Tropomyosin Receptor Kinase Inhibitors for the Treatment of TRK Fusion Cancer

Home , Larotrectinib

Author Manuscript Published OnlineFirst on April 23, 2021; DOI: 10.1158/1078-0432.CCR-21-0465 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Theodore W. Laetsch1 and David S. Hong2

1Department of Pediatrics and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, and the Division of Oncology, Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Running title: TRK inhibitors in TRK fusion cancer

Corresponding author: Dr Theodore W. Laetsch Children’s Hospital of Philadelphia 3501 Civic Center Blvd, CTRB 4016 Philadelphia PA 19104 USA Phone: 267-425-0126 email: [email protected]

Authors’ Disclosures T.W. Laetsch reports consultancy relationships with Novartis, Cellectis, Bayer, Loxo Oncology, Lilly, Deciphera, Jumo Health, and Y-mAbs Therapeutics; and research funding from Pfizer, Novartis, Bayer, Loxo Oncology, AbbVie, Amgen, Atara, Biotherapeutics, Bristol-Myers Squibb, Lilly, Epizyme, GlaxoSmithKline, Janssen, Jubilant Pharmaceuticals, Novella Clinical, and Servier. D.S. Hong reports research/grant funding from AbbVie, Adaptimmune, Aldi-Norte, Amgen, AstraZeneca, Bayer, Bristol- Myers Squibb, Daiichi Sankyo, Eisai, Erasca, Fate Therapeutics, Genentech, Genmab, Ignyta, Infinity, Kite, Kyowa, Lilly, Loxo Oncology, Merck, MedImmune, Mirati, miRNA, Molecular Templates, Mologen, Navier, NCI-CTEP, Novartis, Numab, Pfizer, Seattle Genetics, Takeda, Turning Point Therapeutics, Verstatem, and VM Oncology; travel, accommodations, expenses from: Bayer, Loxo Oncology, miRNA, Genmab, AACR, ASCO, and SITC; consulting, speaker or advisory role with: Alpha Insights, Acuta, Amgen, Axiom, Adaptimmune, Baxter, Bayer, Boxer Capital, COG, Ecor1, Genentech, GLG, Group H, Guidepoint, HCW Precision, Infinity, Janssen, Merrimack, Medscape, Numab, Pfizer, Prime Oncology, Seattle Genetics, ST Cube, Takeda, Tavistock, Trieza Therapeutics, WebMD; and other ownership interests with Molecular Match (advisor), OncoResponse (founder), and Presagia Inc (advisor).

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 23, 2021; DOI: 10.1158/1078-0432.CCR-21-0465 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract Chromosomal rearrangements of NTRK1–3 resulting in gene fusions (NTRK gene fusions) have been clinically validated as oncogenic drivers in a wide range of human cancers. Typically, NTRK gene fusions involve both inter- and intrachromosomal fusions of the 5′ regions of a variety of genes with the 3′ regions of NTRK genes leading to TRK fusion proteins with constitutive, ligand-independent activation of the intrinsic tyrosine kinase. The incidence of NTRK gene fusions can range from the majority of cases in certain rare cancers to lower rates in a wide range of more common cancers. Two small molecule TRK inhibitors have recently received regulatory approval for the treatment of patients with solid tumors harboring NTRK gene fusions, including the selective TRK inhibitor, larotrectinib and the TRK/ROS1/ALK multikinase inhibitor, entrectinib. In this review we consider the practicalities of detecting tumors harboring NTRK gene fusions, the pharmacologic properties of TRK inhibitors currently in clinical development, the clinical evidence for larotrectinib and entrectinib efficacy, and possible resistance mechanisms.

Introduction

The neurotrophic receptor tyrosine kinase (NTRK) genes, NTRK1, NTRK2 and NTRK3, encode the tropomyosin receptor kinase (TRK) family of transmembrane proteins TRKA (high-affinity nerve growth factor receptor), TRKB (BDNF/NT-3 growth factors receptor), and TRKC (NT-3 growth factor receptor). Normal TRK receptor signaling has a role in the development, maintenance, and function of the vertebrate nervous system (1). Ligand binding to the extracellular domain of the wild-type TRK receptor leads to homodimerization, activation of the intrinsic intracellular tyrosine kinase, and the consequent triggering of downstream signaling.

