Emerging Mutations in Lung Cancer and Other Solid Tumors

Special Issues, Focus on Pathways: Emerging Mutations in Lung Cancer and Other Solid Tumors,

There is an evolving role of gene mutations within the cancer treatment spectrum, from discoveries in tumor biology and detection technologies, to the development targeted therapies and the clinical impact of pivotal trial data.

Tumor Biology and Treatment Approaches:Historical Perspective and New Innovations

Advances within the field of oncogenetics over the past several decades have ushered in a new stage in cancer treatment. Continued discoveries in the realm of gene fusions coupled with innovations in detection technology have since fueled development of targeted therapies. Data have increasingly shown the therapeutic potential of the neurotrophic tyrosine receptor kinase (NTRK) gene mutation as a target for inhibition. Preclinical and clinical studies of recently developed NTRK inhibitors have demonstrated robust response in NTRK-dependent tumors. Additionally, NTRK inhibitors have demonstrated activity in inhibiting anaplastic lymphoma kinase- (ALK) and proto-oncogene receptor kinase- (ROS1) dependent tumors. This supplement chronicles the evolving role of gene mutations within the cancer treatment spectrum, from discoveries in tumor biology and detection technologies, to the development targeted therapies and the clinical impact of pivotal trial data.Decisions regarding cancer therapy have historically been based largely on histologic considerations. For example, lung cancers have typically been categorized into small-cell lung cancer (SCLC) and non—small cell lung cancer (NSCLC) types, the latter of which is then further subdivided into squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma.1 In the past, platinum-based doublet chemotherapy was the standard treatment for patients with advanced NSCLC; efficacy was limited.1 However, after a study demonstrated better response to pemetrexed than gemcitabine among patients with nonsquamous NSCLC,1 researchers began evaluating whether genetic variations of cancer have a role in treatment selection.

The treatment of lung cancer has become further refined with the emergence of next-generation sequencing (NGS) techniques, along with the availability of verified in-clinic assays for defining molecular subtypes and mutations that may be targeted with therapy. Diagnostic technologies, such as liquid biopsy2 and transcriptome sequencing,3 can assist clinicians in determining which targeted therapy is optimal for their patients. Genomic studies over the past decade have uncovered additional molecular driver mutations in lung cancer, allowing further subdivision of NSCLC classification based on these driver mutations.1 These findings have assisted in the development of targeted therapy.1 For example, recent findings suggest that presence of mutations in the epidermal growth factor receptors (EGFR) strongly predicts the efficacy of EGFR tyrosine kinase inhibitors (TKIs), wherein response rates greater than 70% were seen in multiple studies.1

Molecular alterations in NSCLC can lead to oncogene activation through multiple mechanisms, including point mutations, insertions, deletions, and gene rearrangements.4 Generally, these alterations are mutually exclusive, but all have the propensity to confer oncogene addiction, which describes the phenomenon by which oncogenesis is driven primarily or exclusively by aberrant oncogene signaling.4 The implications of oncogene arrangements have been well described in NSCLC. With respect to oncogenic fusions, in which a 5-inch partner forms an in-frame gene fusion with a 3-inch proto-oncogene, the kinase domain of the proto-oncogene is typically preserved, and the result is expression of a constitutively activated protein whose downstream signaling promotes cell proliferation and survival in a ligand-independent manner.4 Gene fusions often represent markers for specific cancer subtypes. For example, BCR-ABL1 gene fusion has been implicated in chronic myelogenous leukemia.3 Some gene fusions, however, termed multitumor rearrangements, are nonspecific and have been seen in multiple cancer types.3 For example, ETV6-NTRK3 has been linked to secretory breast cancer, congenital fibrosarcoma, acute myeloid leukemia, and other malignancies.3

In isolation, the occurrence of these genetic events may appear rare; however, these findings may have a significant clinical impact when they are factored into universal, comprehensive molecular profiling.4 Using NGS technology that identifies both gene sequence alterations and gene fusions may increase discovery of targetable mutations for a patient, thereby identifying more patients who may benefit from molecularly targeted therapy.4 Applications of NGS in clinical and research settings continues to identify relevant driver mutations that may be targetable using inhibitory approaches. For example, NTRK fusion molecules were first identified in 1986, but in the nascent days of pharmacogenomics, molecular profiling technology to test for such mutations was unavailable.5 The emergence of new genomic technology, such as NGS, prompted the development of NTRK-targeted inhibitor therapy.5 More recently, the ability to screen a large tumor cohort to identify rare targetable drivers has led to the development of new tailored agents that selectively inhibit such molecular alterations.5 Second generation NTRK inhibitors, such as entrectinib and larotrectinib, have exhibited a high degree of potency against NTRK1/2/3 fusions and in some cases against other mutations, rearrangements, and amplifications within the anaplastic lymphoma kinase ALK and ROS1 pathways.5 Studies of these new agents introduced a new early phase study design, a “basket trial design,” whereby patients are matched with experimental drugs based on their genomic profile rather than the tissue where the tumor originated.5

Parallel to and in concert with the more specified understanding of tumor biology at the individual level is the advancement in treatment approaches predicated on personalized factors. The past decade has witnessed a veritable explosion in advancements in precision therapy approaches that have transformed the treatment of a variety of cancer types. In 2015, there were particularly noteworthy approvals of agents for the treatment of NSCLC, which comprised 7 FDA approvals, including 4 treatments with breakthrough therapy designation, 3 accelerated approvals, and 4 expedited reviews.6 Currently, several TKIs that target specific gene fusions, such as ALK and ROS1, have gained regulatory approval in the United States.4

The time from mutation discovery, to candidate molecule identification, to regulatory approval, to clinical use has also shortened considerably. Just 4 years after the identification of ALK as a major oncogenic driver in NSCLC in 2007, the first targeted therapy, crizotanib, gained approval, which is in sharp contrast to the 13-year time lapse (1985 to 1998) between the discovery of HER2 as an oncogenic driver in breast cancer and the approval of trastuzumab.7 As a result of these and other comparable discoveries in approaches to targeted therapy approaches, the percentage of patients who might be eligible for and benefit from genome-targeted and genome-informed therapy has risen exponentially.4

Results of a 2018 study found that the percentage of patients with advanced cancer eligible for genome-driven therapy and genome-informed therapy increased from 5.09% and 10.50% in 2006, respectively, to 8.33% and 15.44% in 2018.8 In addition, the percentage of patients estimated to benefit from genome-targeted therapy and genome-informed therapy also increased from 0.70% and 1.13% in 2006, respectively, to 4.90% and 6.62% in 2018.8

