History and examination
A 42-year-old gentleman, with a history of smoking and no comorbidities presented to us with a one-month history of painless neck swelling. On examination, he was noted to have non-tender conglomerate lymphadenopathy affecting levels II to IV on the right side of his neck. His Eastern Cooperative Oncology Group performance status (ECOG PS) was 1. There was a family history of lung cancer in the patient’s father and paternal aunt.
Investigations and diagnosis
The whole body fluorodeoxyglucose positron emission tomography (FDG PET)-computed tomography (CT) thorax revealed a hypermetabolic lesion in the right upper lobe measuring 5.6 × 3 × 3.6 cm, standardized uptake value max 24.09, with involvement of the mediastinal and right axillary nodes [Figure 1]. The FDG PET-CT also showed suspicious right sided neck nodes, which were reactive on biopsy. Hence, a CT-guided biopsy from the lung mass was done, which was suggestive of adenocarcinoma lung. Thus, the tumor was staged as cT3 cN3 cM1b as per the American Joint Committee on Cancer (AJCC), 8th edition. Assessment of the epidermal growth factor receptor (EGFR) by quantitative real time polymerase chain reaction (PCR), and anaplastic lymphoma kinase (ALK) by immunohistochemistry (IHC) were negative. On IHC, ~55% to 60% of the tumor cells showed moderate to strong intensity membranous staining for PD-L1 antibody by the Ventana SP263 assay. Additionally, the tumor cells showed membranous staining of moderate intensity for ROS1 by the D4D6 antibody clone. The ROS1 positivity on IHC was confirmed by fluorescence in-situ hybridization (FISH).
On the original biopsy sample, the Illumina Focus panel was used to detect single-nucleotide variations and insertions/deletions in 42 genes, 139 RNA fusions, gene amplification events in 24 genes, and the status of microsatellite instability at six distinct loci. DNA and RNA libraries were created using the Solid Tumor Plus library preparation kit and sequenced on the Illumina MiSeq system. The SOPHiA Data Driven Medicine was used to assess the data. The sample was found to have a pathogenic (tier I) fusion between exon 6 of CD74 and exon 34 of ROS1 (read count of 3162). No other mutations were detected in the next-generation sequencing (NGS) [Table 1].
EXCERPTS FROM THE DISCUSSION IN THE MOLECULAR TUMOR BOARD
In terms of receptor tyrosine kinases, ROS1 and ALK are related. Mutant C-terminal fusion proteins called ROS and ALK have been noted in multiple malignancies, including non-small-cell lung cancer (NSCLC), anaplastic large-cell lymphoma, colorectal cancer, glioblastoma, and cholangiocarcinoma. Similar to other driver mutations, ROS1- rearrangements, with an incidence of 1% to 2%, have been well described in patients with NSCLC. Our patient was diagnosed with ROS1-rearranged (CD74-ROS1 fusion) lung cancer as confirmed by IHC, FISH, and NGS. The molecular tumor board discussed that possible therapeutic options included entrectinib, lorlatinib, ceritinib, and crizotinib. Despite the PD-L1 positivity (55% to 60%), immunotherapy or chemo-immunotherapy were not advised because of the presence of the molecular driver and the expected poor response to immunotherapy. Hence, the recommendation was to discuss the treatment options with the patient and advise the appropriate tyrosine kinase inhibitor (TKI).
The patient was counseled about the disease status, prognosis, treatment options, cost, and side-effects of therapy. He started crizotinib on February 18, 2021. His disease continues to be well controlled with no evidence of disease progression on crizotinib, as of August 25, 2022.
Clinical characteristics of ROS1-rearranged NSCLC
ROS1 fusion positivity in NSCLC has been reported to predominantly occur in younger patients, who are light- or never-smokers, and whose tumors are of adenocarcinoma histology. ROS1 rearrangement can occasionally be seen in large-cell and squamous NSCLC. An increased incidence of venous thromboembolism has been noted in these ROS1 positive NSCLC patients. Hence, primary thromboprophylaxis may be considered in these patients. Metastasis to the brain is not uncommon and has been reported to occur in 3.2% to 36% cases. In patients on first line crizotinib, central nervous system (CNS) is the most frequent site of progression, comprising ~30% of the cases.
How to detect ROS1 fusion positive NSCLC?
Many methods have been utilized for the detection of ROS1 rearrangements, including IHC, FISH, RT-PCR, and NGS. FISH is considered the gold standard tool, while NGS being a multi-gene panel can simultaneously detect alterations in other genes. IHC has several advantages over FISH, including lower cost, higher sensitivity (90% to 100%), less operator needs, and quicker processing time However, IHC has the disadvantages of subjective interpretation, antibody specificity and sensitivity, and the possibility of improper tissue fixation, all of which may affect the accuracy of the results. The histochemical scoring (H-score) assessment incorporates both the staining intensity and the percentage of stained cells at each intensity level in order to interpret immunoreactivity. An H-score of 150 or higher on IHC inside the cytoplasmic compartment substantially correlated with FISH-positivity in a study by Huang et al. The following equation was used to determine the H-score: (% of strongly stained cells × 3) + (% of moderately stained cells × 2) + (% of weakly stained cells × 1). Additionally, 2+ or higher IHC positivity in >30% of the tumor cells exhibited the highest association with FISH-positivity (positive percentage agreement = 97.8%, negative percentage agreement = 89.5%).