Chromosomal rearrangements of NTRK1–3 resulting in gene fusions (NTRK gene fusions) have been shown to be oncogenic drivers in a wide range of human cancers (2). Typically, the chimeric oncoproteins encoded by such gene fusions include an oligomerization domain from one of a wide range of possible 5′ fusion partners, joined in frame with the TRK tyrosine kinase domain of an NTRK 3′ fusion partner. This results in the constitutive ligand- independent activation of the TRK kinase and downstream signaling. Point mutations and amplifications of NTRK genes have also been reported in human tumors (3-6). However, whether these types of mutational events are oncogenic drivers is not currently clear and requires further study (7-9).

Several small molecule TRK inhibitors are currently in clinical development. In particular, two agents have recently received US Food and Drug Administration (FDA) regulatory approval for the tumor agnostic treatment of patients with solid tumors harboring an NTRK gene fusion; these are the selective TRK inhibitor, larotrectinib and the TRK/ROS1/ALK multikinase inhibitor, entrectinib. In this review, we consider the potential of these and other agents in the treatment of patients with TRK fusion cancer.

NTRK Gene Fusions

The detection of tumors harboring particular gene fusions may be undertaken using a range of established techniques including polymerase chain reaction (PCR) assays, reverse transcriptase (RT-)PCR, fluorescence in situ hybridization (FISH), DNA-based next generation sequencing (NGS) and RNA-based NGS. The presence of a gene fusion may also be inferred in certain situations using immunohistochemical approaches (10).

NTRK gene fusions have been identified in the majority of cases of certain rare cancers, including infantile fibrosarcoma (11), secretory carcinomas of the breast (12) and salivary gland (13), and the cellular and mixed subtypes of congenital mesoblastic nephroma (14). NTRK gene fusions have also been identified at lower frequencies in a wide range of more common cancers, including non-small cell lung cancer (15), breast cancer (16), colorectal cancer (17-19), thyroid cancer (20,21), melanocytic lesions (22,23), primary central nervous system (CNS) tumors (20,24), other soft tissue sarcomas (25) and leukemias (26). The distribution of fusions by tumor type, as recently reported in a series of clinical trials for entrectinib and larotrectinib, is shown in Fig. 1A–C (27-30). While NTRK2 is predominantly associated with primary CNS tumors, NTRK1 and NTRK3 are associated with a broad range of different tumor types. Fusion partners for each of the NTRK genes are distributed across the genome (Fig. 1D–F); interestingly, over 70% of the fusions involving NTRK1 are intrachromosomal. In addition, NTRK1 exhibited the greatest diversity in both involvement in different tumor types as well as the number of different fusion partners. It remains to be determined whether this is due to bias in testing (10,31) or a functional difference.

The detection of NTRK gene fusions is complicated by several factors. These include the structure of the NTRK genes (in particular, large repetitive element-rich introns in NTRK2 and NTRK3 within which breakpoints may be located), the large number of possible 5′ fusion partner genes, the positional variability of intronic breakpoints in 5′ fusion partners and the NTRK genes, and the variability in baseline TRK protein expression levels in different tumor types (10). In relation to immunohistochemical approaches, in addition to varying sensitivity and specificity by tumor type, there are likely to be disparities in interpretation across different diagnostic laboratories. Of note, a commercially available pan-TRK in vitro diagnostic immunohistochemical assay directed against the C-terminal region of the TRK proteins has recently been developed (VENTANA pan-TRK (EPR17341) Assay [Roche; Basel, Switzerland]).

The widely different incidence of NTRK gene fusions in certain tumor types and the technical complexity and cost implications of effectively routinely screening potentially large numbers of common tumors to detect the relatively small number harboring NTRK gene fusions in order to identify those patients who will potentially benefit from TRK inhibitor therapy has spurred the development of several novel diagnostic algorithms (10,31-33). These have been proposed by different expert author groups and include recommendations from the European Society for Medical Oncology (ESMO). The proposals reflect a consensus view that it is unlikely that one single currently available diagnostic method will be optimal in terms of the efficient identification of all patients with TRK fusion cancer. In each case therefore,