A 2013 study conducted a breakpoint analysis of transcriptional and genomic profiles to develop a method of detecting “breakpoints” in genomic datasets.3 The researchers identified 198 candidate fusions involving annotated cancer genes out of 974 diverse human cancer samples, including novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L-ROS1), SLC1A2 glutamate transporter in colon cancer (APIP-SLC1A2), RAF1 kinase in pancreatic cancer (ATG7-RAF1) and anaplastic astrocytoma (BCL6-RAF1), EWSR1 in melanoma (EWSR1-CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B-CDK6), and CLTC in breast cancer (CLTC-VMP1).3 The authors noted that the study’s findings may have clinical implications on future drug targets.3 Other study results have similarly demonstrated novel fusion genes in a wide variety of cancer types.9,10



Perhaps most tangible to the field of oncology, though, is the fact that real-world applications of molecular targeted therapy have already demonstrated the positive benefits and feasibility of this approach.11 The Biomarkers France study evaluated the impact and feasibility of routine broad molecular screening for EGFR mutations, ALK rearrangements, and HER2, KRAS, BRAF, and PIK3CA mutations at a national level.12 In a 1-year period, 18,679 molecular analyses were performed in 17,664 patients with NSCLC. Investigators found genetic alterations in 49.5% of the analyses, with EGFR, HER2, KRAS, BRAF, and PIK3CA mutations and ALK rearrangements reported in 11.0%, 0.8%, 28.7%, 1.9%, 2.3%, and 4.8% of the analyses, respectively.12 Of note, the presence of a genetic alteration impacted first-line treatment for 51.3% of patients, and was associated with a significant improvement in the overall response rate for first- and second-line treatment (P = .03% and P <.0001, respectively).12EGFR belongs to a family of receptor tyrosine kinases (RTKs) that includes EGFR, ERBB2 (also known as HER2), ERBB3 (also known as HER3), and ERBB4 (also known as HER4), each of which is composed of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain.13 Under normal physiologic conditions, ligation with any of the aforementioned receptor domains results in conformational changes that facilitate homodimerization or heterodimerization, setting in motion intermolecular autophosphorylation.13 The end result is recruitment of proteins involved in downstream signaling events that control multiple cellular processes, including proliferation and survival.14

Study findings have shown that exon 19 deletion and L858R mutation confer ligand-independent activation and prolonged receptor kinase activity after ligand stimulation, thereby suggesting a mechanism for constitutive activation and oncogenic transformation.13 For example, in vitro studies have demonstrated that selected EGFR mutations (eg, exon 18 G719S, exon 19 deletion, exon 21 L858R, and exon 20 insertion) can transform both fibroblasts and lung epithelial cells.13 Furthermore, EGFR L858R and G719S mutations disrupt autoinhibitor interactions, resulting in a 50-fold increase of TKI activity at the receptor compared with wild-type EGFR.13

Various measurement techniques have been used to detect EGFR, including PCR and FISH (fluorescence in situ hybridization).15 Data showing that somatic mutations in the EGFR gene were associated with clinical response to the EGFR TKIs erlotinib and gefitinib began emerging in 2004.13 The discovery suggested the potential for improved treatment outcomes in this subset of patients, representing the tipping point in how other oncogenic mutations would be researched and eventually managed in the clinic.1 In addition to ushering in the era of tailored molecular therapy, the finding that certain subtypes of lung adenocarcinomas were dependent on EGFR mutations for oncogenesis extended the concept of “oncogene addiction” to human carcionomas.13

Subsequent studies in the context of EGFR mutations would later confirm the “oncogenic shock” model, in which competing proapoptotic and pro-survival signals ultimately determine the fate of oncogenesis.15 The model provides that when an addicting oncogene is active, pro-survival signals suppress pro-apoptotic signals, allowing the cancer cell to survive.15 Conversely, if the oncogene is inactivated (eg, introduction of a TKI) pro-apoptotic signals will predominate, thereby setting in motion signaling pathways that yield cell death.15 However, this apoptotic response to oncogene inactivation is transient as the cell adapts to the signal imbalance over time, eventually negating the apoptotic response.15 Another research development to emerge from EGFR tumor models is the finding that analysis of circulating tumor cell preparations using NGS techniques may be clinically useful diagnostically and for monitoring mutational status of the tumor during treatment.14 One study’s findings showed that CTC preparations are a sufficient source of tumor DNA for efficient detection of EGFR mutations when NGS techniques were used.14 The authors noted that this method may be useful for monitoring acquired TKI resistance as well.

EGFR mutations have been noted in various types of cancer, including those of the head and neck, ovary, cervix, bladder, esophagus, stomach, brain, breast, endometrium, pancreas, colon, and lung.13,14 Within the lung, EGFR may be overexpressed in 62% of cases of NSCLC.15 Histologically, EGFR kinase mutations are primarily seen in a subset of NSCLC, although rare mutations have been detected in patients with other cancers, such as small SCLC.14 For unknown reasons, however, EGFR mutations are more commonly found among individuals of East Asian descent, women, and never-smokers.13

Treatment Considerations. Study results have shown robust response rates to EGFR-targeted TKIs, such as erlotinib and gefitinib, in patients with EGFR-mutant NSCLC compared to those with wild-type EGFR tumors, which confirms that EGFR mutation status can predict response to EGFR-targeted TKIs.13,16 Several factors may contribute to the degree of response. For example, patients with amplification or polysomy of EGFR were more likely to respond to erlotinib or gefitinib compared with those with normal EGFR copy number.13 Additionally, although several different types of EGFR mutations have been described, only certain ones are drug sensitive, although they are all contained within exons 19 and 21.14

Patients with NSCLC harboring EGFR mutations in exon 19 have been noted to exhibit a better response to gefitinib and erlotinib than NSCLC with mutations in exon 21.14 Exon 19 deletions and the exon 21 L858R substitution are the most common, although point mutations in exons 18 (G719A/C) and 21 (L861Q) have also been acknowledged to be drug sensitive.13 Additionally, 3 EGFR mutations, in particular, have been shown to confer primary resistance to TKIs: exon 19 point mutation D761Y, exon 20 point mutation T790M (the so-called gatekeeper mutation), and exon 20 insertion D770_N771insNPG.13 PCR and FISH have been used to measure gene copy number and aneuploidy levels, which may offer insight into the likelihood of disease stabilization via TKI treatment.14

Nevertheless, the introduction of EGFR TKIs has improved treatment outcomes compared with chemotherapy in clinical trials. In the phase III IPASS study, which enrolled East Asian individuals with chemotherapy-naïve lung adenocarcinoma and who were either never-smokers or light smokers, the response rate to gefitinib was 71.2% compared with a 43.0% response rate to chemotherapy among individuals harboring EGFR-mutant tumors.13 Erlotinib was subsequently shown to be at least as effective in patients with mutant EGFR adenocarcinoma.13 Other studies have noted that gefitinib is slightly less effective in the second-line setting compared with first line.13

