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Diagnostics, therapeutics and RET inhibitor resistance for RET fusion–positive non-small cell lung cancers and future perspectives

Open AccessPublished:January 15, 2021DOI:https://doi.org/10.1016/j.ctrv.2021.102153

      Highlights

      • DNA + RNA NGS should be considered in nonsmoker or driver negative NSCLC with low TMB.
      • Selective RET inhibitors have become the preferred treatment for RET + NSCLC.
      • Acquired RET fusions may not resemble de novo RET fusions in NSCLC.
      • Reports of resistance mutation in patient have elevated since selective TKI emerged.
      • Future clinical trials of the existing TKIs should differentiate histology.

      Abstract

      Selective RET inhibitors is the current hot topic, making multikinase inhibitors a thing of the past. However, the limitation of various test approaches, coupled with lack of knowledge of acquired resistance mechanisms, and specific patient groups that bear special consideration, remains a challenge. Herein, we outline utility of various diagnostic techniques, provide evidence to guide management of RET-fusion-positive Non-Small Cell Lung Cancer (NSCLC) patients, including specific patient groups, such as EGFR-mutant NSCLC patients who acquired RET fusions after resisting EGFR TKIs, and offer a compendium of mechanisms of acquired resistance to RET targeted therapies. This review further provides a list of ongoing clinical trials and summarizes perspectives to guide future development of drugs and trials for this population.

      Keywords

      Introduction

      High potency of selective RET (rearranged during transfection) inhibitors have sparked interest on targeted therapies for RET-altered lung cancers, which has long been marked by unsatisfaction. Identified as a proto-oncogene in 1985, RET underwent rearrangement during transfection of DNA extracted from human tumor into NIH-3T3 cells [
      • Takeuchi K.
      Discovery stories of RET fusions in lung cancer: a mini-review.
      ,
      • Takahashi M.
      • Ritz J.
      • Cooper G.M.
      Activation of a novel human transforming gene, ret, by DNA rearrangement.
      ]. Since then, studies have clarified its function and biology. The RET gene encodes a transmembrane glycoprotein receptor tyrosine kinase (RTK) [
      • Takahashi M.
      • Buma Y.
      • Iwamoto T.
      • Inaguma Y.
      • Ikeda H.
      • Hiai H.
      Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains.
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      • Ishizaka Y.
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      • Tahira T.
      • Ikeda I.
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      • Tucker J.
      • et al.
      Human ret proto-oncogene mapped to chromosome 10q11.2.
      ], that indirectly binds to its glial cell line-derived neurotrophic factor family ligands (GFLs) [
      • Wang X.
      Structural studies of GDNF family ligands with their receptors-Insights into ligand recognition and activation of receptor tyrosine kinase RET.
      ,
      • Goodman K.M.
      • Kjær S.
      • Beuron F.
      • Knowles P.P.
      • Nawrotek A.
      • Burns E.M.
      • et al.
      RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association.
      ], leading to RET homodimerization and subsequent activation of downstream pathways [
      • Worby C.A.
      • Vega Q.C.
      • Chao H.H.
      • Seasholtz A.F.
      • Thompson R.C.
      • Dixon J.E.
      Identification and characterization of GFRalpha-3, a novel Co-receptor belonging to the glial cell line-derived neurotrophic receptor family.
      ,
      • Andreozzi F.
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      • Carlomagno F.
      • Oriente F.
      • Miele C.
      • Fiory F.
      • et al.
      Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.
      ,
      • Fukuda T.
      • Kiuchi K.
      • Takahashi M.
      Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase.
      ,
      • Arighi E.
      • Borrello M.G.
      • Sariola H.
      RET tyrosine kinase signaling in development and cancer.
      ]. RET fusions play key roles in multiple cancer types. Generally, tumorigenesis begins with ligand-independent homodimerization and constitutive activation of the RET tyrosine kinase domain, through chromosomal rearrangements that form chimeric fusion gene [
      • Mizukami T.
      • Shiraishi K.
      • Shimada Y.
      • Ogiwara H.
      • Tsuta K.
      • Ichikawa H.
      • et al.
      Molecular mechanisms underlying oncogenic RET fusion in lung adenocarcinoma.
      ]. Consequently, multiple cellular proliferation pathways are activated, culminating in promotion of cell proliferation and survival [
      • Borrello M.G.
      • Alberti L.
      • Arighi E.
      • Bongarzone I.
      • Battistini C.
      • Bardelli A.
      • et al.
      The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.
      ,
      • Verbeek H.H.
      • Alves M.M.
      • de Groot J.W.
      • Osinga J.
      • Plukker J.T.
      • Links T.P.
      • et al.
      The effects of four different tyrosine kinase inhibitors on medullary and papillary thyroid cancer cells.
      ,
      • Suzuki M.
      • Makinoshima H.
      • Matsumoto S.
      • Suzuki A.
      • Mimaki S.
      • Matsushima K.
      • et al.
      Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo.
      ,
      • Kohno T.
      • Ichikawa H.
      • Totoki Y.
      • Yasuda K.
      • Hiramoto M.
      • Nammo T.
      • et al.
      KIF5B-RET fusions in lung adenocarcinoma.
      ,
      • Takeuchi K.
      • Soda M.
      • Togashi Y.
      • Suzuki R.
      • Sakata S.
      • Hatano S.
      • et al.
      RET, ROS1 and ALK fusions in lung cancer.
      ,
      • Lipson D.
      • Capelletti M.
      • Yelensky R.
      • Otto G.
      • Parker A.
      • Jarosz M.
      • et al.
      Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.
      ,
      • 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.
      ].
      However, it was not until two dacades later, in 2012, that the first RET fusion, KIF5B-RET, was reported in lung cancer (Fig. 1). RET fusions, mainly identified as solid therapeutic targets, are found in 1–2% of non-small cell lung cancers (NSCLC), implying that RET-addicted malignancies are sensitive to targeted inhibition. The first glimmer of hope came with the discovery of multikinase inhibitors (MKIs), such as cabozantinib and vandetanib, with nonselective RET activity. However, all of these drugs provided suboptimal RET inhibition, coupled with significant off-target adverse effects. Consequently, none of them has been approved for RET-altered NSCLC [
      • Drilon A.
      • Rekhtman N.
      • Arcila M.
      • Wang L.
      • 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.
      ,
      • Lee S.H.
      • Lee J.K.
      • Ahn M.J.
      • Kim D.W.
      • Sun J.M.
      • Keam B.
      • et al.
      Vandetanib in pretreated patients with advanced non-small cell lung cancer-harboring RET rearrangement: a phase II clinical 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.
      ,
      • 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.
      ,
      • Horiike A.
      • Takeuchi K.
      • Uenami T.
      • Kawano Y.
      • Tanimoto A.
      • Kaburaki K.
      • et al.
      Sorafenib treatment for patients with RET fusion-positive non-small cell lung cancer.
      ,
      • Drilon A.
      • Fu S.
      • Patel M.R.
      • Fakih M.
      • Wang D.
      • Olszanski A.J.
      • et al.
      A phase I/Ib trial of the VEGFR-sparing multikinase RET inhibitor RXDX-105.
      ]. Clinical data from two neck and neck novel RET inhibitors, pralsetinib (BLU-667) and selpercatinib (LOXO-292), provided insights into the development of RET-targeted therapies. Specifically, both agents selectively inhibited RET activation, enhanced efficacy while circumventing toxicity, thereby becoming the preferred treatment option for RET fusion-positive NSCLC [
      • FDA approves first RET inhibitor
      ,
      • Subbiah V.
      • Gainor J.F.
      • Rahal R.
      • Brubaker J.D.
      • Kim J.L.
      • Maynard M.
      • et al.
      Precision targeted therapy with BLU-667 for RET-driven cancers.
      ] (see Table 1).
      Figure thumbnail gr1
      Fig. 1Milestones in the discovery and clinical studies of RET fusion-positive NSCLC. Timeline illustrates some of the pivotal discoveries and clinical trials of RET fusion-positive NSCLC. *cabozantinib, vandetanib, lenvatinib.
      Table 1Results from phase I/II trials of pralsetinib (BLU-667) and selpercatinib (LOXO-292) in patients with advanced RET fusion-positive non-small cell lung cancer.
      Pralsetinib (BLU-667)
      • Gainor J.F.
      • Curigliano G.
      • Kim D.-W.
      • Lee D.H.
      • Besse B.
      • Baik C.S.
      • et al.
      Registrational dataset from the phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET fusion+ non-small cell lung cancer (NSCLC).
      Selpercatinib (LOXO-292)
      • Subbiah V.
      • Gainor J.F.
      • Oxnard G.R.
      • Tan D.S.-W.
      • Owen D.H.
      • Cho B.C.
      • et al.
      Intracranial activity of selpercatinib (LOXO-292) in RET fusion-positive non-small cell lung cancer (NSCLC) patients on the LIBRETTO-001 trial.
      ,
      • 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.
      Previous Platinum Chemotherapy (n = 92)Treatment Naïve (n = 29)Previous Platinum Chemotherapy (n = 105)Treatment Naïve (n = 39)
      Median age—yr60 (28–85)65 (30–87)61 (23–81)61 (23–86)
      Male50%48%41%44%
      Race53% White, 35% Asian59% White, 34% Asian52% White, 38% Asian72% White, 18% Asian
      RET fusion partner74% KIF5B, 17% CCDC669% KIF5B, 10% CCDC656% KIF5B, 23% CCDC667% KIF5B, 21% CCDC6
      Brain metastases at baseline41%31%36%18%
      Prior therapy45% PD-1/PD-L1 inhibitor, 45% prior kinase inhibitors55% PD-1/PD-L1 inhibitor, 48% prior kinase inhibitors
      Objective response
      Independent review.
      61%
      Response-evaluable population (previously platinum chemotherapy treated patients = 80; treatment naïve patients = 26).
      73%
      Response-evaluable population (previously platinum chemotherapy treated patients = 80; treatment naïve patients = 26).
      64%85%
      CNS evaluable response
      Independent review.
      922
      CNS objective response
      Independent review.
      56%82%
      Median duration of response
      Independent review.
      —mo (95% CI)
      NR (11.3-NR)17.5 (12.0–NE)NR (12.0-NE)
      Median progression-free survival
      Independent review.
      — mo
      16.5 (13.7–NE)NE (13.8–NE)
      Drug discontinuation rate4%
      Of all patients who received pralsetinib or selpercatinib.
      2%
      Of all patients who received pralsetinib or selpercatinib.
      CNS: central nervous system; NR: not reached; NE: not evaluable.
      a Independent review.
      b Response-evaluable population (previously platinum chemotherapy treated patients = 80; treatment naïve patients = 26).
      c Of all patients who received pralsetinib or selpercatinib.
      The advent of highly effective selective RET inhibitors has necessitated tailoring of treatment strategies for patients diagnosed with advanced RET fusion-positive NSCLC. In this review, we discuss strengths and pitfalls of contemporary diagnostic approaches in RET fusion-positive NSCLC, present current evidence of therapeutic strategies and resistance mechanisms of RET targeted therapies.