Who to test for ROS1?
All patients with metastatic non-squamous NSCLC, squamous NSCLC (small biopsy specimen), mixed histology NSCLC, and NSCLC, not otherwise specified (NOS) should undergo ROS1 testing, according to the Indian guidelines and the National Comprehensive Cancer Network panel.
ROS1 signaling pathway
The ROS1 or the ROS proto-oncogene 1 is located on chromosome 6q22.1 and codes for a receptor tyrosine kinase. As members of the insulin receptor superfamily, ALK and ROS1 proteins exhibit substantial homology (84% = ATP-binding region; 64% = kinase domains). ROS1 genomic alterations are widely documented and often result in gene fusions with their partners [Table 2]10] These gene fusions are strong oncogenic drivers. In turn, this results in a constitutively active ROS1 kinase, which leads to upregulation of the MAPK/ERK, PI3K/AKT, and JAK/STAT pathway. A number of malignancies, including NSCLC, have been related to ROS1 which has been found to have tumor-promoting potential.
Treatment for ROS1-positive lung cancer
ROS1-directed oral TKIs
Table 3 provides a summary of the ROS1 inhibitors and Table 4 gives their toxicities.
- Crizotinib: Crizotinib was granted United States Food and Drug Administration (US FDA) approval in 2016 for the treatment of ROS1-fusion positive NSCLC, after PROFILE 1001, a phase I trial reported the efficacy of crizotinib. The data of 53 patients with histologically confirmed ROS1 positive NSCLC were analyzed. Patients with ECOG PS 0-1 were included. Patients with ECOG PS 2 were also considered for the study after obtaining the approval of the investigator and sponsor. The median number of prior regimens in the advanced/metastatic setting was 2 (range, 1 – 6). The updated results of this study reported a remarkable median overall survival (OS) of 51.4 [95% confidence intervals (CI), 29.3—not reached] months and a median progression free survival (PFS) of 19.3 (95% CI, 15.2 – 39.1) months. Several other studies have also reported the efficacy of crizotinib; the important ones are discussed in Table 3.
- Ceritinib: Ceritinib is a highly selective and potent ROS1 inhibitor that was found to be 20 times more efficacious than crizotinib in a rat model. Because of good CNS penetration, it is also used in patients with CNS metastasis. Ceritinib has demonstrated activity in a phase II trial in 32 patients with ROS1 positive NSCLC (all but two were crizotinib-naive) who were given a daily dose of 750 mg per day after 2 h of fasting. The median PFS in the crizotinib-naïve, and the overall patient cohorts were 19.3 (95% CI, 1 – 37), and 9.3 months (95% CI, 0 – 22), respectively. Of the eight patients with CNS metastases, disease control was noted in five patients (63%) and an intracranial response occurred in two of eight patients (25%). Fatigue was the commonest non-laboratory grade ≥3 adverse event occurring in 12 patients (37%). Serious adverse events occurred in 16 patients (50%). Ceritinib in combination with immunotherapy is being tested in clinical studies to find a way to mitigate the toxicities. Furthermore, ceritinib’s efficacy is confined to patients who have not previously received crizotinib; it failed to show activity against crizotinib-resistant tumors.
- Entrectinib: Entrectinib is an orally administered TKI that was particularly developed to cross the blood-brain barrier. Entrectinib was granted US FDA approval in 2019 to treat ROS1-positive lung cancer. In preclinical studies, entrectinib failed to show effectiveness against certain ROS1 mutations such as D2033N, G2032R, and L2026M. An integrated analysis was performed on patients with ROS1 rearranged advanced NSCLC who had participated in three phase I-II studies (ALKA-372-001, STARTRK-1, and STARTRK-2). The median PFS was 19 months (95% CI, 12.2 to 36.6) in the entire cohort of 53 patients, and 13.6 months (95% CI, 4.5—not reached) in patients with brain metastasis. Moreover, entrectinib’s toxicities were manageable with drug discontinuations and dose reductions required in 27% and 4% of patients, respectively.
- Lorlatinib: This is a potent FDA approved oral TKI efficacious against ALK- and ROS1-rearranged NSCLC, with activity against several ROS1 mutations (S1986Y, D2033N, and G2032R) which impart resistance to ceritinib and crizotinib. It also has the ability to cross the blood–brain barrier. A phase I-II study in 69 patients with ROS1-positive NSCLC, 30% of whom were TKI-naive, reported promising results. More than half (57%) the patients had baseline CNS involvement and 49% had been treated with radiotherapy. The objective response rate (ORR) and median PFS for TKI-naïve versus crizotinib pre-treated patients were 62% and 21 months (95% CI, 4.2–31.9) months, respectively, and 35% and 8.5 months (95% CI, 4.7–15.2) months, respectively. Interestingly, intracranial response rates of 50% and 64% were noted in patients who had previously received crizotinib and those who were TKI-naïve, respectively. Notably, lorlatinib maintained efficacy in the presence of resistance mechanisms mediated by bypass signaling pathways, while it had minimal effect in individuals with the G2032R mutation.