the proposed algorithms seek to triage tumors to different diagnostic routes according to the prevalence of NTRK gene fusions in individual tumor types, with a distinction drawn between tumor histologies in which NTRK gene fusions are highly recurrent genetic alterations, and those in which they are relatively uncommon. For the latter group, there is a consensus that a broad NGS testing approach that includes the detection of the full spectrum of NTRK gene fusions (most likely a combined approach of analyzing both tumor DNA and RNA) will be ideal. However, in indications or settings where NGS testing is not already a routine diagnostic approach, pan-TRK immunohistochemistry and genomic-based triaging may have a role in prioritizing tumors for broad NGS analysis. In relation to genomic triaging, NTRK gene fusions are enriched in tumors lacking other canonical driver mutations (34). Notably, however, in colorectal cancer, it has been established that tumors showing mismatch repair deficiency (MMRd) due to MLH1 promoter methylation may be more likely to harbor NTRK gene fusions (32,35,36).The ESMO recommendations note that the appropriate population to be tested for TRK fusion cancer should include those with any malignancy at an advanced stage, in particular if it has been proven wild-type for other known genetic alterations tested in routine practice, and especially if diagnosed in young patients (33).

TRK Inhibitors – Pharmacologic Properties

Several first- and next-generation, ATP-competitive, small molecule inhibitors with potent activity against TRK kinases are currently in clinical development. In addition, there are a number of other multikinase inhibitors which have a variable degree of activity against TRK kinases, and which may therefore have some activity in relation to TRK fusion cancer (Table 1).

First-Generation TRK Inhibitors

Larotrectinib is a potent, orally available TRK inhibitor with biochemical IC50 values for TRKA and C of 0.9 and 2.8 nm, respectively and cellular IC50 values in the range of 5–18.8 nM against TRKA, TRKB, and TRKC, with potent antiproliferative activity against TRKA/B/C fusions in the nanomolar range (Table 1). Larotrectinib is highly selective and demonstrated greater than a 100-fold selectivity against non-TRK protein kinases and greater than a 1000- fold selectivity against a wide variety of non-kinase receptors, channels and transporters (37,38). Larotrectinib may be administered in a capsule or liquid formulation, with the later particularly important for treating infants and very young children as well as patients with difficulty in swallowing capsules. Entrectinib is an orally available, TRK inhibitor with biochemical IC50 values in the range of 1–5 nM for TRKA–C. In addition to the TRK kinases, entrectinib also potently inhibits ROS1 (enzymatic IC50 of 0.2 nM) and ALK (enzymatic IC50

of 1.6 nM) kinases (39). Entrectinib is administered as a capsule formulation. It is notable that the half-life of larotrectinib (2.9 hours) is markedly shorter than that of entrectinib (20–22 hours), characteristics which may impact on the safety and efficacy profiles of these agents (40,41).

Next-Generation TRK Inhibitors As with other tyrosine kinase inhibitors (TKIs) that target oncogenic drivers, despite durable initial disease control, acquired on-target or off-target resistance to first-generation TRK inhibitors may eventually lead to their therapeutic failure (37). Next-generation TRK inhibitors which maintain activity in the presence of on-target resistance mutations have therefore been sought and are currently undergoing clinical development. Selitrectinib is an orally available, next-generation selective TRK inhibitor, which retains potency in the presence of TRK kinase domain mutations. While showing strong binding to wild-type TRKA, TRKB and TRKC (<1 nM potency in kinase enzyme assays), selitrectinib also achieves low nanomolar inhibitory activity against TRK proteins harboring different resistance mutations, with IC50 values ranging from 2–10 nM (42) (Table 1). A further next-generation agent in development is repotrectinib, an orally available inhibitor that was rationally designed to potently inhibit ROS1, TRKA–C, and ALK kinases harboring solvent front and other clinically relevant non- solvent front resistance mutations, in addition to the wild-type ROS1, TRKA–C, and ALK kinases (43) (Table 1).

Other Multikinase Inhibitors with Anti-TRK Activity Several other multikinase inhibitors approved for indications other than NTRK gene fusion- positive tumors have some measure of activity against TRK kinases, including crizotinib, cabozantinib, ponatinib, and nintedanib (39). In addition, several other inhibitors are in variable stages of clinical development, including PLX7486, MGCD516, TSR-011, DS- 6051b, atiratinib, and merestinib. While sporadic responses have been reported (44,45), the clinical activity of these multikinase inhibitors in patients with NTRK gene fusion-positive cancers has so far not been well characterized, and whether their development will be further explored in this setting following regulatory agency approval of larotrectinib and entrectinib and the clinical development of potent next-generation TRK inhibitors is currently not clear.