      The challenge of molecular diagnostics

      Testing for RET fusions is highly recommended in NSCLC, since it can predict benefits from targeted inhibition. Specifically, a variety of diagnostic tools, including fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and next-generation sequencing (NGS), are used to detect gene fusions at the DNA, RNA, and protein levels.
      Classical methods such as FISH [
      • Chen F.
      • Clark D.P.
      • Hawkins A.L.
      • Morsberger L.A.
      • Griffin C.A.
      A break-apart fluorescence in situ hybridization assay for detecting RET translocations in papillary thyroid carcinoma.
      ], RT-PCR, and IHC have been traditionally used for detection of RET fusions. Although these conventional techniques are fast and cheap, they are limited to detection of one fusion and often fail to provide information associated with fusion partners and the breakpoint [
      • Ferrara R.
      • Auger N.
      • Auclin E.
      • Besse B.
      Clinical and translational implications of RET rearrangements in non-small cell lung cancer.
      ,
      • Go H.
      • Jung Y.J.
      • Kang H.W.
      • Park I.K.
      • Kang C.H.
      • Lee J.W.
      • et al.
      Diagnostic method for the detection of KIF5B-RET transformation in lung adenocarcinoma.
      ,
      • Wang R.
      • Hu H.
      • Pan Y.
      • Li Y.
      • Ye T.
      • Li C.
      • et al.
      RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
      ]. Consequently, many oncologists have shifted to NGS for detection.

