The therapeutic landscape of non-small-cell lung cancer (NSCLC) has witnessed a dramatic revolution since the discovery of targetable molecular alterations and the rapid development of small molecule inhibitors. ROS1 (c-ros oncogene) is one of the well-known alterations. ROS1 alterations are mainly genomic rearrangements present in up to 2% of cases of NSCLC. They can be targeted by various tyrosine kinase inhibitors (TKIs) like crizotinib, the most widely used TKI for this alteration. However, after an initial response, resistance develops because of various on-target and off-target mechanisms. This phenomenon has been widely elucidated in the literature. In this review article, we aim to describe the molecular biology, alterations, clinical and pathological profiles, diagnostic methods, treatment options, and outcomes of ROS1 rearranged NSCLC.
In this narrative review, we identified the relevant articles by using various search engines and searching in scientific databases like PubMed, Scopus, Embase, and MyCancerGenome using the keywords “ROS1”, “NSCLC”, “ROS1 crizotinib”, “Oncogene NSCLC,” and “ROS1 resistance”. Since this was not intended to be a systematic review or a meta-analysis, we did not perform a formal statistical analysis and did not pre-specify any inclusion or exclusion criteria for the articles to be included in the review. We included 72 articles for the preparation of this review.
ROS1 was first identified in 1986 during pre-clinical research involving the chicken sarcoma ribonucleic acid (RNA) UR2 tumor virus. The first ROS1 rearrangement was detected in the U118MG glioblastoma cell line.[3,4] Subsequently, the oncogenic potential of these rearrangements has been widely elucidated in many pre-clinical studies. Finally, in 2007, the first case of ROS1 rearranged NSCLC was identified in a proteomic screen.
ROS1 or c-ROS (cellular ROS) is a receptor tyrosine kinase encoded by ROS1, which maps to chromosome 6q22.1. The structure and details of signaling are discussed below.
ROS1 exists in two alternative isoforms, one with 43 exons and one with 44 exons.[6,7] The extracellular N terminal domain contains exons 1-34, with 9 fibronectin type III repeats and 3 beta-propeller domains.[8,9] The tyrosine kinase domain is intracellular and along with the C terminal, it is connected to the extracellular domain via a single transmembrane domain, homologous to that seen in other receptor tyrosine kinases like anaplastic lymphoma kinase (ALK).[6,10,11] ALK and ROS1 are considerably homologous in structure. The structure is depicted in Figure 1a.
The ligand for ROS1 is neural epidermal growth factor-like like 2 (NEFLL2), which is a testicular germ cell-secreted lumicrine factor. This was discovered initially in the testes, and additional validation reports for other organs are awaited. It has been postulated that ROS1 is activated by the binding of specific tyrosine residues in the intracellular domain, which serve as docking sites for adaptor proteins like SHP2. This stimulates the RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT pathways, thus regulating cell survival, cell growth, and proliferation.[6,10,13,14] The signaling schema is depicted in Figure 1b.
ROS1 ALTERATIONS IN CANCER
Various types of alterations can occur in the ROS1 gene, which may cause oncogenesis. These are described in detail below.
ROS1 fusions/gene rearrangements
ROS1, analogous to ALK, is a promiscuous gene, with at least 55 different 5’ fusion partners described in the literature, across all malignancies.[15,16] The first partner reported was in glioblastoma, the so-called “FIG-ROS1” (Fused in glioblastoma). The other partners are listed in Table 1.[7,17] The frequency or prevalence of each partner varies with the different cancer types and a schematic depiction is provided in Figure 2.[7,17–19] In NSCLC, the most common is CD74-ROS1, reported in 44% of cases of ROS1 rearranged NSCLC, followed by EZR in 16%, SDC4 in 14%, and SLC34A2 in 10% of cases. On the contrary, GOPC is the most common partner in glioblastoma cases, reported in approximately 77% of cases of ROS1-positive glioblastoma [Figure 3].[7,17,19,20]
The fusions in ROS1 can occur through intrachromosomal or interchromosomal mechanisms. The majority of ROS1 fusions in NSCLC occur as a result of interchromosomal alterations, in contrast to glioblastoma, where most fusions occur as a result of microdeletions of 6q, and thus are intrachromosomal.[7,21] This distinction is important as it suggests that a distinct variation exists in both the dynamics as well as the chromatin architecture between different tumor types with respect to ROS1 rearrangement. Commonly, the extracellular domain is lost and there is an in-frame fusion between the N terminal of the partner gene and the intracellular tyrosine kinase domain of ROS1. The ROS1 fusion eventually leads to ligand-independent constitutive catalytic activity with unabated downstream signaling through RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, or the JAK/STAT pathways.