Resistance Mutations. Despite the high efficacy associated with EGFR TKIs, both primary and acquired (secondary) resistance has been seen.13,14 Some patients who do not exhibit a response to TKI may appear to have a drug-resistant tumor; however, the lack of response is more attributable to the reduction in tumor size not meeting the Response Evaluation Criteria in Solid Tumors (RECIST), which requires minimum shrinkage of 30% to be considered a partial response.13 True primary resistance has been associated with mutations in exons 18 to 21 that cause the receptors to be less drug-sensitive.14,16 As noted earlier, rare insertions or duplication in exon 20 may confer primary resistance to EGFR TKIs, as they reduce the receptor’s sensitivity to EGFR TKIs approximately 100-fold.15,16 In in vitro studies, constitutively active PI3K mutants have shown to be resistant to gefitinib, likely due to inhibition of downstream signaling of EGFR.16

By comparison, development of acquired, or secondary, resistance is more common than primary resistance and is usually seen within 1 year of initiating therapy.16 Within this category, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain, is the predominant mechanism of resistance to gefitinib or erlotinib.17 Subsequent studies have identified T790M mutations in about half of cases of secondary resistance13 and that this mutation confers the highest degree of drug resistance among known secondary mutations.16 Other second-site EGFR mutations have been associated with acquired resistance, including L747S on exon 19, D761Y on exon 19, and T854A on exon 21 in the activation loop.16 In addition to these mutations, amplification of the mutant EGFR or hyperactivation of downstream signaling components that circumvent EGFR inhibition, may increase expression of signal-attenuating molecules or induce cellular changes that alter bioavailability of EGFR TKIs.14 For example, amplification of the MET oncogene was observed in patients with EGFR-mutant NSCLC who failed TKI therapy.13


Overcoming Resistance. There are novel EGFR TKIs that have been developed to overcome drug-resistant EGFR mutants currently being studied for clinical efficacy.16 These form irreversible bonds as opposed to first-generation EGFR TKIs, which only form reversible bonds.15,16 There are several “second-generation” agents currently in phase II and III trials.16 Afatinib and osimertinib have shown efficacy in overcoming resistance in earlier trials.16 Utilizing the knowledge of the mechanisms of primary and acquired resistances, researchers are investigating the efficacy of using combination therapy; for example, afatinib with a mechanistic target of rapamycin (mTOR) inhibitor or afatinib with cetuximab.16 Because amplification of the MET oncogene has been associated with acquired resistance, MET inhibitors are also being investigated.16ALK rearrangements were first described in the cancer literature in 1994 in anaplastic large-cell lymphoma (ALCL). The occurrence of an echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion was later reported in NSCLC in 2007.18 Reverse-transcriptase PCR, FISH, and immunohistochemistry (IHC) have all been used to detect EML-ALK.18 Among these, IHC may present the most practical option for detecting ALK-positive NSCLCs, as it is not associated with the cumbersome technical challenges and operator dependence of FISH.21

Because it was first recognized as having a role in oncogenesis, ALK fusions have been identified in a number of cancer types: 3% to 7% of patients with NSCLC, approximately 55% of cases of ALCL, and up to 50% of inflammatory myofibroblastic tumors.18 Although far less frequently, ALK rearrangements have also been seen in various other cancers, such as diffuse large B-cell lymphoma cancers, colorectal cancers, renal cell carcinomas, breast cancer, renal medullary carcinoma, esophageal cancer, and ovarian cancer.18 In other series, ALK has been reported in 0.05% to 2.5% of patients with colorectal cancers and to be largely mutually exclusive of KRAS, NRAS, or BRAF alterations.19 In a study of 1,683 patients, ALK mutations was also been found to be mutually exclusive of EGFR and KRAS mutations.20

Subtypes of ALK fusions are noted to differ among cancer types. For example, whereas NPM1-ALK fusion is the predominant subtype in ALCL, EML4-ALK fusions are enriched in NSCLC cases.18 Within NSCLC, a variety of EML4-ALK rearrangements have been reported, and several other fusion partners have been identified, such as NPM and tropomyosin.21 Whereas ALK is acknowledged to be a member of the RTK insulin receptor superfamily,21 its biological function in humans is not completely understood.18 Its role has been elucidated in lower-order animals.

For example, in Drosophila melanogaster, ALK regulates the development of the gut musculature and neuronal circuitry within the visual system, and in mice, it is expressed in the nervous system at the embryonic and neonatal stage.18 Some common features of ALK fusions have been noted across different ALK fusion types: (1) the entire ALK kinase domain is preserved; (2) the N-terminal partner contributes its promoter and oligomerization domain to the ALK fusion protein, leading to constitutive activation of ALK; and (3) ALK fusion proteins’ interactions with downstream pathways, Janus kinase/signal transducer and activator of transcription (JAK/STAT), phosphatidylinositol 3-kinase (PI3K)/AKT, and MEK/extracellular signal-regulated kinase (ERK).18


Immediately after EML4-ALK fusion was established as a molecular target in NSCLC, agents targeting the ALK fusion were developed and their efficacy studied in clinical trials.18 Early clinical trials of the first agent targeting ALK fusions, crizotinib, showed such promising results that they were granted accelerated approval by the FDA for use in patients with ALK-positive NCSLC. Thereafter, second generation ALK inhibitors were developed with the intent of overcoming crizotinib resistance.18 ALK inhibitors have not yet been approved in non-lung ALK-driven cancers; however, studies are underway to evaluate the efficacy of ALK inhibitors in other cancer types.18Crizotinib was the first ALK inhibitor granted FDA approval, receiving an accelerated approval in 2011 based on phase I and II clinical trials, and full approval in 2013 based upon the results of phase III clinical trials.21 In the PROFILE 1001 phase I clinical trial, 60.8% of 143 patients evaluated achieved partial or complete response to crizotinib, with a median time to first documented response of 7.9 weeks.22 Median progression free survival (PFS) was 9.7 months; however, in patients who did not receive prior cancer treatment (16% of the patient population), the median PFS was 18.3 months compared with 9.2 months in patients who were previously treated (84% of the patients).22 Treatment-related adverse events (AEs) were reported in 97% of patients; the most common AEs were gastrointestinal events (nausea, vomiting, diarrhea), visual effects, and peripheral edema.22 The PROFILE 1007 phase III clinical trial evaluated crizotinib versus conventional chemotherapy among patients with locally advanced or metastatic NSCLC who had progressive disease after receiving platinum-based chemotherapy.23 Of the patients treated with crizotinib, 66% achieved partial or complete response, compared with 20% of the patients who received chemotherapy.23 Median PFS in the crizotinib group was 7.7 months versus 3.0 months in the chemotherapy group. 23 Crizotinib was also studied as a first-line option in the parallel phase III PROFILE 1014 clinical trial.21 In that study, PFS was significantly longer with crizotinib compared with platinum-pemetrexed chemotherapy (11 months vs 7 months, respectively); the response rate was also improved (74% vs 45%, respectively).21