Clinical Findings with TRK Inhibitors

Larotrectinib and entrectinib have now received tumor agnostic regulatory approval in several major jurisdictions for the treatment of patients with solid tumors harboring NTRK

gene fusions. These approvals were based on pooled analyses of ongoing phase I/II studies in both adults and children (larotrectinib) and adults only, with extrapolation of efficacy to adolescents based on pharmacokinetic data (entrectinib). Other than the patient age range, key differences in the baseline characteristics of patients included in these registrational analyses were that a higher proportion of larotrectinib-treated patients were heavily pretreated and a higher proportion of entrectinib treated patients had CNS metastases at baseline. Baseline characteristics and efficacy outcomes for these two study populations are summarized in Table 2.

Larotrectinib The initial FDA approval of larotrectinib was based on a pooled analysis of 55 adult and pediatric patients with a range of TRK fusion cancers enrolled onto one of three phase I or II clinical trials. An objective response rate for larotrectinib of 80% according to investigator assessment was reported (37). Responses were seen across a wide range of different tumor types. An updated pooled efficacy analysis was subsequently carried out on an expanded combined population of 159 adult and pediatric patients with TRK fusion cancer. This represented a population almost 3 times larger than the initial analysis, and with longer follow-up. Included patients were mostly pretreated, with 78% having received ≥1 prior systemic anticancer regimens (27). The objective response rate in this expanded population (the primary endpoint) was 79% according to investigator assessment, with 16% of patients having complete responses. Responses were again seen across a wide range of different tumor types.

High objective response rates were demonstrated in both adult (73%) and pediatric (92%) patient subgroups. Responses were mostly rapid and durable; in the overall population, the median time to response was 1.8 months in line with the first protocol mandated response assessment at 8 weeks, and the median duration of response was 35.2 months. For the entire cohort, the median progression-free survival (PFS) was 28.3 months and median overall survival was 44.4 months. In the pediatric patient subset, the median duration of response had not been reached at the time of reporting.

Entrectinib The regulatory approval of entrectinib by the FDA for adult and pediatric patients 12 years of age and older with TRK fusion cancer was based on a similar pooled analysis of 54 adults with a range of advanced or metastatic TRK fusion cancers enrolled onto one of three phase I or II clinical trials. Most included patients had previously been treated, with 63% having received ≥1 prior line of systemic therapy (28). The objective response was 57% according

to independent review, of which 7% were complete responses. Similar to larotrectinib, responses were seen across a wide range of different tumor types. Responses tended to be durable with a median duration of response of 10.4 months and a median progression survival time of 11.2 months.

CNS Activity Both larotrectinib and entrectinib have been associated with CNS activity, with responses reported in CNS metastases and primary CNS tumors in patients with TRK fusion cancer (46-49). In the updated larotrectinib pooled analysis, intracranial responses were not a pre- specified endpoint. However, in 12 patients with CNS metastases, 75% had an objective response when considering all disease sites. Three of these patients had measurable CNS metastases as target lesions at baseline and intracranial responses were achieved in two patients with the remaining patient having stable disease (Table 2) (27). Furthermore, only one patient with non-target intracranial disease at baseline had progression in the brain during treatment. In addition, in 14 evaluable patients with primary CNS tumors harboring NTRK gene fusions who were treated with larotrectinib, the response rate was 36%, including 14% of patients with complete responses. The remaining 64% of patients had a best response of stable disease (24).

In the entrectinib pooled analysis, intracranial responses were a pre-specified endpoint for patients with brain metastases at baseline. In a subset of 11 (20%) of 54 patients who had brain metastases at baseline, six (55%) of these patients had an intracranial response, according to independent central review (Table 2). When considering all disease sites, as assessed by investigators, 12 of the 54 patients had metastatic CNS disease at baseline; six of these patients (50%) had a partial response according to independent central review, and four had stable disease (28). In addition, entrectinib reportedly induced rapid and durable responses in three children with primary refractory CNS tumors harboring NTRK gene fusions (50).

Safety Pooled analyses of adult and pediatric patient populations treated with larotrectinib and entrectinib demonstrate that both agents are generally well tolerated, with the majority of adverse events being of grade 1 or 2 (27,28,37,51).

The safety profile of larotrectinib was investigated in a combined population of 260 adult and pediatric patients treated regardless of TRK fusion status and a subpopulation of 52 pediatric patients aged less than 18 years. The adverse event profiles of these two populations were

similar (27). The most common grade 3 or worse events regardless of attribution were anemia (10% of patients) and decreased neutrophil count (5%). The most common grade 3 or 4 treatment-related adverse events were increased alanine aminotransferase (3%), anemia (2%), and decreased neutrophil count (2%). In the pediatric population, 17% of patients had grade 3 or 4 treatment-related adverse events, with the most common being decreased neutrophil count (10%). Adverse events potentially attributable to TRK inhibition, such as weight gain and dizziness, were not common and mostly of grade 1 or 2. In pediatric patients, neurological toxicities were rarely reported (27,51). Dose discontinuation due treatment-related adverse events occurred in only 2% of patients.

