clearly established. The future of precision tration of these drugs into certain organs, particularly into the brain, medicine will likely integrate comprehensive genomic tumor charac­ poses additional challenges. This is particularly important for patients terization, dynamic monitoring of liquid biopsy and/or tissue guided re- with NSCLCs harboring oncogenic driver fusions, who have an increased biopsies, and the enrollment of patients into innovative clinical trials. 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) [100]. CRediT authorship contribution statement Since brain metastases constitute an independent prognostic factor for worse overall survival and poorer quality of life, novel generation TKIs Maria Saigí: Conceptualization, Writing – original draft, Writing – with improved intracranial activity are being designed. These TKIs need review & editing, Supervision. Enric Carcereny: Writing – review & to demonstrate higher efficacy in reducing brain metastases than pre­ editing. Teresa Morán: Writing – original draft, Writing – review & vious generations, to prevent the onset of new metastases, and to delay editing. Marc Cucurull: Writing – review & editing. Marta Domènech: local therapeutic strategies, such as whole-brain radiotherapy and its Writing – original draft, Writing – review & editing. Ainhoa Hernan­ related toxicity [101]. dez: Writing – review & editing. Anna Martinez-Cardús: Writing – Of note, a small proportion of NSCLC patients with targetable fusions review & editing, Supervision. Eva Pros: Conceptualization, Writing – can present oligometastatic disease at diagnosis or undergo oligoprog­ original draft, Writing – review & editing, Supervision. Montse San­ ression under TKI treatment [102]. This means that, while maintaining a chez-Cespedes: Conceptualization, Writing – original draft, Writing – systemic overall response to TKI, a subset of tumor clones has become review & editing, Supervision. resistant and has progressed into specific locations. Therefore, adding local ablative strategies should be considered to increase the disease’s Declaration of Competing Interest control rate with prognostic implications [103]. Novel and specific inhibitors are the preferred therapeutic options The authors declare that they have no known competing financial for NSCLC patients with actionable driver oncogenes. Consequently, interests or personal relationships that could have appeared to influence most immunotherapy trials exclude these patients, and the benefits of the work reported in this paper. these treatments remain unclear for them. The majority of the currently available evidence comes from subgroup analysis from real-world data Acknowledgements and clinical trials or from small trials specifically designated to address this issue [104]. Actionable driver fusions are commonly found in never- This work was supported by Spanish grant from ICAPEM (ICAPEM smoker patients, which tend to be associated with a low tumor mutation becas 2021), to EP, and by Spanish grant form Asociación Española burden and a less inflamed tumor microenvironment. The multicenter Contra el Cancer (AECC) (grant number GCB14142170MONT) to MSC. study Immunotarget retrospectively collected the clinical outcomes of We thank M Rey providing language help and O Romero for data NSCLC patients harboring driver mutations who were treated with analysis support. immunotherapy. Its results showed low efficacy in patients harboring oncogenic driver fusions (e.g., ALK or RET), regardless of the levels of PD-L1 [105]. Funding Finally, the clinical management of lung cancer patients, as for many other cancers, would need to consider genetic predisposition as another MS is supported by a Juan-Rodés contract from the Instituto de Salud variable. Most NSCLCs with actionable oncogenic drivers, including Carlos III (JR20/00015). fusions and gene amplification, are found in never-smokers and, often, in young individuals. To date, the explanation is unclear, and the pos­ References sibility that these cancers arise within hereditary cancer syndromes [1] Campbell JD, Alexandrov A, Kim J, Wala J, Berger AH, Pedamallu CS, et al. should be considered. In this regard, the Li-Fraumeni syndrome is the Distinct patterns of somatic genome alterations in lung adenocarcinomas and most common hereditary syndrome associated with lung cancer devel­ squamous cell carcinomas. Nat Genet 2016;48(6):607–16. https://doi.org/ opment, especially EGFR-mutant tumors [97,106]. The occasional as­ 10.1038/ng.3564. 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