ROS1 mutations and amplifications
Mutations in ROS1 have been reported throughout the gene, as noted in cBioportal, however, they have not been deemed pathogenic by any functional assays. On the other hand, missense mutations in the tyrosine kinase domain have been reported to be important, as they confer resistance to ROS1-tyrosine kinase inhibitors (TKIs). Likewise, splice variants have been reported resulting in exon skipping,[15,22] analogous to the alteration that occurs in the MET gene, however, the clinical significance of these is yet to be established. Amplification and copy number gains in ROS1 have been reported in NSCLC as well as in other malignancies like breast cancer, and sarcomas[7,23]; their effect on pathogenesis and the treatment efficacy of ROS1-TKIs is unclear.
Concomitant alterations with ROS1
ROS1 rearranged NSCLC is a distinct molecular entity, and is, by and large mutually exclusive of the other known driver alterations like EGFR and ALK; some exceptions have been reported as anecdotal cases.[25,26] In a study by Zhang et al. on 235 patients with ROS1 rearranged NSCLC, next-generation sequencing (NGS) done in 54 (22.9%) patients revealed more than one genomic alteration in 38.9% (n = 21) cases, with the commonest alteration being TP53 mutations in 13% (n = 7) cases. Other co-mutations included KRAS and EGFR mutations and MET amplification. However, the number of patients harboring co-mutations was too small to draw definitive conclusions.
CLINICAL AND PATHOLOGIC FEATURES OF ROS1 REARRANGED NSCLC
Although rare, ROS1 rearranged NSCLC has been studied in both controlled trials as well as in real-world series. A meta-analysis of 9898 patients with NSCLC, concluded that ROS1 is associated with young age, female sex, never smoking status, and adenocarcinoma histology. A study by Joshi et al. from India, reported a 4% prevalence (22 cases positive of the 535 tested) of ROS1 rearranged NSCLC. The median age of these 22 patients with ROS1 rearranged NSCLC was 53 years (range, 45-70), similar to that reported in the PROFILE 1001 (NCT00585195) registration trial. The proportion of never-smokers was 75% (n = 40) in the ROS1 positive group. Similar findings were reported in another study from the Indian subcontinent by Mehta et al. in 20 patients with ROS1 rearranged NSCLC. Other clinical aspects which were reported included a higher incidence of venous thromboembolism. This occurs owing to the mucin present in both the extracellular and intracellular compartments which binds the P or L selectins, thus resulting in platelet activation and subsequently embolization. Brain metastases at diagnosis have been reported in 35% (n = 12) of ROS1 rearranged NSCLC in a study by Patil et al. Histologically, tumors with ROS1 rearrangement are noted to have adenocarcinoma morphology with predominantly acinar and lepidic patterns, with more than 90% of cases expressing TTF1. Imaging may show lymph node tropism characterized by a higher predilection for lymph node metastases, with fewer extrathoracic metastases when compared to ALK-positive cancers.
Alterations in ROS1 have been reported across various tumor types in almost 20 different adult and pediatric malignancies. The reported frequencies are provided in Figure 2.[7,35–40]
DETECTION OF ROS1 ALTERATIONS
Various methods have been described for the detection of ROS1 fusions. A detailed comparison of each is shown in Table 2 along with the advantages and disadvantages of each.[41–48]
Kinase domain mutations
Kinase domain mutations are usually missense mutations in the tyrosine kinase domain of ROS1. The canonical G2032R (G: glycine, R: Arginine) has been well documented. This mutation can be detected using any specifically designed single-gene polymerase chain reaction (PCR). However, since resistance is an ever-evolving phenomenon with the ongoing discovery of new mutations and variants, NGS-based assays designed to cover the entire tyrosine kinase domain of the gene are the preferred modality.