The overall safety profile of entrectinib was investigated in a combined population of 355 adult and pediatric patients treated regardless of gene rearrangement, and a subpopulation of 68 patients with TRK fusion cancer. The adverse event profiles of these two populations were similar (28). In the overall safety population, the most frequently reported all-causality grade 3 or 4 adverse events were anemia (11% of patients), increased weight (7%), dyspnea (6%), and fatigue (4%); a full listing of treatment-emergent adverse events has not been reported. The most common grade 3 or 4 treatment-related adverse events were increased weight (5%), anemia (5%), fatigue and neutropenia (both 3%). Dose discontinuation due to treatment-related adverse events occurred in only 4% of patients. Entrectinib administration was however associated with an increased risk of fractures. According to the label, in an expanded safety population that included 338 adult patients and 30 pediatric patients who received entrectinib, 5% of adult and 23% of pediatric patients experienced fractures (52). Although some fractures in adult patients occurred in the setting of a fall or other trauma to the affected area, all fractures in pediatric patients were reported to have occurred in patients with minimal or no trauma.

In addition to these prospective analyses, Liu and colleagues carried out a retrospective analysis of adverse events in 96 patients who had received a TRK inhibitor for the treatment of advanced cancer (53). The focus of this study was on-target adverse events likely to have arisen secondary to TRK inhibition, including weight gain (53% of patients at any grade), dizziness (41%; 6% of patient concurrently with ataxia), paresthesia (18%), and pain associated with treatment withdrawal (34%). This on-target safety profile was deemed to be unique relative to other anticancer agents. While warranting careful monitoring, these adverse events were found to be manageable either with pharmacologic intervention and/or dose modification, or TRK inhibitor re-initiation (53).

Other Agents

Efficacy data for selitrectinib and repotrectinib and other agents in patients with TRK fusion cancer are currently limited. Both next-generation TRK inhibitors have shown efficacy in individual patients who have progressed following the acquisition of on-target resistance during treatment with larotrectinib or entrectinib (42,43).

Mechanisms of Acquired Resistance

Tumors may become resistant to TKIs either through on-target mechanisms, such as mutations affecting the coding sequence of the tyrosine kinase domain of the driver, or off- target mechanisms, such as changes affecting bypass pathways, for example BRAF or KRAS activating mutations. Both mechanisms may allow escape of the tumor from driver kinase inhibition. For first-generation TRK inhibitors, on-target resistance mechanisms so far described include mutations in NTRK1 and NTRK3 leading to amino acid substitutions in the solvent front (TRKA G595R and TRKC G623R), activation loop xDFG motif (TRKA G667S and TRKC G696A), or gatekeeper residue (TRKA F589L) of the tyrosine kinase domains (Table 1). To our knowledge, resistance mutations in NTRK2 have not yet been reported, most likely due to the relatively low number patients with NTRK2 gene fusions who have so far been treated with TRK inhibitors (39).

In some cases, on-target NTRK resistance mutations have not been identified in the tumors of patients who have progressed on TRK inhibitors, including following treatment with next- generation agents. Sequencing of pre-treatment and post-progression tumor biopsies in such patients has in some cases revealed the presence of new off-target driver mutations in TRK inhibitor resistant tumors. Emergent off-target TRK inhibitor resistance mechanisms commonly involve components of the MAPK pathway, including known activating mutations of BRAF, KRAS, ERBB2, and high-level amplification of MET (54). However, it should be noted that some patients with TRK fusion-positive tumors have additional tumor driver mutations at initial presentation and yet still respond to TRK inhibitors (49,55,56), suggesting that the NTRK fusion is the predominant oncogenic alteration. The presence of other oncogenic mutations at initial diagnosis should therefore not automatically preclude TRK inhibitor treatment, with further work necessary to understand when such alterations might confer primary resistance to TRK inhibitors.