Similar to other targeted therapies, acquired resistance was seen in patients treated with crizotinib, with disease progression typically seen in 1 to 2 years.18Ceritinib was developed to overcome acquired resistance to ALK inhibitors and was the first second-generation ALK inhibitor granted FDA approval. Initially ceritinib was granted accelerated approval in April 2014 for patients with metastatic ALK-positive NSCLC with disease progression on or intolerance to crizotinib , based on results from the ASCEND-1 trial. FDA broadened the indication in 2017 to use in patients with metastatic ALK-positive NSCLC, based upon the ASCEND-4 trial results.24 In ASCEND-1, 72.3% of patients who were ALK inhibitor—treatment naïve (ALKi-naïve) had partial or complete response, while 56% of patients who previously received ALK inhibitor treatment (ALKi-experienced) exhibited partial or complete response.25 The duration of response (DOR) and PFS for ALKi-naïve patients was 17.0 months and 18.4 months, respectively.22 Median DOR and PFS for ALKi-experienced patients was 8.3 months and 6.9 months, respectively.26


In the phase III ASCEND-5 trial, ceritinib was compared with chemotherapy in patients with NSCLC who had progressed on crizotinib and platinum-based chemotherapy.21 In this study, superior PFS and response rates were noted with ceritinib compared with single-agent chemotherapy, thus suggesting that additional ALK inhibition is more beneficial than chemotherapy when NSCLC is crizotinib-resistant.27 The most common AEs associated with ceritinib include elevations in transaminases, diarrhea, nausea, vomiting, fatigue, abdominal pain, decreased appetite, constipation, rash, dyspnea, musculoskeletal pain, and arthralgia. Moreover, the dose recommended in phase II studies (ie, 750 mg) was shown to be difficult to tolerate, with 62% of patients treated at this dose level requiring dose reduction.21Alectinib is a newer “second generation” ALK inhibitor that is more potent and specific than crizotinib and has activity against some ALK mutations that are resistant to crizotinib.21 Similar to ceritinib, alectinib was granted accelerated FDA approval in 2015 for patients with metastatic ALK-positive NSCLC previously treated with crizotinib, based upon results from two phase II trials, and was granted a broader indication for use in patients with metastatic ALK-positive NSCLC in November 2017 based upon ALEX trial results.28 The 2 phase II clinical trials studied alectinib in patients with locally advanced or metastatic ALK-positive NSCLC who had disease progression while taking crizotinib. The pooled analysis of the two trials showed an overall response rate (ORR) of 51.3% in all patients, 49.3% in ALKi-experienced patients, and 58.5% in ALKi-naïve patients.26 The median DOR was 14.9 months: 14.9 months in ALKi-experienced patients and 11.2 months in ALKi-naïve patients.26 The ALEX study, comparing alectinib with crizotinib in ALKi-naïve patients, showed that alectinib was associated with a 53% reduction in risk of progressive disease overcrizotinib and had a significantly longer median PFS of 25.7 months compared with 10.4 months with crizotinib.26

Pipeline Agents. A robust pipeline of ALK inhibitors is currently in development; relevant data are presented in Table 1.21,31

Table 1. Select ALK Inhibitors in Development21,31

AE indicates adverse event; ALK, anaplastic lymphoma kinase; AXL, tyrosine-protein kinase receptor UFO; EPHA2, epithelial cell receptor protein tyrosine kinase; FMS/CFS1, protooncogene fms in concert with colony stimulating factor 1; ROS1, proto-oncogene recepto kinase; MET, mesenchymal-epithelial transition factor; TRKA, TRKB, TRKC, tropomyosin receptor kinase.

Table adapted from: Dagogo-Jack I, Shaw AT, Riely GJ. Optimizing treatment for patients with anaplastic lymphoma kinase-positive lung cancer. Clin Pharmacol Ther. 2017;101(5):625-633; and from Mok TSK, Crino L, Felip E, et al. The accelerated path of ceritinib: translating pre-clinical development into clinical efficacy. Cancer Treat Rev. 2017;55:181-189.

Mechanisms of Resistance and Implications for Therapy. Resistance is a common issue with first and second generation ALK-inhibitors; however, it appears that the rate of acquired resistance and mechanism of resistance may differ according to the particular ALK inhibitor.18 This differs from the mutation spectrum for resistance among patients with EGFR-mutant lung adenocarcinoma who develop resistance to ge&#64257;tinib, erlotinib, or afatinib, in which the majority of secondary mutations are the T790M gatekeeper mutation.21 Measuring resistance patterns has proven to be complicated, however, because of a paucity of available of on-treatment samples and the inherent difficulty involved in obtaining multiple biopsies for assessment. As such, in vitro and in vivo models have become popular for studying resistance patterns, although their presumed applicability to clinical practice is not easy to ascertain.18 More recently, the potential to use liquid biopsy samples has generated a new mechanism for the discovery and identification of both primary and secondary resistance patterns.18

Nevertheless, 2 broad categories of resistance have been identified: (1) ALK-dependent, “on-target” mechanisms, including secondary resistance mutations or amplifications, such that ALK addiction persists, and (2) ALK-independent mechanisms that include bypass tracks and/or lineage changes facilitating tumor escape from ALK inhibition.18 Analogous to imatinib resistance in patients with chronic myelogenous leukemia, the largest category of secondary resistance mechanisms are mutations within the target kinase that re-induce kinase activation.18,21 The 2 most frequently observed resistance mutations to date among individuals on crizotinib are the L1196M gatekeeper mutation and G1269A mutation; both of these are located in the ATP-binding pocket and inhibit crizotinib binding.18,21

In an analysis of 100 repeat biopsies from patients who progressed on first- and second-generation ALK inhibitors, acquired ALK mutations were seen in about 20% to 30% of patients progressing on crizotinib, compared with 56% in patients progressing on second-generation ALK inhibitors.18 L1196M and G1269A are most commonly associated with crizotinib resistance, while G1202R is the predominant resistance mechanism among patients progressing on ceritinib, alectinib, and brigatinib.18 For example, study results have shown that ceritinib can overcome resistance in mutations of L1196M, G1269A, I1171T, and S1206Y, although crizotinib cannot.29 However, mutations of G1202R and F1174C were found to be resistant to ceritinib.29 A third-generation pan-inhibitory ALK inhibitor, lorlatinib, has been shown to G1202R cell lines and in patients.18 Additionally, ALK amplification has been recognized as a cause of acquired resistance to crizotinib, but has not been seen in second- and third-generation ALK inhibitors, reflecting that more potent ALK inhibition may overcome these less potent mutations that induce resistance.18

Based on the findings that different ALK inhibitors have different mutation spectrums, researchers are starting to study whether the use of combination therapy may reduce risk of resistance.18 A number of combination strategies, including ALK inhibitor + MEK inhibitor, ceritinib + CDK4/6 inhibition, ceritinib + everolimus (an mTOR inhibitor), and alectinib with bevacizumab (a VEGF mediator), are in various stages of clinical study.18 Combination ALK TKI and immunotherapy has also been investigated, although patients with ALK-positive NSCLC tend to be never-smokers with low tumor mutational burden, both of which are proposed to limit the response to immune checkpoint therapy.18