      DNA-based hybrid capture NGS

      A widespread approach for identifying RET fusion, as well as a broader range of genomic aberrations, such as translocation, deletion, inversion, and mutation, is through a comprehensive molecular diagnostic technique, such as NGS. However, numerous studies have recently called for revisiting of this prevailing “one-size-fits-all” view of oncogene fusion detection before its application in clinical practice. Herein, we focus on DNA-based hybrid capture NGS, since it is likely to be the first option owing to its high throughput and convenience [
      • Jennings L.J.
      • Arcila M.E.
      • Corless C.
      • Kamel-Reid S.
      • Lubin I.M.
      • Pfeifer J.
      • et al.
      Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the association for molecular pathology and college of American Pathologists.
      ], albeit with a number of caveats. Overall, DNA-based NGS may miss driver fusions. For instance, Benayed et al. [
      • Benayed R.
      • Offin M.
      • Mullaney K.
      • Sukhadia P.
      • Rios K.
      • Desmeules P.
      • et al.
      High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden.
      ] used RNA-sequencing to identify actionable kinase fusions, involving three RET fusions, in lung adenocarcinoma cases who had been deemed driver-negative by previous DNA-sequencing. In fact, most patients exhibited clinical benefits that matched targeted therapies. Similarly, Solomon et al. [
      • Solomon J.P.
      • Linkov I.
      • Rosado A.
      • Mullaney K.
      • Rosen E.Y.
      • Frosina D.
      • et al.
      NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.
      ] and Davies et al. [
      • Davies K.D.
      • Le A.T.
      • Sheren J.
      • Nijmeh H.
      • Gowan K.
      • Jones K.L.
      • et al.
      Comparison of molecular testing modalities for detection of ROS1 rearrangements in a cohort of positive patient samples.
      ] compared different platforms for NTRK fusion and ROS1 fusion assessment, and confirmed the lower sensitivity of DNA-sequencing. Several features of gene fusions may result in the finite capacity of DNA-based NGS. Firstly, fusion breakpoints sometimes happen in the intronic regions, and requires tilling of the regions of interest. However, some involved introns are very long, making the progress challenging [
      • Benayed R.
      • Offin M.
      • Mullaney K.
      • Sukhadia P.
      • Rios K.
      • Desmeules P.
      • et al.
      High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden.
      ]. Secondly, some introns contain highly repetitive elements, that are poorly covered by DNA-based assays owing to their recurring presence in other areas of the genome. Therefore, such sequencing data cannot be specifically mapped onto the desired target region of the genome and may be excluded by software programs [
      • Treangen T.J.
      • Salzberg S.L.
      Repetitive DNA and next-generation sequencing: computational challenges and solutions.
      ,
      • Garcia E.P.
      • Minkovsky A.
      • Jia Y.
      • Ducar M.D.
      • Shivdasani P.
      • Gong X.
      • et al.
      Validation of OncoPanel: a targeted next-generation sequencing assay for the detection of somatic variants in cancer.
      ].
      Apart from generating false negative-results, DNA-based NGS can confound treatment strategies as it may detect non-productive bystander events, which means fusions that do not generate functional fusion transcripts. Li et al. [
      • Li W.
      • Liu Y.
      • Li W.
      • Chen L.
      • Ying J.
      Intergenic breakpoints identified by DNA sequencing confound targetable kinase fusion detection in NSCLC.
      ,

      Li W, Guo L, Liu Y, Dong L, Yang L, Chen L, Liu K, Shao Y, Ying J. Potential unreliability of uncommon ALK/ROS1/RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC. LID - S1556-0864(20)31023-6 [pii] LID - 10.1016/j.jtho.2020.10.156 [doi]. 2020.

      ] used RNA-based NGS, IHC, and FISH technique to evaluate the function of DNA-seq-identified intergenic-breakpoint fusions—one or both genomic breakpoints localize to intergenic regions—in NSCLC. Their results indicated that the genomic breakpoint position did not logically predict breakpoint of the fusion transcript in these cases, possibly owing to complex and reciprocal rearrangements, as well as alternative splicing. Similarly, Yang et al. [
      • Yang Soo-Ryum
      • Mata Douglas
      • Benayed Ryma
      • Mullaney Kerry
      • Frosina Denise
      • Rekhtman Natasha
      • et al.
      Detection of RET kinase fusions using DNA, RNA, and protein-based methods.
      ] used a targeted DNA-based sequencing assay to screen tumors for RET structural variants. Specifically, SVs of unknown significance (SVUS), defined as RET SVs without known 5′ partners, were analyzed by a targeted RNA-based sequencing assay. They found several RNA-seq-only fusions from SVUS, which produced kinase fusion transcripts with known or novel 5′ partners, while more than half of the SVUS did not produce kinase fusion transcripts. Notably, Supplee et al. [
      • Supplee J.G.
      • Milan M.S.D.
      • Lim L.P.
      • Potts K.T.
      • Sholl L.M.
      • Oxnard G.R.
      • et al.
      Sensitivity of next-generation sequencing assays detecting oncogenic fusions in plasma cell-free DNA.
      ] reported discordance between two plasma cell-free DNA-based NGS assays during detection of oncogenic fusions. Based on these findings, the researchers cautioned the ability of DNA-based NGS for detection of novel gene fusion partners and highlighted the need for confirmatory testing in certain settings [
      • Davies K.D.
      • Aisner D.L.
      Wake up and smell the fusions: single-modality molecular testing misses drivers.
      ,
      • Sholl L.M.
      Recognizing the challenges of oncogene fusion detection: a critical step toward optimal selection of lung cancer patients for targeted therapies.
      ,
      • Wang Q.
      • Xia J.
      • Jia P.
      • Pao W.
      • Zhao Z.
      Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives.
      ,
      • Heyer E.E.
      • Deveson I.W.
      • Wooi D.
      • Selinger C.I.
      • Lyons R.J.
      • Hayes V.M.
      • et al.
      Diagnosis of fusion genes using targeted RNA sequencing.
      ].

      RNA-based assays

      Currently, there are several technologies for RNA-based multiplexed fusion sequencing. For instance, hybridization- or amplicon-based RNA sequencing, sequencing coding sequences with quick turnaround time [
      • Heyer E.E.
      • Deveson I.W.
      • Wooi D.
      • Selinger C.I.
      • Lyons R.J.
      • Hayes V.M.
      • et al.
      Diagnosis of fusion genes using targeted RNA sequencing.
      ,
      • Beadling C.
      • Wald A.I.
      • Warrick A.
      • Neff T.L.
      • Zhong S.
      • Nikiforov Y.E.
      • et al.
      A Multiplexed Amplicon approach for detecting gene fusions by next-generation sequencing.
      ,
      • Vaughn C.P.
      • Costa J.L.
      • Feilotter H.E.
      • Petraroli R.
      • Bagai V.
      • Rachiglio A.M.
      • et al.
      Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.
      ,
      • Levin J.Z.
      • Berger M.F.
      • Adiconis X.
      • Rogov P.
      • Melnikov A.
      • Fennell T.
      • et al.
      Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence variants and novel fusion transcripts.
      ], and anchored multiplexed PCR (AMP), using one gene-specific primer combined with one universial primer to identify gene fusion agnostic of 5′ partner [
      • Zheng Z.
      • Liebers M.
      • Zhelyazkova B.
      • Cao Y.
      • Panditi D.
      • Lynch K.D.
      • et al.
      Anchored multiplex PCR for targeted next-generation sequencing.
      ,
      • Sussman R.T.
      • Oran A.R.
      • Paolillo C.
      • Lieberman D.
      • Morrissette J.J.D.
      • Rosenbaum J.N.
      Validation of a next-generation sequencing assay targeting RNA for the multiplexed detection of fusion transcripts and oncogenic isoforms.
      ]. In these techniques, RNA is used as a template to form complimentary DNA (cDNA) through reverse transcription reaction. Absence of introns in mRNA circumvents the aforementioned challenges posed by large introns or highly repetitive elements in the introns. Moreover, RNA sequencing provides direct evidence of fusion transcript, implying that the gene rearrangement generates fusion expression at the mRNA level. Therefore, RNA sequencing is considered a complement for DNA-based assay, although it counts on high quality RNA to generate cDNA libraries, which is usually hard to obtain especially in archived tumour samples.
      Oncologists have now turned to combining DNA and RNA-based NGS, owing to its efficiency in generating information regarding fusions, mutations, and microsatellite instability with minimal specimen requirements [
      • Sholl L.M.
      Recognizing the challenges of oncogene fusion detection: a critical step toward optimal selection of lung cancer patients for targeted therapies.
      ,

      Cohen D, Hondelink LM, Solleveld-Westerink N, Uljee SM, Ruano D, Cleton-Jansen AM, von der Thüsen JH, Ramai SRS, Postmus PE, Graadt van Roggen JF, et al. Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing. J Thorac Oncol 2020; 15: 1000–1014. doi: 10.1016/j.jtho.2020.01.019.