Strategies to Overcome Acquired Resistance Anticipating that acquired resistance or intolerance to TRK inhibitors would arise in some treated patients, and extrapolating from precedent resistance mechanisms in structurally similar oncogenic kinases, the development of a next-generation TRK inhibitor was

undertaken in parallel with the clinical development of first-generation agents. This led to the identification of a novel TRK inhibitor, selitrectinib, which demonstrated pre-clinical activity against all NTRK resistance mutations that had at that time been identified in patients. The unprecedented parallel development programs of first- and next-generation agents facilitated rapid entry into the clinic of the next-generation agent and allowed for selitrectinib treatment of the first two patients with TRK fusion cancer to develop acquired resistance to larotrectinib (42). The next-generation TRK inhibitor repotrectinib was specifically designed to overcome clinical resistance mutations, in particular, TRK solvent front mutations. In a proof-of-concept case study, a rapid and dramatic response to repotrectinib was achieved in a patient with an NTRK3 resistance mutation acquired during prior entrectinib treatment (43). Clinical trials of both selitrectinib and repotrectinib are ongoing in both children and adults.

In the case of off-target resistance mechanisms, while next-generation TRK inhibitors are likely to be ineffective in such patients, a subset of the bypass pathway mutations may be themselves actionable, as has been confirmed in proof-of-concept studies in two patients (54).

The longitudinal analysis of circulating tumor DNA (ctDNA) using sensitive digital PCR or NGS technologies provides a potentially valuable approach to the detection and tracking of on-target and off-target tumor resistance mutations in patients receiving targeted agent therapy (57-59). Deriving such information from the analysis of ctDNA has several advantages over conventional tissue biopsy approaches. In particular, the collection of plasma samples is a simple, non-invasive procedure that can be carried out at any time during or after treatment and at regular intervals. In addition, a single tissue biopsy may contain only a proportion of the genetic alterations present within a patient’s tumor and may miss mutations that vary by geographical distribution (60). By contrast, ctDNA analyses can provide a more comprehensive view of the heterogeneity of resistance mechanisms present within a disseminated tumor. In relation to NTRK resistance mutations, ctDNA analyses have been shown to be useful in the detection and monitoring of tumor resistance mutations in separate case studies of patients treated with selitrectinib (42) and entrectinib (61).

Overview of Clinically Approved First-Generation TRK Inhibitors

The registrational analyses confirm that both larotrectinib and entrectinib are active in adult and adolescent patients with TRK fusion cancer. In addition, larotrectinib is highly active in children less than 12 years of age, including infants. Both agents have demonstrated activity in patients with CNS metastases and in patients with primary CNS disease. It is notable that

for the overall study populations, notwithstanding the caveats associated with cross study comparisons, the median duration of response and median PFS were markedly longer and the response rate appeared higher for larotrectinib-treated patients compared with entrectinib-treated patients (Table 2). Whether these data are indicative of a more durable antitumor activity of larotrectinib over entrectinib in patients with TRK fusion cancer and/or are related to baseline differences in study populations will require confirmation in future comparative studies. In this context, it should be borne in mind that the entrectinib study population included a higher percentage of patients with CNS metastases at baseline (22%) compared with the larotrectinib study population (8%). Furthermore, in relation to duration of response and PFS outcomes, it should also be noted that the larotrectinib but not the entrectinib study population included pediatric patients, although the response rate nevertheless remained higher in the adult subgroup of the larotrectinib population (73%) (27), compared with the adult population of the entrectinib study (57%) (28). For both agents, the majority of adverse events were mild, with those deemed to be associated with TRK inhibition manageable. However, entrectinib may be associated with an increased risk of fractures, particularly in pediatric patients.

Areas for Further Consideration

In addition to more data on the activity of TRK inhibitors and the durability of benefit in specific indications and genomic subgroups of patients such as those with MMRd colorectal cancers harboring TRK fusions, other issues relating to optimal treatment strategies may become clearer as more patients are treated with these agents over longer periods of time. In particular, the long-term safety profiles of TRK inhibitors will become better defined. Given their normal role in the maintenance and functioning of the nervous system, whether there is any impact on neurocognitive function and development in pediatric patients will need to be further explored with longer follow up.

The optimum treatment duration is also an area for further consideration. Especially in relation to young patients, whether TRK inhibitor therapy needs to be continued indefinitely in the setting of ongoing response needs to be explored. In particular, it may be that some patients can discontinue therapy with response maintained, as has been seen for TKIs in other settings (62,63). A further aspect of treatment duration which might be investigated is whether continuing treatment beyond progression in certain patients might confer a tangible clinical benefit. For example, if progression occurs at a single site that is amenable to local therapy, maintenance of TRK inhibitor treatment might be justified in order to control lesions at other sites. Similarly, continuing TRK inhibition with the addition of other kinase inhibitors

blocking treatment emergent resistance mutations has shown proof of concept and deserves further study.