On the other hand, about 40% to 50% of cases of resistance to second-generation secondary resistance appear to be caused by non—ALK dependent mechanisms, such as activation of bypass signaling tracks and/or phenotypic changes in the primary tumor.18 Another ALK-independent mechanism of resistance that has been identified involves P-glycoprotein (P-gp), which is encoded by the multidrug resistance 1 (MDR1) gene.18 In particular, P-gp may limit diffusion of crizotinib and ceritinib through blood-brain barrier to the central nervous system (CNS).18 Specific to CNS metastases, because alectinib and lorlatinib are not P-gp substrates, higher CNS levels of these agents can be acheived.18 In general, though, designing treatment strategies for these off-target, ALK-independent resistance profiles would seem to be predicated on systematic assessment of paired pre- and post-treatment biopsies to identify therapeutic targets.18

Implications for CNS Disease. A likely site of progression among patients with ALK-positive lung cancer is to the CNS.21 Case reports and laboratory studies report that crizotinib poorly penetrates the blood brain barrier into the CNS. 21 One retrospective study evaluated the clinical benefits of crizotinib in patients with brain metastases from the PROFILE 1005 and 1007 studies.30 Patients that were not previously treated for brain metastases (treatment-naïve) had an intracranial median time to progression (TTP) of 7.0 months compared to systemic TTP of 12.5 months.30 Patients that were previously treated for brain metastases (treatment-experienced) had a TTP of 13.2 months compared with systemic TTP of 14.0 months.30 It is also noted that 20% of the patients without baseline brain metastases developed brain lesions.30 These data seem to suggest that an alternative treatment approach is required to forestall CNS metastatic spread and/or to manage such cases when they arise.



A pooled analysis of the 2 alectinib phase II studies recently reported an intracranial response rate of 64% and CNS DCR of 90% among 50 patients not treated with radiation with baseline measurable CNS disease.21 Additionally, in the J-ALEX study, alectinib signi&#64257;cantly improved PFS relative to crizotinib, with a hazard ratio (HR) of 0.08, and in separate studies, escalation of alectinib to 900 mg twice daily or switching to lorlatinib have each proven successful for recovery of CNS response.21 A retrospective study evaluated patients with baseline brain metastases in the ASCEND-1 trial and reported intracranial DCR of about 62% to 78% with ceritinib.31 However, it is noted that ceritinib is a P-gp substrate, similar to crizotinib and may thus have lower potential to penetrate the blood-brain barrier.18,21Over the last 10 years, gene infusion research has expanded significantly, leading to the discovery of not potential pathways and therapeutic targets for the treatment of various tumors. These include ROS1, RET, and arguably most notable, NTRK.As an oncogenic driver, the role of the ROS1 gene, first identified in 2007,32 is implicated in approximately 0.5% to 2% of NSCLC9,33,34 cases and is generally associated with a more favorable prognosis compared with lung cancers harboring other mutations.33 Patients with ROS1 mutations appear to exhibit unique clinical features, including being never-smokers or having a light smoking history, younger age, Asian ethnicity, advanced stage at the time of diagnosis, and predominant occurrence in adenocarcinoma while also being observed less frequently in large cell and squamous cell carcinomas.34

ROS1 is located at chromosome 6q22 and encodes for an RTK belonging to the insulin receptor family. In oncogenic models, rearrangements of ROS1 with CD74, EZR, FIG1, CCD6, KDELR2, LRI3, SDC4, SLC34A2, TPM3 and TPD52L1 have been identified,9,33,34 all of which may result in constitutive activation of all or some of the following signaling pathways: ERK, PI3K, mTOR, and JAK, with implications for driving tumor cell differentiation, growth, and survival.33,34 No specific ligand for ROS1 has yet been identified in humans, and its role in native biology is poorly understood. 34 In mice, ROS1 expression has been noted in the kidney, lung, heart, intestine, and testis. 34 ROS1 rearrangements have been identified in glioblastoma cell lines and in other malignancies as well, including cholangiocarcinoma, gastric adenocarcinoma, ovarian serous carcinoma, colonic adenocarcinoma, inflammatory myofibroblastic tumor, angiosarcoma, epithelioid hemangioendothelioma, and spitzoid melanocytic tumors,33,34 ROS1 rearrangements are rarely found with other genetic alterations, however, such as EGFR, KRAS, or ALK.34

Targeted Therapy. In addition to a favorable prognosis being associated with ROS1 rearrangements, ROS1 mutations may also be a biomarker for improved response to cytotoxic treatment. In clinical trials, patients with NSCLC harboring ROS1-addicted tumors demonstrate superior responses to pemetrexed-based chemotherapy in terms of response and long-term outcomes.33 Despite this, selected TKIs active against ALK have been evaluated in NSCLC patients harboring ROS1-rearranged tumors.

The rationale for targeting ROS1-positive tumors with known ALK inhibitors is supported by the fact that the kinase domains of ALK and ROS1 share 77% amino acid identity within the ATP-binding sites.35 Moreover, similar binding affinity has been demonstrated in preclinical studies.35 In addition, the shared homology in the kinase domains of ALK and ROS1 suggests that ROS-positive tumors may also respond to such other inhibitors as ceritinib, lorlatinib, and entrectinib.33

In general, ROS1-positive status is suggestive of an even greater response to ALK TKIs compared with the treatment of similar patients with ALK-addicted tumors. In some studies, PFS following crizotinib therapy was longer than 19 months for patients identified as ROS1 positive, which exceeds the 9 to 12 months typically observed with treatment directed at other relevant oncogenes.33 In an expansion cohort of a phase I study of crizotinib, which enrolled patients with advanced NSCLC who tested positive for a ROS1 rearrangement, the ORR was 72% with 3 complete responses and 33 partial responses observed in 50 patients; the median DOR was 17.6 months.35 The safety profile was comparable to what has been previously observed with crizotinib in studies among patients with ALK-positive NSCLC.35

Resistance. Some cancers eventually develop crizotinib resistance.36,37 In particular, ROS1-rearranged NSCLC has been associated with crizotinib resistance, resulting in molecular changes such as ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.37 In a study evaluating patients with ALK and ROS1 NSCLC progressing on different types and/or lines of ROS1/ALK-targeted therapy, researchers used a combination of NGS, multiplex mutations assay, direct DNA sequencing, PCR, and FISH to identify mutations serving as resistance mechanisms to therapies.38 They found that 50% of patients with ROS1 and 86% of patients with ALK were associated with a putative TKI resistance mechanism, suggesting that a focus on kinase domain mechanisms will miss most resistance mechanisms.38