      ,
      • Volckmar A.L.
      • Leichsenring J.
      • Kirchner M.
      • Christopoulos P.
      • Neumann O.
      • Budczies J.
      • et al.
      Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.
      ]. Concerns regarding cost-effectiveness has prompted comparisons between parallel (using a combination of DNA NGS and RNA NGS) and sequential (performing RNA NGS only when no certain pathogenic driver mutation were found in DNA NGS) approaches, for fusion detection in NSCLC. Results revealed enrichment of fusions and exon-skipping events in samples from non-smokers, indicating the superiority of the parallel approach for its shorter median turnaround time. Conversely, additional RNA NGS resulted in a low yield in former and current smokers, implying that sequential approach may be the most efficient strategy [

      Cohen D, Hondelink LM, Solleveld-Westerink N, Uljee SM, Ruano D, Cleton-Jansen AM, von der Thüsen JH, Ramai SRS, Postmus PE, Graadt van Roggen JF, et al. Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing. J Thorac Oncol 2020; 15: 1000–1014. doi: 10.1016/j.jtho.2020.01.019.

      ].
      In summary, the inherent strengths and pitfalls of each diagnostic approach in RET fusion-positive NSCLC have necessitated examination of the current “one-size-fits-all” view of molecular workup. When making a “lean and mean” diagnostic option, oncologists need to take into account patient characteristics, specimens, turnaround time, and costs [

      Cohen D, Hondelink LM, Solleveld-Westerink N, Uljee SM, Ruano D, Cleton-Jansen AM, von der Thüsen JH, Ramai SRS, Postmus PE, Graadt van Roggen JF, et al. Optimizing Mutation and Fusion Detection in NSCLC by Sequential DNA and RNA Sequencing. J Thorac Oncol 2020; 15: 1000–1014. doi: 10.1016/j.jtho.2020.01.019.

      ]. Fig. 2 shows an algorithm for testing RET fusion in non-small cell lung cancer. It should be noted that never smoked NSCLC patients and patients harboring driver negative tumors with low TMB (0–5 mutations/Megabase) are enriched for gene fusions, and should therefore be prioritized for RNA-based sequencing [
      • Benayed R.
      • Offin M.
      • Mullaney K.
      • Sukhadia P.
      • Rios K.
      • Desmeules P.
      • et al.
      High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden.
      ]. In addition, a validated method should be considered during analysis of driver-negative samples, and novel fusions identified using DNA based-NGS. Furthermore, it is imperative to accurately interpret any unexpected findings (see Fig. 3).
      Figure thumbnail gr2
      Fig. 2An algorithm for testing RET fusion in non-small cell lung cancer. NGS, next-generation sequencing; FISH, fluorescence in situ hybridization; RT-PCR, reverse transcriptase-polymerase chain reaction.
      Figure thumbnail gr3
      Fig. 3Mechanisms of acquired resistance to multikinase and selective RET tyrosine kinase inhibitors in RET fusion-positive non-small cell lung cancer.