TREATMENT OF ROS1 REARRANGED NSCLC
Conventional chemotherapy and response
Systematic studies have assessed the responsiveness to chemotherapy of ROS1-rearranged NSCLC. In a study by Xu et al. first-line platinum-based chemotherapy was administered to 46 patients with ROS1-rearranged tumors (detected and confirmed by FISH), of which 35 (76.1%) patients also received pemetrexed; the median progression-free survival (PFS) was 8.8 months (95% CI, 6.8-10.3). Another study by Chen et al. reported an objective response rate (ORR) of 47% (95% CI, 29.8-57.4) with a median PFS of 8.6 months (95% CI, 6.9-10.3) in patients with ROS1 rearranged NSCLC who were treated with first-line platinum-pemetrexed combination chemotherapy (n = 47). With pemetrexed monotherapy, Park et al., reported an ORR of 53.3% in 90 cases, and a PFS of 8 months (95% CI, 6.4-11.7). ROS1 rearranged cancers have low thymidylate synthase messenger RNA (mRNA) levels, which may explain the benefit noted from pemetrexed-based therapy, when compared to that in other oncogene-addicted tumors.
In the PROFILE 1001 (NCT00585195) trial, crizotinib, a first-generation ALK-TKI, showed efficacy in 53 pretreated patients with ROS1 rearranged NSCLC; the reported ORR was 72% (95% CI, 58-83), the median PFS was 19 months (95% CI, 15.2-45.3), and the median overall survival (OS) was 51.4 months (95% CI, 29.3-not reached [NR]). In the AcSé study on 37 patients, the ORR was 47.2% (95% CI, 30.4-64.5) with median PFS and OS of 5.5 months (95% CI, 4.2-9.1) and 17.2 months (95% CI, 6.8-32.8), respectively. Thus, the AcSé results were strikingly worse as compared to those of PROFILE 1001 owing to a higher proportion of patients with Eastern Cooperative Oncology Group (ECOG) performance status (PS) 2 in the AcSé study. The other prospective studies, including the European EUROCROSS (NCT02183870) and METROS (NCT02499614) reported results that were more similar to those of the PROFILE study with ORRs of 70% (95% CI, 51-85) and 65% (95% CI, 44-82), respectively. However, despite the remarkably high reported responses, there was poor central nervous system (CNS) penetration, especially considering the fact that brain metastases have been reported in approximately 35% of cases of ROS1 rearranged NSCLC at diagnosis. Newer TKIs have been developed to tackle this problem. The commonly reported adverse events with crizotinib include vision disturbances (82%), diarrhea (44%), nausea and peripheral edema (40% each), deranged liver function tests (22%), fatigue (20%), and dysgeusia (18%), mainly grade 1 and 2.[30,32,57]
This is a second-generation TKI which has been approved for patients with TKI-naive and crizotinib-resistant ALK-positive NSCLC. Ceritinib is also a selective inhibitor of ROS1. In the Pan-Korean study, the ORR was 62% (95% CI, 45-77) with a median PFS of 19.3 months. The intracranial ORR was 25% (95% CI, 7-59). However, ceritinib is not active against several mutations like G2032R, D2033N, L1951R, and S1986F/Y that confer resistance to crizotinib. The adverse events reported include diarrhea (78%), nausea (59%), anorexia (56%), and vomiting (53%).
This is a multi-kinase inhibitor which the targets ALK, ROS1, and pan-tropomyosin receptor kinase (TRK). Entrectinib can penetrate the blood-brain barrier. In vitro studies have shown that the anti-ROS1 activity of entrectinib is 40 times greater than that of crizotinib. Two landmark trials that investigated the efficacy and outcomes of entrectinib were STARTRK-1/ALKA-372-001 (NCT02097810) and STARTRK-2 (NCT02568267). In 53 evaluable patients, the reported ORR was 67% (95% CI, 59-74) with a median PFS of 16 months (95% CI, 11-21) and a one-year survival rate of 81%. Among the 17 patients with brain metastases at diagnosis, the intracranial response rate to entrectinib was 55% (95% CI, 32-68). Thus, due to favorable CNS responses, entrectinib gained FDA (United States Food and Drug Administration) approval in August 2019. However, owing to activity against pan-TRK, it can lead to peculiar side effects like fatigue (46%), dizziness (16%), dysgeusia (42%), paresthesia (29%), nausea (28%), and myalgia (23%).[60,61]