In another study seeking to identify novel crizotinib-resistance mutations in the ROS1 kinase domain, investigators found that the cMET/RET/VEGFR inhibitor cabozantinib (XL 184) effectively inhibited the survival of CD74-ROS1 wild-type and resistant mutants harboring Ba/F3 and MGH047 cells.36The RET proto-oncogene is expressed in the developing central and peripheral nervous systems, as well as in the excretory system, of mice.39 Moreover, RET plays an acknowledged role in kidney morphogenesis and embryonic development of the enteric nervous system in humans.40 Gain-of-function mutations in RET are associated with constitutive activation and have been implicated in several cancer types, including medullary and papillary thyroid carcinoma, multiple endocrine neoplasia types 2A and B, neuroblastoma, and pheochromocytoma.39,40 RET mutations are also an acknowledged oncogenic driver in a subset of patients with NSCLC.41 RET kinase activation may result from RET point mutations or RET gene rearrangements.40 RET fusions are generally reported to occur in 10% to 20% of papillary thyroid cancer (with CCDC6 and NCOA4 as the predominant fusion partners), in 1% to 2% of case of NSCLC (in which KIF5B is the most common fusion partner), and at variable rates in other cancer subtypes, including colorectal and breast cancers.38-42 Patients with RET-positive NSCLC tumors are believed to display unique histopathologic characteristics. Compared with patients with EGFR-, KRAS-, or ALK-mutant adenocarcinomas, patients with RET fusions are more likely to be never-smokers and are typically younger than patients with EGFR.10 RET-positive adenocarcinomas are generally small and poorly differentiated at the time of identification (ie, <3 cm), but patients often present with ≥ N2 disease.10 Early lymph node metastases have also been noted as a defining feature.10

Treatment Approaches. Because of the rarity of RET-mutant cancers, patient recruitment for clinical trials has proven challenging. As a result, several plausible treatment strategies have been demonstrated in vitro and in vivo. Using a NGS assay to identify potentially druggable mutations in tissue specimens, Lipson and colleagues identified KIF5B-RET fusions in 2% of cases and subsequently determined that they were sensitive to multi-kinase inhibitors.43 Preliminary data from human studies show confirmed partial responses associated with cabozantinib44 and vandetanib.45 Data from multicenter registries demonstrate the plausibility of TKIs that target RET in real world settings outside of clinical trials. In a population of 165 patients from Asia, Europe, and the United States, the majority were never-smokers (63%), adenocarcinoma was the predominant histology (98%), and most patients had stage IV disease at time of diagnosis (72%).40 KIF5B, CCDC6, NCOA4, EPHA5, or PICALM fusions were identified in 81 of the tumor samples.40


Among TKI—naïve patients, the optimal response to any kind of single-agent RET inhibition was a complete response in 2 patients (4%), a partial response in 11 patients (22%), stable disease in 16 patients (32%), and progressive disease in 20 patients (40%); 1 patient (2%) was not evaluable. Responses were achieved with cabozantinib, vandetanib, sunitinib, lenvatinib, and nintedanib, but they were not observed with sorafenib, alectinib, ponatinib, or regorafenib.40 BLU-667, a next-generation small-molecule RET inhibitor, which was expressly created for highly potent and selective targeting of oncogenic RET alterations such as fusions and mutations, has been studied in vivo and in vitro, as well as in humans.41 In a study, BLU-667 inhibited RET without modulating VEGFR-2 in vivo.41 Early results from phase 1 human studies have demonstrated the therapeutic benefit of BLU-667 in patients with medullary thyroid cancer and NSCLC.41NTRK rearrangements were one of the original oncogenic fusion products mentioned in the literature. Martin-Zanca and colleagues are widely credited with describing the first such rearrangement involving NTRK1 in a tumor sample from a patient with colorectal cancer.46,47 Subsequent work would help elucidate that the NTRK genes (NTRK1, NTRK2, and NTRK3) encode the proteins TRKA, TRKB, and TRKC, respectively, and play roles in neuronal development, cell survival, and cellular proliferation.48-51

All 3 of these proteins share structural similarities, including an extracellular domain for ligand binding, a transmembrane region, and an intracellular domain with a kinase domain.47 Ligation triggers oligomerization of the receptors and phosphorylation in the intracytoplasmic domain, thereby setting in motion a signal transduction pathway that ultimately plays a role in neuronal cell proliferation, differentiation, and survival.5,47,52,53 Whereas nerve growth factor binding to TRKA causes activation of the RAS/MAPK pathway, leading to propagation and cellular growth through ERK signaling, and with implications for the PLCγ and PI3K pathways, TRKB ligation activates RAS-ERK, PI3K, and PLCγ, leading to neuronal differentiation and survival.47 Finally, TRKC pairing with NT3 causes preferred activation of the PI3/AKT pathway, thereby inhibiting cell death.47 The inherent regulation of TRKC receptor levels is essential for cell function, which is underscored by the fact that TRK receptors appear to be upregulated in several neurological disorders, such as TRKB in epilepsy, neuropathic pain, and depression.47 As such, NTRK chromosome rearrangements, which give rise to oncogenic fusions, which are marked by TRK overexpression and constitutive activation of the RTK domain, have consequences for activation of RAS/MAPK and/or PI3K/AKT, resulting in tumor cell transformation, proliferation, and survival.5


Despite its recognition as a potential oncogenic driver more than 3 decades ago, it was not until the recent introduction of NGS techniques that NTRK fusions could be identified on a routine basis.5 Moreover, until the discovery of TKIs that exhibit activity in the NTRK pathway,54 the discovery of NTRK fusions remained opportunistic and incidental.5 However, NTRK fusions have now been identified in a number of cancer types (Table 2).47-53Since the first NTRK gene fusions was identified in colon cancer, other tumors have been investigated for potential NTRK expression. Beyond NSCLC, increasing amounts of tumor-sequencing studies have revealed a wide variety of cancer types associated with NTRK gene fusions.47-53

Table 2. Reported Gene Fusions Involving NTRK1, NTRK2, and NTRK3, and Cancer Types in Which They Have Been Identified.57

Table adapted from Amatu A, Sartore-Bianchi A, Siena S. NTRK gene fusions as novel targets of cancer therapy across multiple tumour types. ESMO Open. 2016;1(2):e000023.