Further questions for consideration relate to the most appropriate line of therapy for TRK inhibitor treatment, whether it might be optimal to initially administer first- or next-generation agents in certain or all patients, and whether these considerations might vary between different indications. Currently ongoing studies will provide more data to address such questions.

Conclusions

The high response rates and durable disease control achieved with TRK inhibitors across a wide range of tumor types have validated NTRK gene fusions as actionable, tumor agnostic oncogenic drivers. TRK inhibitors appear to be generally very well tolerated and are amenable to long-term administration. Although resistance may be acquired, strategies are being developed to maintain disease control over extended periods of time, including the development of next-generation agents and genomically directed combination therapy. The establishment of diagnostic algorithms to allow for the efficient and cost-effective identification of tumors harboring NTRK gene fusions across different settings remains an ongoing clinical priority.

Acknowledgements Medical writing support was provided by Jim Heighway of Cancer Communications and Consultancy (Knutsford, UK), with funding from Bayer. Circos plots were kindly provided by Justyna Wierzbinska (Bayer).

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Table 1. Reported IC50 values of selective and multikinase TRK inhibitors Larotrectinib Entrectinib Selitrectinib Repotrectinib Kinase (IC50 nM) Enzyme Activity Enzyme Activity Enzyme Activity Enzyme Activity Biochemical Cellular Antiproliferative Biochemical Cellular Antiproliferative Biochemical Cellular Antiproliferative Biochemical Cellular Antiproliferative Ba/F3 4c LMNA-TRKA

KM12 12.3c Ba/F3 0.5 KM12 Ba/F3 <0.2 <5 TPM3-TRKA LMNA-TRKA TPM3-TRKA LMNA-TRKA TRKA NIH 3T3 NIH 3T3 1.6 0.9 5a 1 -- 0.6 0.5 -- WT ΔTRKA* ΔTRKA* KM12 3.5d KM12 9.2 CUTO-3 KM12 0.2 <5 TPM3-TRKA TPM3-TRKA MPRIP-TRKA TPM3-TRKA

Cuto3.29 59d MPRIP-TRKA NIH 3T3 Ba/F3 Ba/F3 NIH 3T3 TRKA G595R 69.0 819a 1024c -- -- 705 2.0 7 -- -- 2.7 -- 0.2 ΔTRKA* LMNA-TRKA LMNA-TRKA ΔTRKA* NIH 3T3 NIH 3T3 TRKA G667C 45.5 315a ------9.8 64 ------ΔTRKA* ΔTRKA* Ba/F3 ETV6-TRKB 10.9c Ba/F3 TRKB Ba/F3 -- 8.1b 3 -- ETV6-TRKB 0.5 ------0.3 -- <0.2 WT ETV6-TRKB BAF TRIM24-TRKB 35d Ba/F3 Ba/F3 -- Ba/F3 TRKB G639R -- -- 3000c -- -- 1834 ------2.7 -- 0.6 ETV6-TRKB ETV6-TRKB ETV6-TRKB Ba/F3 10.2c ETV6-TRKC TRKC NIH 3T3 Ba/F3 NIH 3T3 MO-91 Ba/F3 2.8 18.8a 5 -- 0.6 <2.5 2.0 <1 0.2 -- <0.2 WT ETV6-TRKC ETV6-TRKC ETV6-TRKC ETV6-TRKC ETV6-TRKC Mo91 1d ETV6-TRKC NIH 3T3 Ba/F3 Ba/F3 NIH 3T3 Ba/F3 TRKC G623R 48 >10,000a 3293c -- -- 1623 2.3 45.5 -- -- 4.5 -- 0.4 ETV6-TRKC ETV6-TRKC ETV6-TRKC ETV6-TRKC ETV6-TRKC TRKC Ba/F3 Ba/F3 Ba/F3 742.3c -- 1351 ------1.4 G623E ETV6-TRKC ETV6-TRKC ETV6-TRKC NIH 3T3 NIH 3T3 -- TRKC G696A 4.5 172a ------<2.5 17.5 ------ETV6-TRKC ETV6-TRKC Drilon et al, aDrilon et al, 2017; cDrilon et al, 2018; (suppl) Ardini et al, 2016; Drilon et al, 2018; (suppl) Drilon et al, Drilon et al, 2017; Drilon et al, 2017; Drilon et al, 2018; (suppl) Drilon et al, 2018; 2017; Figure 2A Table S6B Table 1 Table S6B 2017; Figure 2A (suppl) Table S3 (suppl) Figure 1B (42) (43) (64) (43) Figure 1B (42) Figure S3A (43) Table S6B References (42) bGhilardi JR et al, 2010 dDrilon et al, 2018; (42) (42) (43) Figure 1 (suppl) Table S1 (38) (37)