      Therapeutic strategies of de novo RET fusions

      As previously described, traditional MKIs show modest clinical benefit in RET-positive NSCLC. In contrast, next-generation, highly potent RET inhibitors such as pralsetinib and selpercatinib selectively inhibited RET activation, thereby becoming the preferred treatment option for RET fusion-positive NSCLC [
      • FDA approves first RET inhibitor
      ,
      • Subbiah V.
      • Gainor J.F.
      • Rahal R.
      • Brubaker J.D.
      • Kim J.L.
      • Maynard M.
      • et al.
      Precision targeted therapy with BLU-667 for RET-driven cancers.
      ]. Notably, both agents have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with metastatic RET fusion-positive NSCLC.
      The highly selective RET inhibitor is setting a new paradigm for personalized treatment in RET-positive NSCLC, including subsets of patients that bear special consideration. Previous studies have shown that brain metastases (BM) frequently occur in RET fusion-positive NSCLC patients. For instance, in a significant subset of RET fusion-positive NSCLC patients, who had BM, approximately 25% (95%CI 18–32%) present at diagnosis and 46% (95%CI 34–58%) would eventually develop during lifetime, based on a combined global, multi-institutional registry and bi-institutional cohort study [
      • Drilon A.
      • Lin J.J.
      • Filleron T.
      • Ni A.
      • Milia J.
      • Bergagnini I.
      • et al.
      Frequency of brain metastases and multikinase inhibitor outcomes in patients with RET-rearranged lung cancers.
      ]. Subsequent MKIs trials have yielded limited intracranial efficacy [
      • Drilon A.
      • Rekhtman N.
      • Arcila M.
      • Wang L.
      • 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.
      ,
      • Lin J.J.
      • Kennedy E.
      • Sequist L.V.
      • Brastianos P.K.
      • Goodwin K.E.
      • Stevens S.
      • et al.
      Clinical activity of Alectinib in advanced RET-rearranged non-small cell lung cancer.
      ]. For instance, the aforementioned retrospective study found that the MKI therapy resulted in a median progression-free survival (PFS) of 2.1 months and an intracranial response of 18% in patients with in patients with BM [
      • Drilon A.
      • Lin J.J.
      • Filleron T.
      • Ni A.
      • Milia J.
      • Bergagnini I.
      • et al.
      Frequency of brain metastases and multikinase inhibitor outcomes in patients with RET-rearranged lung cancers.
      ].
      In contrast to MKIs, selective RET TKIs have been much heralded for their impressive and durable responses in patients in recent years. Vivek and colleagues [
      • Subbiah V.
      • Gainor J.F.
      • Oxnard G.R.
      • Tan D.S.-W.
      • Owen D.H.
      • Cho B.C.
      • et al.
      Intracranial activity of selpercatinib (LOXO-292) in RET fusion-positive non-small cell lung cancer (NSCLC) patients on the LIBRETTO-001 trial.
      ] analyzed selpercatinib’s (LOXO-292) intracranial activity in RET fusion-positive NSCLC patients during a phase 1/2 LIBRETTO-001 trial. They found that 18 of 22 (81.8%, 95% CI 59.7–94.8%) subjects had CNS response with a median duration of response of 9.4 months (95%CI 7.4 - NE). Notably, previous studies have documented intracranial disease control to selpercatinib in patients who developed symptomatic progression of brain metastases on alectinib [
      • Subbiah V.
      • Velcheti V.
      • Tuch B.B.
      • Ebata K.
      • Busaidy N.L.
      • Cabanillas M.E.
      • et al.
      Selective RET kinase inhibition for patients with RET-altered cancers.
      ], as well as those who exhibited progression of brain metastases and new leptomeningeal disease on RXDX-105 [
      • Guo R.
      • Schreyer M.
      • Chang J.C.
      • Rothenberg S.M.
      • Henry D.
      • Cotzia P.
      • et al.
      Response to selective RET inhibition with LOXO-292 in a patient with RET fusion-positive lung cancer with leptomeningeal metastases.
      ]. On the other hand, findings from the ARROW study, targeting patients with baseline brain metastases, revealed radiographic response with pralsetinib in 56% of 9 subjects with measurable BM at baseline [
      • Gainor J.F.
      • Curigliano G.
      • Kim D.-W.
      • Lee D.H.
      • Besse B.
      • Baik C.S.
      • et al.
      Registrational dataset from the phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET fusion+ non-small cell lung cancer (NSCLC).
      ](see Table 1). Moreover, intracranial disease control was presented in one patient with RET fusion-positive, AKT2 amplified lung adenocarcinoma with brain metastases receiving a combination of vandetanib and everolimus, owing to the putative assistance of brain penetrance from the mTOR inhibitor [
      • Subbiah V.
      • Berry J.
      • Roxas M.
      • Guha-Thakurta N.
      • Subbiah I.M.
      • Ali S.M.
      • et al.
      Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.
      ]. Efficacy of this combination is expected to be validated by results from an ongoing trial (NCT01582191). Nevertheless, a betther understanding of CNS metastasis and the associated biological behaviour of RET fusion is desperately needed to guide development of more effective therapies for BM. For example, presence of KIF5B fusion partner is a common occurrence in patients with brain metastases [
      • 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.
      ,
      • Drilon A.
      • Lin J.J.
      • Filleron T.
      • Ni A.
      • Milia J.
      • Bergagnini I.
      • et al.
      Frequency of brain metastases and multikinase inhibitor outcomes in patients with RET-rearranged lung cancers.
      ].
      Recently, a retrospective analysis indicated improved survival outcomes in selective RET TKI treated versus untreated patients (median 49.3 vs. 15.3 months; p < 0.001) [
      • Tan A.C.
      • Seet A.O.L.
      • Lai G.G.Y.
      • Lim T.H.
      • Lim A.S.T.
      • San Tan G.
      • et al.
      Molecular characterisation and clinical outcomes in RET rearranged non-small cell lung cancer (NSCLC).
      ]. Interestingly, in this retrospective cohort, OS was also prolonged in CCDC6-RET fusion versus KIF5B-RET fusion positive NSCLC patients (median 113.5 vs. 37.7 months; p = 0.009). Similarly, an analysis of clinical trials of MKIs and results from a phase I/Ib trial of RXDX-105 suggested lower response rate and PFS in KIF5B-RET-containing tumors compared with non-KIF5B-RET containing tumors [
      • Drilon A.
      • Fu S.
      • Patel M.R.
      • Fakih M.
      • Wang D.
      • Olszanski A.J.
      • et al.
      A phase I/Ib trial of the VEGFR-sparing multikinase RET inhibitor RXDX-105.
      ,
      • Ferrara R.
      • Auger N.
      • Auclin E.
      • Besse B.
      Clinical and translational implications of RET rearrangements in non-small cell lung cancer.
      ,
      • Drilon A.
      • Hu Z.I.
      • Lai G.G.Y.
      • Tan D.S.W.
      Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes.
      ]. However, this divergence in clinical outcomes dependent on the gene fusion partner needs additional investigation in larger sample sizes.
      Although RET fusions are thought to be mutually exclusive with other oncogenic drivers such as EGFR mutations, ALK and ROS1 rearrangements, concurrent activation or genomic alterations of other pathways have been identified. This underlines the rationale for combination therapies. For example, the aforementioned patient with RET fusion-positive, AKT2 amplified lung adenocarcinoma responded to the combination of vandetanib and everolimus [
      • Subbiah V.
      • Berry J.
      • Roxas M.
      • Guha-Thakurta N.
      • Subbiah I.M.
      • Ali S.M.
      • et al.
      Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.
      ]. Preclinical data have also shown enhanced antitumor effect of alectinib in combination with cyclin-dependent kinase 4/6 inhibitor against RET-fusion–positive non–small cell lung cancer cells [
      • Fujimura T.
      • Furugaki K.
      • Harada N.
      • Yoshimura Y.
      Enhanced antitumor effect of alectinib in combination with cyclin-dependent kinase 4/6 inhibitor against RET-fusion-positive non-small cell lung cancer cells.
      ]. Additional research is required to evaluate the optimal approach for patients in this rare fusion subset.

      Management of acquired RET fusions after resistant to EGFR TKIs

      Tumors harboring sensitizing EGFR mutations are sensitive to targeted therapy, whereas those with acquired receptor tyrosine kinase (RTK) fusions are resistant due to activated bypass signaling pathways. A previous literature review [
      • Zhu V.W.
      • Klempner S.J.
      • Ou S.I.
      Receptor tyrosine kinase fusions as an actionable resistance mechanism to EGFR TKIs in EGFR-mutant non-small-cell lung cancer.
      ], indicated that the acquired RET fusion appeared as the most common acquired RTK fusion in EGFR-mutant—mostly EGFR 19del—NSCLC, especially to third generation EGFR TKIs.
      Post-progression management of such fusion events matters. Notably, more than half of the acquired RTK fusions were identified through liquid biopsy [
      • Zhu V.W.
      • Klempner S.J.
      • Ou S.I.
      Receptor tyrosine kinase fusions as an actionable resistance mechanism to EGFR TKIs in EGFR-mutant non-small-cell lung cancer.
      ]. However, as previously discussed, the unreliable capture of fusion events limits the sensitivity of this approach, and may underestimate the true frequency of acquired RET fusions. A tumor genotyping therefore remains the gold standard approach for examining the mechanisms of resistance, and an additional RNA-based fusion panel could be helpful.
      Given the lack of approved therapy currently, combined targeted therapies and non-precision therapies remain to be the mainstay of treatment modality applied in clinical practice. So far, four patients have shown good response to a combination of EGFR TKI and RET TKI [
      • Schrock A.B.
      • Zhu V.W.
      • Hsieh W.S.
      • Madison R.
      • Creelan B.
      • Silberberg J.
      • et al.
      Receptor tyrosine kinase fusions and BRAF kinase fusions are rare but actionable resistance mechanisms to EGFR tyrosine kinase inhibitors.
      ,
      • Piotrowska Z.
      • Isozaki H.
      • Lennerz J.K.
      • Gainor J.F.
      • Lennes I.T.
      • Zhu V.W.
      • et al.
      Landscape of acquired resistance to Osimertinib in EGFR-mutant NSCLC and clinical validation of combined EGFR and RET inhibition with osimertinib and BLU-667 for acquired RET fusion.
      ]. Whether this co-target strategy is a safe and effective treatment option remains to be validated. Results from an on-going open-label, multicentre, multi-drug phase II platform ORCHARD trial which is designed to prospectively examine the efficacy of targeted combination strategies in EGFR-mutant patients who acquired RET fusions after resistant to EGFR TKIs will be informative.
      Futher investigation are needed to trace RET fusions in the mutational history of the founder EGFR-mutant lung adenocarcinoma, as well as to understand whether its biological behavior is the same as de novo RET fusion-positive NSCLC. For example, KIF5B-RET is the most common fusion variant in de novo RET fusion-positive NSCLC, but it has only been reported in 2% of the acquired RET fusions in EGFR-mutant NSCLC, in which CCDC6-RET is the actual dominant [
      • Zhu V.W.
      • Klempner S.J.
      • Ou S.I.
      Receptor tyrosine kinase fusions as an actionable resistance mechanism to EGFR TKIs in EGFR-mutant non-small-cell lung cancer.
      ].