NSCLS. The implications of gene mutations in NSCLC have been well documented. With the development of various gene-based therapies for NSCLC based on ALK and ROS1, low-frequency rearrangements of NTRK mutations have been observed in NSCLC, as well.52 Specifically, NSCLC has been associated with NTRK1 and NTRK1-3 expression.65

Thyroid Cancer. After the discovery of TRK oncogenes in human papillary thyroid carcinoma nearly 30 years ago, new research continues to elucidate new insights into the role of NTRK fusion genes in thyroid cancer. For example, investigators have learned that the ETV6-NTRK3 rearrangement in thyroid cancers is more common in tumors with exposure to (131) I and can be directly induced in thyroid cells by ionizing radiation in vitro.67-68

Pancreatic Cancer. NTRK expression also been found in pancreatic cancer cells.69 In a 2001 study, researchers noted that the expression and distribution of neurotrophins and their receptors suggest their role in the potential of pancreatic cancer cells for neural invasion. More findings revealed that TRKA potentially mediates the proliferation and invasiveness of pancreatic cancer cells.70

Colorectal Cancer. The somatic rearrangement TPM3-NTRK1 has been identified in colorectal cancer using quantitative reverse transcriptase PCR and IHC, suggesting the potential of TRK inhibition as a targeted therapy in patients harboring the NTRK1 rearrangement.71,72

Breast cancer. NTRK gene fusions have also been expressed in difference forms of breast cancer. In particular, TRKC is often overexpressed in human breast cancers and is thought to play an essential role in tumor growth and metastases.73 Moreover, suppression of TRKC expression in highly metastatic breast cancer cells appears to inhibit their growth in vitro and decreases their ability to metastasize from the mammary gland to the lung in vivo.73 In addition, TRKA is also a potential target in breast cancer therapy, as its overexpression has been shown to enhance tumorigenic properties of breast cancer cells.74



Other Rare Tumors. Although NTRK fusions are found at low frequency in patients with some forms of cancer, such as lung and gastrointestinal tumors, they are observed in many rare tumors.53,75 For example, NTRK1 gene fusions have been found in soft tissue sarcomas such as spindle cell sarcoma.76 Additionally, mammary analogue secretory carcinoma was discovered to harbor an ETV6-NTRK3 translocation bearing similarities to secretory carcinoma of the breast, suggesting the potential for the use of molecular testing for diagnosis.77The emergence of inhibitor molecules that are active within the NTRK pathway has generated considerable excitement, both because thus far they have demonstrated robust responses in preclinical and clinical studies among patients with NTRK-dependent tumors and because some of these molecules demonstrate activity in inhibiting ALK- and ROS1-dependent tumors.54Entrectinib is an oral inhibitor that was selected for development because of its promising pharmacokinetic, safety, and tolerability profile.54 It has also demonstrated an ability to cross the blood-brain barrier, therefore suggesting a role for both primary brain tumors and brain metastases in patients with NTRK1-, NTRK2-, NTRK3-, ROS1-, and ALK-rearranged cancers.52 In preclinical studies, entrectinib demonstrated high biochemical potency against TRKA, ROS1, and ALK in cellular models.52 These benefits were later confirmed in patients with NSCLC harboring NTRK fusions that were identified with the use of advanced NGS techniques.78 Treatment of cells expressing NTRK1 fusions led to inhibition of autophosphorylation and cell proliferation,59 and a case report of a patient with stage IV lung adenocarcinoma with an SQSTM1-NTRK1 fusion transcript expression who was treated with entrectinib verified the presence of significant anti-tumor activity.79 A separate case report also noted tumor reduction in a patient with a glioneuronal tumor with a BCAN-NTRK1 fusion, in addition to maintenance of radiologic and clinical response for 11 months on treatment.78

Entrectinib has also been tested in phase I and II clinical trials (ALKA-371-001 and STARTRK-1) in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions who had not been previously treated with a TKI.75 In the phase I studies, responses were observed in patients with NSCLC, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma as early as 4 weeks after initiating treatment lasting as longer than 2 years in some patients.75 One noteworthy patient with SQSTM1-NTRK1 rearranged lung cancer and secondary brain involvement demonstrated a complete resolution of all brain metastases.75 Treatment-related AEs (TRAEs) of any grade that occurred most frequently included fatigue/asthenia (46%), dysgeusia (42%), paresthesias (29%), nausea (28%), and myalgias (23%); most TRAEs were grade 1 or 2 in severity and resolved with dose modi&#64257;cations, and no dose-limiting toxicities were reported.75

Data from 25 patients from the phase I studies were available for analysis based on phase II eligibility criteria, which were defined as the following: (1) patients whose tumors harbored a recurrent gene fusion involving any of the 5 genes of interest, (2) patients with no prior TKI treatment targeting the fusion of interest, and (3) patients who were treated with daily doses of 600 mg.75 Within this cohort, the ORR was 100% in 3 patients with NTRK1/2/3-rearranged advanced solid tumors with RECIST-measurable disease, 86% among patients (n = 14) with ROS1-rearranged solid tumors, and 57% in patients (n = 7) with ALK-rearranged solid tumors.75 Responses to entrectinib therapy were noted as early as cycle 1 (ie, within 4 weeks of initiating treatment). Median PFS in 4 patients harboring NTRK1/2/3-rearranged malignancies, 14 patients harboring ROS1-rearranged malignancies, and 7 patients harboring ALK-rearranged malignancies was not reached, 19.0 months, and 8.3 months, respectively.75 Eight of 25 patients had known primary or metastatic brain involvement prior to treatment. Among these, responses were noted in 4 patients (1 patient with NTRK1-rearranged NSCLC, 2 patients with ROS1-rearranged NSCLC, and 1 patient with and ALK-rearranged NSCLC), as well as 1 additional patient with ALK-rearranged colorectal cancer.75

Investigators also evaluated responses in patients with malignancies involving the brain, because entrectinib crosses the blood-brain barrier.75 Among 25 patients deemed eligible for phase II study, 8 (32%) had a primary tumor or metastatic disease involving the brain. Responses were observed in 5 of these 8 patients (63%), including 4 patients with NSCLC (1 with NTRK1-, 2 with ROS1-, and 1 with ALK-1 gene fusions) and 1 patient with ALK-rearranged colorectal cancer.

Despite the relatively small number of treated patients, ALKA and STARTRK-1 offer proof-of-concept data supporting further investigation of tyrosine kinase inhibitors in patients with tumors harboring gene fusions involving ALK, ROS1, and NTRK1/2/3.75 Based on current data, treatment response with entrectinib may occur as early as 4 weeks, and the longest DOR approached 2.5 years. Notably, patients who have been previously treated with other tyrosine kinase inhibitors did not respond to entrectinib, suggesting the possibility of potential resistance mutations. Nevertheless, the ALKA and STARKTRK-1 trials provide a glimpse of the potential of molecular biomarkers for targeted therapies in patients with genomic alterations.

The phase II STARTRK-2 basket study (NCT02568267), which is evaluating entrectinib for the treatment of solid tumors that harbor NTRK1/2/3, ROS1, or ALK gene rearrangements is currently underway.80 The study is evaluating patients with NSCLC, metastatic colorectal cancer, or other solid tumors divided according to ALK, NTRK1/2/3, and ROS1. Primary outcome will be ORR at 24 months according RECIST version 1.1 criteria.