Lorlatinib is a third-generation ALK inhibitor with anti-ROS1 activity, specifically developed for CNS penetration by decreasing the efflux mediated by P-glycoprotein 1. In a phase I/II trial (NCT101970865) the anti-tumor activity in ROS1 rearranged NSCLC was demonstrated in 21 TKI treatment-naive and 40 crizotinib-pretreated patients. In the TKI-naive group, the ORR was 62% (95% CI, 38-82) with a median PFS of 21 months (95% CI, 4.2-31.9) and an intracranial ORR of 64% (95% CI, 31-89). The median intracranial PFS was not reached, thus endorsing the remarkable efficacy of lorlatinib against CNS metastases. In the crizotinib-pretreated patients, the ORR was 35% (95% CI, 21-52). Grade 3 and 4 adverse events included hypercholesterolemia (65%), hypertriglyceridemia (42%), peripheral edema (39%), peripheral neuropathies (35%), weight gain (16%), and mood disturbances (16%). In the presence of resistance mutations in ROS1, p.K1991E and p.S1986F, lorlatinib has been proven to have superior efficacy after pretreatment with crizotinib. However, patients with the canonical G2032R mutation do not show a response to lorlatinib.[7,63]
Also known as TPX-0005, this is a next-generation low molecular weight macrocyclic TKI which can target ROS1-TKI, and ALK and has the ability to cross the blood-brain barrier. The FDA has granted a breakthrough therapy designation to repotrectinib for the treatment of patients with ROS1-positive metastatic NSCLC who have previously been treated with one ROS1 TKI and have not received prior platinum-based chemotherapy. Repotrectinib has shown activity against a variety of solvent-front substitutions, including p.G2032R and D2033N. Repotrectinib is being investigated in a dose-escalation phase I/II clinical trial TRIDENT-1 (NCT03093116) in patients with advanced ALK-, ROS1-, or NTRK1–3-rearranged cancers. This trial included 33 patients. In the TKI-naive subgroup (n = 11), the ORR was 82% (95% CI, 44-97), and the intracranial ORR (n = 3 of those with brain metastases) was 100% (95% CI, 29-100).
This is a dual inhibitor of ROS1 and pan-TRK, with activity against L1951R, L2026M, S1986F, and G2032R mutations proved in pre-clinical studies. A phase I study is ongoing (DS-6051b [NCT02279433])[66,67] and the preliminary ORR reported was 58% (95% CI, NR).
Drugs in development
Preliminary data from the phase I portion of the ARROS-1 phase I/II trial (NCT05118789) presented at the 34th European Organisation for Research and Treatment of Cancer (EORTC)-National Cancer Institute (NCI)-American Association for Cancer Research (AACR) Symposium on Molecular Targets and Cancer Therapeutics, reported that NVL-520, a new drug in development, showed early signs of activity in the 35 patients enrolled in the study, with good CNS penetration as well as commendable activity against solvent-front mutations, with no dose-limiting toxicities (DLTs) or side effects that led to dose reductions or discontinuation of treatment.
In the IMMUNOTARGET study seven patients with ROS1-positive NSCLC were included, and the reported ORR was 17%. In a Japanese retrospective study, 15 patients with ROS1-altered NSCLC were enrolled. High expression of programmed death ligand 1 (PD-L1), i.e., >50% of tumor cells stained by 22C3 was observed in 53% of cases, however, no responses to immunotherapy were noted. Therefore, there is not much evidence to support the use and efficacy of immunotherapy in this group of patients.
Resistance mechanisms to ROS1 TKIs
Despite remarkable responses, resistance eventually develops to targeted ROS1 inhibition. Almost 60% of crizotinib-resistant mutations are on-target and occur in the kinase domain. Of these, G2032R is a canonical mutation, which is analogous to G1202R of ALK. This is a solvent-front alteration and the arginine results in steric hindrance with the crizotinib piperidine ring. This mutation is also capable of inducing an epithelial-mesenchymal transition. Second-generation TKIs like ceritinib, entrectinib, brigatinib, and even lorlatinib are not active against this mutation. Repotrectinib is one of the few drugs that has shown activity against this.[71,72] Other resistance mutations described include D2033, L1951, S1986F/Y, and the gatekeeper mutation L2026M. Rarer mutations include L1982F, E1990G, and F1994L. Activation of bypass pathways with mutations in EGFR, ERBB2, and PIK3CA has been described as well. Concomitant alterations in TP53 and other tumor suppressors like PTEN and RB1 have also been reported in anecdotal cases. The effect of these mutations requires further validation and larger studies.[7,30,71,72]
CONCLUSIONS AND FUTURE DIRECTIONS
ROS1 rearranged NSCLC is a distinct molecular entity, with available targeted therapy resulting in remarkable outcomes. Myriad detection methods are available which are all well-established and validated. It is necessary for clinical laboratories to include ROS1 in the initial testing battery. Additionally, the use of liquid biopsy can be investigated in future trials for monitoring of response to therapy and developing a BCR-ABL1-like treatment response parameter for the same.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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