      Acquired resistance to RET inhibitors

      Understanding resistance mechanisms of targeted therapies is imperative to prolonging the remissions of these agents. In cases where progression is observed, full restaging and rebiopsy should be performed to unravel the underlying molecular mechanisms driving the resistance and guide post-progression management. Although tissue biopsy is the gold standard, it can be surrogated by liquid biopsy, but only when tissue is scarce.
      Currently, only a handful of reports have described patients who acquired resistance to prior RET MKIs. This may be attributed to inhibition of non-RET kinases, also known as non-kinase targets, by MKIs. Firstly, drug related toxicities associated with this ‘off-target’ activity have resulted in treatment discontinuation, implying that some patients are likely to stop dosages before drug resistance occurs [
      • Drilon A.
      • Rekhtman N.
      • Arcila M.
      • Wang L.
      • 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.
      ,
      • Lee S.H.
      • Lee J.K.
      • Ahn M.J.
      • Kim D.W.
      • Sun J.M.
      • Keam B.
      • et al.
      Vandetanib in pretreated patients with advanced non-small cell lung cancer-harboring RET rearrangement: a phase II clinical 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.
      ]. Moreover, the incapability of RET inhibition may also prevent these agents from generating certain kinds of resistance mechanisms, for the optimal RET-inhibitory plasma concentrations is unachieved [
      • Drilon A.
      • Hu Z.I.
      • Lai G.G.Y.
      • Tan D.S.W.
      Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes.
      ]. Conversely, pralsetinib and selpercatinib decouple RET inhibition from the inhibition of non-RET kinases, and therefore might be more likely to explain the underlying mechanisms of acquired resistance [
      • Drilon A.
      • Hu Z.I.
      • Lai G.G.Y.
      • Tan D.S.W.
      Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes.
      ].

      Reported mechanisms of on-target drug resistance in patients

      Preclinical studies have shown that mutations in the RET kinsase domain that could induce resistance to RET MKIs are located in the Gly-rich loop (L730, E732 and V738), the gatekeeper residue (V804), the hinge strand (Y806, A807 and G810), or distant sites in the large C-terminal lobe away from the TKI binding pocket (V871I and F998V), mostly due to the direct interference from the drug binding site [
      • Liu X.
      • Shen T.
      • Mooers B.H.M.
      • Hilberg F.
      • Wu J.
      Drug resistance profiles of mutations in the RET kinase domain.
      ,
      • Carlomagno F.
      • Guida T.
      • Anaganti S.
      • Vecchio G.
      • Fusco A.
      • Ryan A.J.
      • et al.
      Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors.
      ,
      • Huang Q.
      • Schneeberger V.E.
      • Luetteke N.
      • Jin C.
      • Afzal R.
      • Budzevich M.M.
      • et al.
      Preclinical modeling of KIF5B-RET fusion lung adenocarcinoma.
      ]. It was not untill recently that several second-site mutations have been identified in samples from patients with RET fusion-positive NSCLCs who develop acquired resistance to targeted therapies, including the gatekeeper RET V804M/L mutations [
      • Huang Q.
      • Schneeberger V.E.
      • Luetteke N.
      • Jin C.
      • Afzal R.
      • Budzevich M.M.
      • et al.
      Preclinical modeling of KIF5B-RET fusion lung adenocarcinoma.
      ], the non-gatekeeper mutation RET S904F [
      • Nakaoku T.
      • Kohno T.
      • Araki M.
      • Niho S.
      • Chauhan R.
      • Knowles P.P.
      • et al.
      A secondary RET mutation in the activation loop conferring resistance to vandetanib.
      ], and the solvent-front RET G810R/S/C mutations [
      • 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.
      ].

      Gatekeeper mutations

      RET gatekeeper mutation-mediated resistance to MKI (RET V804M/L mutation) has been reported independently in two patients [
      • Dagogo-Jack I.
      • Stevens S.E.
      • Lin J.J.
      • Nagy R.
      • Ferris L.
      • Shaw A.T.
      • et al.
      Emergence of a RET V804M gatekeeper mutation during treatment with vandetanib in RET-rearranged NSCLC.
      ,
      • Wirth L.J.
      • Kohno T.
      • Udagawa H.
      • Matsumoto S.
      • Ishii G.
      • Ebata K.
      • et al.
      Emergence and targeting of acquired and hereditary resistance to multikinase RET inhibition in patients with RET-altered cancer.
      ]. Interestingly, both patients progressed on treatment with vandetanib. Structural modeling has demonstrated that RET V804M/L mutation—the conserved gatekeeper residue which is altered by mutations—introduces steric clashes between the leucine and methionine side chains and the 4-bromo-2-fluorophenyl group of vandetanib, an anti-RET MKI. Moreover, the RET V804M mutation is analogous to ALK L1196M and ROS1 L2026M which confer resistance to ALK, and ROS1 TKIs by increasing adenosine triphosphate affinity [
      • Subbiah V.
      • Velcheti V.
      • Tuch B.B.
      • Ebata K.
      • Busaidy N.L.
      • Cabanillas M.E.
      • et al.
      Selective RET kinase inhibition for patients with RET-altered cancers.
      ,
      • Drilon A.
      • Hu Z.I.
      • Lai G.G.Y.
      • Tan D.S.W.
      Targeting RET-driven cancers: lessons from evolving preclinical and clinical landscapes.
      ,
      • 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.
      ,
      • Facchinetti F.
      • Loriot Y.
      • Kuo M.S.
      • Mahjoubi L.
      • Lacroix L.
      • Planchard D.
      • et al.
      Crizotinib-resistant ROS1 mutations reveal a predictive kinase inhibitor sensitivity model for ROS1- and ALK-rearranged lung cancers.
      ].
      The selective RET inhibitors have been designed to overcome gatekeeper mutations (Fig. 4). One patient with RET V804L mutation mentioned above showed a rapid clinical and biochemical response to subsequent LOXO-292, which indicates a better accommodation of the bulky leucine and methionine side chains in the gatekeeper residues without any steric interactions [
      • Wirth L.J.
      • Kohno T.
      • Udagawa H.
      • Matsumoto S.
      • Ishii G.
      • Ebata K.
      • et al.
      Emergence and targeting of acquired and hereditary resistance to multikinase RET inhibition in patients with RET-altered cancer.
      ].
      Figure thumbnail gr4
      Fig. 4Summary of next-generation inhibitors activity against known RET inhibitor resistance mutations in in vitro assays. ATP, adenosine triphosphate. *Ba/F3 cell-based assays.