Resistance. Resistance to NTRK inhibition with TKIs is inevitable for most patients, as with other gene fusions. New NTRK-TKI resistance mutations in the NTRK1 kinase domain include G595R and insulin growth factor receptor type 1 (IGF1R) bypass pathway-mediated resistance.81

One case report evaluated LMNA-NTRK1—positive metastatic colorectal cancer treated with entrectinib developed who acquired resistance.64 Circulating tumor DNA profiling revealed 2 new NTRK1 genetic alterations in the kinase domain: G595R and G667C.64 Longitudinal analysis of circulating tumor DNA revealed that these mutated alleles were not present at baseline before treatment but appeared in circulation 4 weeks after treatment initiation with entrectinib. Clinical progression was confirmed radiologically at 16 weeks, at which point mutation frequencies in circulating tumor DNA peaked.64

The investigators also explored a dose-dependent effect leading to the emergence of the mutations. In an in vitro model, NTRK1 G677C emerged during exposure to low concentrations of entrectinib and was absent with high doses. At high concentrations of entrectinib, the investigators detected only the NTRK1 G595R mutation, which was absent at low doses.64 More investigation is required to determine whether continuous or intermittent dosing affects the emergence or type of acquired mutation.47 NTRK1 G595R and G677C mutations have also shown resistance to larotrectinib and TSR-011.64


In a second reported case of acquired resistance with entrectinib, a patient with ETV6-NTRK3 fusion—positive metastatic mammary analogue secretory carcinoma of the salivary gland was evaluated.82 The patient was treated with crizotinib prior to entrectinib, resulting in modest inhibitory activity against TRKC. Within 4 weeks of initiating treatment, the patient experienced a rapid partial response to entrectinib. Acquired resistance and disease progression were seen at 7 months, allowing investigators to identify the NTRK3 G623R mutation, which is homologous to a TRK1 G595 mutation.82,83 Structural analysis revealed that the NTRK3 G623R mutation created steric hindrance and reduced the binding of entrectinib with the ATP binding.82A second pan-TRK molecule with suspected activity for inhibition of NTRK-rearranged tumors has also been evaluated in phase I and II studies. In a study that enrolled pediatric patients with infantile fibrosarcoma, soft tissue sarcomas, and papillary thyroid tumors harboring TRK fusions, 14 of 15 patients (93%) with TRK fusion—positive cancers achieved an objective response as per RECIST version 1.1, whereas 0 of 7 patients with TRK fusion—negative cancers had attained an objective response.84 In the study, larotrectinib was associated with a favorable safety profile, with most AEs reported as grade 1 or 2. The most common TRAEs reported included increased alanine and aspartate aminotransferase elevations (42% each), leucopenia (21%), decreased neutrophil count (21%), and vomiting (21%).84 Only 2 serious AEs, observed in 1 patient each, were reported during the 28-day follow-up period after discontinuing larotrectinib treatment: grade 3 nausea and grade 3 ejection fraction decrease.84

In a separate set of phase I and II studies that enrolled adult and pediatric patients with consecutively and prospectively identified TRK fusion-positive cancers detected by molecular profiling, the primary endpoint was ORR determined by independent review.85 Among 55 patients who ranged in age from four months to 76 years, ORR was 75% and 80% according to independent review and investigator assessment, respectively.85 Most of the adverse events reported were grade 1 or 2 in severity, and no TRAEs grade 3 occurred in > 5% of patients. No patients discontinued larotrectinib therapy due to drug-related AEs.85


Resistance. Primary and acquired resistance associated with larotrectinib treatment has been correlated with lack of response and disease progression. In clinical trials, researchers identified an NTRK3 G623R mutation in the kinase domain’s ATP-binding site in 1 patient who had previously been treated with another TRK inhibitor. 85 Other patients with acquired resistance to larotrectinib displayed kinase domain mutations after disease progression, such as solvent front mutations and mutations at the gatekeeper and the xDFG position, both of which interfere with the binding of larotrectinib.85 Notably, these mutations are paralogues of acquired resistance mutations that have been reported for other classes of kinase inhibitors. 85The possibility of redesigning treatment approaches for individual patients based on their distinct genomic profile offers tremendous potential for improved outcomes in a variety of cancer types. Available clinical evidence demonstrates robust efficacy associated with TKI strategies, and ongoing clinical trials offer to further refine treatment approaches.

Many challenges must be faced to realize the full potential of targeted therapy in oncology. From the perspective of the practicing clinician, although the large number of new medicines in the ever-expanding therapeutic armamentarium is a boon to patient care, yet they also add inherent complexity to patient management. Newly emerging clinical trial results will have to be effectively communicated, and clinicians in turn will be faced with integrating rapidly shifting treatment paradigms in the face of new understandings and information. How the plethora of new information is codified into evidence-based practice will likely determine whether patients derive the fullest benefit possible from breakthrough therapies.

More specifically, the increasing number of targeted drugs available for use engenders critical questions regarding the optimal sequencing of these agents, particularly in the face of resistance mechanisms that may obviate subsequent lines of therapy.6 Questions remain about the management of brain metastases in patients who are candidates for targeted therapy, both in the context of clinical care and in terms of clinical trial development.6 Equally as critical, however, is to increase patient enrollment in treatment trials. It is estimated that < 5% of adult patients with cancer participate in clinical trials.6 Many potential reasons have been offered to explain this conundrum, including practical concerns (eg, patients having to travel long distances to a clinic), methodologies of trial design (eg, previous cancer is often an exclusion criterion for study participation), and the increasing complexity and cost associated with conducting clinical research.6

Molecular profiling offers some solutions to these barriers while, at the same time, presenting a different set of challenges and obstacles. On the one hand, it is becoming simpler to conduct targeted testing to identify actionable genomic mutations, thereby making it easier to identify potential patients and discover opportunities for treatment. A consequence of improved testing capacity is the fact that shared molecular determinants of oncogenesis have been recognized in different types of cancer, which has, in turn, led to the concept of basket trials. These factors have made it easier to accrue sufficient numbers of patients to demonstrate safety and efficacy, even among rare and orphan cancers.7

Yet, integrating pharmacogenomics is inherently more costly than current approaches, and there are several expenses associated with genetic testing that cannot be explained. In an era of tightly regulated healthcare resources, the concept of targeted therapy will likely face pressure to demonstrate cost-effectiveness to garner support from payers and institutions.7 Another important barrier to integration is the knowledge and confidence gap created by new approaches to care. Surveys show that clinicians on the whole support the concept of pharmacogenomics, but many do not use pharmacogenomics in regular practice because of a lack of both knowledge and strong clinical evidence.7 Additionally, testing algorithms designed to detect actionable mutations are not standardized.2 For example, several NGS methods can be used to detect ALK rearrangements; however, sensitivity with NGS methods is generally considered to be lower compared with the use of IHC or FISH.2 In principle, NGS approaches can detect mutations and rearrangements, but it is unclear whether they possess sufficient sensitivity and specificity for routine application.2 More fundamentally, mastering the pre-analytical phase is a major issue that directly affects the reliability of study results.2

These obstacles do little to dampen the laudable concept of precision medicine within oncology. Regarding the concept of targeting actionable mutations, such as NTRK, across multiple cancer types, including several considered to be orphan diseases, the results that have been demonstrated to date far exceed what might be considered incremental.&ensp;


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