*ΔTRKA second Ig domain deletion. WT, wild-type.

Table 2. Summary of baseline characteristics and efficacy in key clinical studies of TRK inhibitors Larotrectinib Larotrectinib Entrectinib

Data cutoff: July 17, 2017 Data cutoff: February 19, 2019 Data cutoff: March 27, 2018 Baseline characteristics N=55 N=159 N=54 Age, years: median (range) 45.0 (0.3–76.0) 43.0 (<1 month–84 years) 58 (48–67) Prior lines of therapy, n (%) Previous systemic chemotherapies: Prior systemic treatments: 0 or 1 27 (49) 83 (52) 31 (57) 2 9 (16) 34 (21) 14 (26) ≥3 19 (35) 42 (26) 9 (17) CNS metastases at baseline, n (%) Yes 1 (2) 13 (8) 12 (22) No 54 (98) 146 (92) 42 (78) Efficacy (all patients) ORR (95% CI) INV (n=55) IRC (n=55) INV (n=153 evaluable) BICR (n=54) 80% (67–90) 75% (61–85) 79% (72–85) 57% (43.2–70.8) DoR, (95% CI) months 35.2 (21.2–NE)* 35.2 (22.8–NE) 10.4 (7.1–NE) Median TTR (range), months 1.8 (0.9–6.4) 1.8 (0.9–6.1) -- Median PFS (95% CI), months 25.8 (9.9–NE)* 28.3 (22.1–NE) 11.2 (8.0–14.9) Median OS (95% CI), months 44.4 (36.5–NE)* 44.4 (36.5–NE) 21.0 (14.9–NE) Efficacy (pediatric) INV; (n=51 evaluable) <1+ years old -- -- ORR (95% CI) 92% (81–89) Efficacy (adult) INV; (n=107, 102 evaluable) BICR (n=54) -- ORR (95% CI) 73% (63–81) 57% (43.2–70.8) Efficacy (CNS) All target lesions with brain met INV (n=13; 12 evaluable) INV assessment (n=12) ORR (95% CI) 75% (43–95) BICR ORR 50% --

Intracranial response Measurable at baseline (n=3) BICR confirmed with measurable CNS disease (n=11) ORR (95% CI) 67% 55% (23.4–83.3) Drilon et al (37) References Hong et al (27) Doebele et al (28) Hong et al (27)

*Data cutoff: February 19, 2019. BICR, blinded independent central review; CNS, central nervous system; DoR, duration of response; INV, investigator; NE, not evaluable; ORR, objective response rate; PFS, progression-free survival; TTR, time to response.

Figure 1. The NTRK gene fusion landscape based on data from larotrectinib (27,30) and entrectinib studies (28,29). A–C: Chord diagrams summarizing the frequencies of NTRK fusions (NTRK1–3) with their partners, per tumor type. Tracks from outside to inside: gene involved in the fusion, frequency of reported fusions, and fusions visualized as ribbons connecting fusion partners. Cumulative frequencies of fusions are color coded by cancer type. D–F: Circular plots show a genome-wide overview of NTRK genes (NTRK1–3) and their fusion partners. Circular tracks from outside to inside: genome positions by chromosome (1–Y), chromosomal cytobands, annotation of the fusion type (inter- or intrachromosomal fusions), and fusions visualized as links/arcs connecting fusion partners. Each fusion partner is annotated with its HUGO Gene Nomenclature Committee approved symbol. Plots were generated using the circlize package in R (65). Genome-scale plots and chord diagrams were generated using a modification of circos.genomicLink and chordDiagram functions from the circlize package, respectively.

A B C

D E F

Tropomyosin Receptor Kinase Inhibitors for the Treatment of TRK Fusion Cancer

Theodore W. Laetsch and David S. Hong

Clin Cancer Res Published OnlineFirst April 23, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-21-0465

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