      Solvent-front mutations

      Gly-810 is located at the solvent front of ATP binding pocket. The RET G810A/S/R mutations are paralogous to the osimertinib-resistant EGFR G796S/R mutations, crizotinib-resistant ALK G1202R and ROS1 G2032R mutations, and entrectinib-resistant NTRK1 G595R/NTRK3 G623R mutations [
      • Ou S.I.
      • Cui J.
      • Schrock A.B.
      • Goldberg M.E.
      • Zhu V.W.
      • Albacker L.
      • et al.
      Emergence of novel and dominant acquired EGFR solvent-front mutations at Gly796 (G796S/R) together with C797S/R and L792F/H mutations in one EGFR (L858R/T790M) NSCLC patient who progressed on osimertinib.
      ,
      • Drilon A.
      • Li G.
      • Dogan S.
      • Gounder M.
      • Shen R.
      • Arcila M.
      • et al.
      What hides behind the MASC: clinical response and acquired resistance to entrectinib after ETV6-NTRK3 identification in a mammary analogue secretory carcinoma (MASC).
      ,
      • Tang Z.H.
      • Lu J.J.
      Osimertinib resistance in non-small cell lung cancer: Mechanisms and therapeutic strategies.
      ,
      • Gainor J.F.
      • Tseng D.
      • Yoda S.
      • Dagogo-Jack I.
      • Friboulet L.
      • Lin J.J.
      • et al.
      Patterns of metastatic spread and mechanisms of resistance to Crizotinib in ROS1-positive non-small-cell lung cancer.
      ,
      • Terzyan S.S.
      • Shen T.
      • Liu X.
      • Huang Q.
      • Teng P.
      • Zhou M.
      • et al.
      Structural basis of resistance of mutant RET protein-tyrosine kinase to its inhibitors nintedanib and vandetanib.
      ]. Recently Solomon et al. [
      • 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.
      ] reported that RET G810 solvent front mutations emerge develop during treatment with selpercatinib in three RET fusion–positive NSCLC patients, which represent the mechanism of resistance to this selective RET inhibitior. Interestingly, RET V804M gatekeeper mutation that emerged simultaneously in trans with the G810 solvent-front mutation in one NSCLC patient reported by Solomon et al. is reminiscent of compound ALK mutations in patients who have failed prior ALK TKIs treatment [
      • Yoda S.
      • Lin J.J.
      • Lawrence M.S.
      • Burke B.J.
      • Friboulet L.
      • Langenbucher A.
      • et al.
      Sequential ALK inhibitors can select for Lorlatinib-resistant compound ALK mutations in ALK-positive lung cancer.
      ]. Whether this indicates that treatment with this selective RET inhibitor is unable to prevent the emergence of gatekeeper mutations will be identified through additional studies.
      A preclinical study has shown that solvent-front mutations remain sensitive to a next generation RET inhibitor, TPX-0046, which also demonstrated low nanomolar potency against WT and many RET mutations [
      • Drilon A.
      • Rogers E.
      • Zhai D.
      • Deng W.
      • Zhang X.
      • Lee D.
      • et al.
      TPX-0046 is a novel and potent RET/SRC inhibitor for RET-driven cancers.
      ].
      Besides, in a patient from the LURET study, an acquired RET S904F mutation in the activation loop of the RET kinase domain was observed after the development of resistance to vandetanib, and was proved to increase kinase activity and confer drug resistance through allosteric effects [
      • 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.
      ,
      • Nakaoku T.
      • Kohno T.
      • Araki M.
      • Niho S.
      • Chauhan R.
      • Knowles P.P.
      • et al.
      A secondary RET mutation in the activation loop conferring resistance to vandetanib.
      ]. Additional studies are advocated to establish the frequency of acquired RET mutations in NSCLC.

      Reported mechanisms of off-target drug resistance

      For patients in which mutations in the RET kinase domain responsible for drug resistance were not detected, evidence exist mostly for the activation of bypass signaling pathways. MET dependence has been reported as a recurring and potentially targetable mechanism of resistance to selpercatinib and pralsetinib [

      Zhu VW, Madison R, Schrock AB, Ignatius Ou SH. Emergence of High Level of MET Amplification as Off-Target Resistance to Selpercatinib Treatment in KIF5B-RET NSCLC; 2020.

      ,

      Rosen EY, Johnson ML, Clifford SE, Somwar R, Kherani JF, Son JA-O, et al. Overcoming MET-Dependent Resistance to Selective RET Inhibition in Patients with RET Fusion-Positive Lung Cancer by Combining Selpercatinib with Crizotinib. LID - 10.1158/1078-0432.CCR-20-2278 [doi]; 2020.

      ,

      Lin JJ, Liu SV, McCoach CE, Zhu VW, Tan AC, Yoda S, et al. Mechanisms of resistance to selective RET tyrosine kinase inhibitors in RET fusion-positive non-small-cell lung cancer; 2020.

      ]. Preclinical studies have demonstrated that EGFR transduced bypass signals to critical downstream pathways which regulate cell proliferation and survival via ERK and AKT, enabling cancer cells to evade RET inhibitors. EGF-mediated EGFR activation also disrupted fusion kinase inhibitor binding and restored fusion kinase-adaptor signaling complexes. This process can be inhibited with EGFR TKI [
      • Chang H.
      • Sung J.H.
      • Moon S.U.
      • Kim H.S.
      • Kim J.W.
      • Lee J.S.
      EGF induced RET inhibitor resistance in CCDC6-RET lung cancer cells.
      ,
      • Vaishnavi A.
      • Schubert L.
      • Rix U.
      • Marek L.A.
      • Le A.T.
      • Keysar S.B.
      • et al.
      EGFR mediates responses to small-molecule drugs targeting oncogenic fusion kinases.
      ]. Finally, AXL overexpression and RAS mutation were reported in two RET fusion-positive cell line which resisted to multi-kinase inhibition, respectively [
      • Nelson-Taylor S.K.
      • Le A.T.
      • Yoo M.
      • Schubert L.
      • Mishall K.M.
      • Doak A.
      • et al.
      Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling.
      ]. Interestingly, acquired BRAF V600E mutation in a patient with KIF5B-RET fusion–positive NSCLC who progressed on selpercatinib was reported recently, albeit lacked validation of this putative mechanism of resistance [

      Justin F. Gainor GC, Robert C. Doebele Jessica J. Lin, Sai-Hong I. Ou, Stephen Miller, et al. Analysis of resistance mechanisms to pralsetinib (BLU-667) in patients with ret fusion–positive non-small cell lung cancer (NSCLC) from the arrow study. In: IASLC North America Conference on Lung Cancer; 2020.

      ].
      Acquired non-RET alterations might also at play in driving resistance. For example, acquired amplification of MDM2 has been has been reported and validated to engender resistance to cabozantinib in RET fusion-positive NSCLC patients [
      • Somwar R.
      • Smith R.
      • Hayashi T.
      • Ishizawa K.
      • Snyder Charen A.
      • Khodos I.
      • et al.
      MDM2 amplification (Amp) to mediate cabozantinib resistance in patients (Pts) with advanced RET-rearranged lung cancers.
      ].
      In summary, reports of mechanisms of resistance to RET TKIs in patients have elevated since selective TKIs emerged. For on-target resistance, pralsetinib and selpercatinib have been designed to overcome gatekeeper mutations. And TPX-0046, whereas is less potent against gatekeeper mutation KIF5B-RET V804M, has demonstrated promising activity against solvent-front mutations based on in vitro assays. For off-target resistance, previous study reported that MET dependence is potentially targetable, and preclinical studies have shown that co-targeting PI3K-AKT [
      • Subbiah V.
      • Berry J.
      • Roxas M.
      • Guha-Thakurta N.
      • Subbiah I.M.
      • Ali S.M.
      • et al.
      Systemic and CNS activity of the RET inhibitor vandetanib combined with the mTOR inhibitor everolimus in KIF5B-RET re-arranged non-small cell lung cancer with brain metastases.
      ,
      • Gild M.L.
      • Landa I.
      • Ryder M.
      • Ghossein R.A.
      • Knauf J.A.
      • Fagin J.A.
      Targeting mTOR in RET mutant medullary and differentiated thyroid cancer cells.
      ], EGFR [
      • Chang H.
      • Sung J.H.
      • Moon S.U.
      • Kim H.S.
      • Kim J.W.
      • Lee J.S.
      EGF induced RET inhibitor resistance in CCDC6-RET lung cancer cells.
      ] and MDM2 [
      • Somwar R.
      • Smith R.
      • Hayashi T.
      • Ishizawa K.
      • Snyder Charen A.
      • Khodos I.
      • et al.
      MDM2 amplification (Amp) to mediate cabozantinib resistance in patients (Pts) with advanced RET-rearranged lung cancers.
      ] may decrease the risk of drug resistance. The combination of RET inhibitors with other targeted agents is currently under investigation. Still, no clear mechanism of resistance was identified in most of the patients whose disease progressed on selpercatinib or pralsetinib.

      Future perspectives on drugs and clinical trials

      Several novel RET inhibitors are at early stages of development. Table 2 shows the ongoing clinical trials for advanced NSCLC patients harboring RET fusions. Future design of next-generation RET TKIs should not only cover the drug-resistant site, both on-target mutations and other RTKs that can activate parallel signalling pathways (for example, TPX-0046 inhibits both RET and SRC kinase while sparing VEGFR kinase), but also improve brain penetration and efficacy.
      Table 2Ongoing clinical trials for advanced NSCLC patients harboring RET fusions.
      Drug regimenPhaseClinicaltrials.gov identifier
      Further details for trials with NCT numbers can be accessed at the clinicaltrials.gov website.
      Second-generation RET inhibitors
      TPX-0046I/IINCT04161391
      BOS-172738INCT03780517
      PralsetinibI/II

      III
      NCT03037385

      NCT04222972
      SelpercatinibI/II

      II

      II

      III
      NCT03157128

      NCT04280081

      NCT04268550

      NCT04194944/NCT03906331
      Multikinase RET inhibitors
      RXDX-105INCT03784378
      AlectinibII

      II
      NCT03445000

      NCT02314481
      CabozantinibII

      II
      NCT04131543

      NCT01639508
      Multikinase RET inhibitor + MTOR inhibitor
      Vandetanib + everolimusINCT01582191
      * Further details for trials with NCT numbers can be accessed at the clinicaltrials.gov website.
      Currently, several clinical trials are investigating whether the existing potent and selective drugs improve clinical outcomes when compared to a list of standard of care treatments [
      • Besse B.
      • Felip E.
      • Clifford C.
      • Louie-Gao M.
      • Green J.
      • Turner C.D.
      • et al.
      AcceleRET Lung: A phase III study of first-line pralsetinib in patients (pts) with RET-fusion+ advanced/metastatic non-small cell lung cancer (NSCLC).
      ,

      Solomon BA-O, Zhou CC, Drilon A, Park K, Wolf J, Elamin Y, et al. Phase III study of selpercatinib vs chemotherapy +/- pembrolizumab in untreated RET positive non-small-cell lung cancer. LID - 10.2217/fon-2020-0935 [doi]; 2020.

      ]. Whether the mechanism of resistance differs between selpercatinib and pralsetinib require further clarification [
      • Xia B.
      • Ou S.I.
      Simultaneous RET solvent-front and gatekeeper resistance mutations in trans: a rare TKI-specific therapeutic challenge?.
      ]. Although cancers sharing driver RET alterations, termed “REToma” [
      • Kohno T.
      • Tabata J.
      • Nakaoku T.
      REToma: a cancer subtype with a shared driver oncogene.
      ], can be treated with tissue-agnostic RET TKIs, drug resistance may occur via different mechanisms. Though scarce in NSCLC, acquisition of a RET gatekeeper mutation accounted for 75% of patients who progressed on vandetanib or cabozantinib in a small proportion of RET M918T-mutant MTC patients [
      • Busaidy N.L.C.M.E.
      • Sherman S.I.
      • Habra M.
      • Dadu R.
      • Hu M.I.
      • Jimenez C.
      • et al.
      Emergence of V804M resistance gatekeeper mutation in sporadic medullary thyroid carcinoma patients treated with TKI tyrosine kinase inhibitors.
      ]. Future clinical trials of the existing selective drugs should aim to differentiate histologies, earlier testing and initiation of treatment during the disease course might increase the number of eligible patients [
      • Drilon A.
      • Hellmann M.D.
      An umbrella approach to test lung cancer therapies.
      ]. Moreover, as discussed in the previous section, further clinical trials of drugs, registries and collaborative studies are encouraged to study the efficacy of combinations of RET inhibitors with other targeted agents are advocated to include baseline and serial brain imaging in patients to characterize baseline intracranial disease, response to therapy, and patterns of disease progression [
      • Drilon A.
      • Lin J.J.
      • Filleron T.
      • Ni A.
      • Milia J.
      • Bergagnini I.
      • et al.
      Frequency of brain metastases and multikinase inhibitor outcomes in patients with RET-rearranged lung cancers.
      ,
      • Tan A.C.
      • Itchins M.
      • Khasraw M.
      Brain metastases in lung cancers with emerging targetable fusion drivers.
      ].
      We are on the threshold of a new era in targeting RET fusion-positive NSCLCs. The effective selective RET inhibitors is setting a new paradigm for treating RET-altered lung cancers. However, we are confronted with emerging challenges that need to be addressed to optimize the benefits of current treatment options. To this end, we suggest accurate molecular diagnosis and development of effective therapeutic strategies.

      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

      We thank the patients and their families for participating in RET inhibitor clinical trials. Fig. 3 was created with BioRender.com.

      Funding

      This research was supported by National Key R&D Program of China (Grant No. 2016YFC1303800 to QZ), National Natural Science Foundation of China (No. 82072562 to QZ), and High-level Hospital Construction Project (Grant No. DFJH201810 to QZ).

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