- Brigatinib: This ALK and EGFR inhibitor has shown activity in patients with ROS1 fusion, even in those with crizotinib-resistant disease though clinical efficacy against G2032R was lacking. Of the three patients with ROS1-rearranged NSCLC, two patients had an objective response (ORR = 66%). The toxicities of brigatinib were manageable with 5% of patients developing serious adverse events like dyspnea and pneumonia. The results of a phase II study (Barossa cohort 2) of brigatinib in 19 crizotinib pre-treated patients presented at the American Society of Clinical Oncology 2021 annual meeting showed moderate activity of brigatinib with an ORR of 26.3%, disease control rate of 57.9%, and a median PFS of 7.3 months (95% CI, 1.3–9.3).
- Cabozantinib: This is a multi-kinase inhibitor, having activity against TIE2, VEGFR2, ROS1, RET, MET, KIT, and ALX. It is approved for use in renal cell carcinoma and medullary thyroid carcinoma. Drilon et al. and Sun et al. have reported the efficacy of cabozantinib in patients with crizotinib-resistance. Because of challenging side-effects including pulmonary embolism, xeroderma and neutropenia, and the availability of relatively safer drugs, clinicians have been hesitant to use cabozantinib.
- Repotrectinib (TPX-0005): Repotrectinib is 90 times more efficacious than crizotinib with activity against ALK and tropomyosin receptor kinase (TRK) receptors in addition to ROS1 mutations including D2033N, L2026, S1986F, L1951R, and G2032R. Repotrectinib was granted an orphan drug designation by the FDA in Jun 2017 for the treatment of NSCLC harboring any one of the above ROS1 mutations. There were 33 patients with ROS1-rearranged NSCLC included in a larger phase I trial in patients with other solid tumors and mutations/fusions (such as TRK and ALK). Repotrectinib was administered at escalating doses. There was a remarkable intracranial response rate of 75% in TKI pre-treated patients and 100% in the TKI-naive patients; the corresponding ORRs were 39% and 82%, respectively. However, most patients experienced drug-related toxicities with dizziness (57%) and dysgeusia (51%) being the commonest.
- Taletrectinib: Taletrectinib has activity against NTRK and ROS1 fusions. Its efficacy against the ROS1 resistance mutation, G2032R, has been demonstrated in preclinical studies. A Japanese phase I trial evaluated taletrectinib in patients with NSCLC harboring ROS1 rearrangements; the ORR was 66.7% in crizotinib-naive patients. Two phase I trials have proven the clinical activity of taletrectinib, with fatigue, abdominal pain and diarrhea being the most commonly encountered grade 3 or higher adverse effects. The evidence is still evolving, and phase II studies are presently underway, similar to those evaluating repotrectinib.
- Ensartinib: Ensartinib (X-396) is a second-generation ALK inhibitor that has CNS activity regardless of prior ALK TKI use and is 10times more potent than crizotinib. In a recently reported phase II trial on ROS1-rearranged NSCLCs (NCT03608007), this drug demonstrated modest efficacy (ORR = 27%, 95% CI = 13.8-44.1) but promising control of brain disease that was noted in three out of four patients. However, the use of ensartinib in the therapy of ROS1-fusion positive NSCLC is currently being phased out because of its relatively limited efficacy, and the availability of more highly efficacious ROS1 TKIs.
- Foretinib: Foretinib is a more potent ROS1 inhibitor compared to crizotinib as demonstrated in preclinical studies. It is effective against G2032R-mutated ROS1.
- AZD3463: In a preclinical study, brigatinib and AZD3463 displayed near equipotency for the inhibition of both CD74-ROS1 and EML4-ALK cells (IC50 of 7.5 versus 9.8 nM and 10.2 versus 39.4 nM, respectively).
The above oral TKIs can be categorized with respect to their selectivity for ROS1 and ALK. These categories include ALK-selective inhibitors (alectinib), dual ROS1/ALK inhibitors (lorlatinib, brigatinib, AZD3463, ceritinib, crizotinib) and ROS1-selective inhibitors (foretinib, cabozantinib).
Chemotherapy in ROS1-fusion positive lung cancer
TKIs are still beyond the reach of the majority of patients in underdeveloped and developing nations, due to the lack of availability, affordability and accessibility. In such cases, chemotherapy is the standard of care for ROS1-fusion positive lung cancer. A retrospective Asian study compared the efficacy of pemetrexed-based chemotherapy given in any line during the course of treatment to patients with ROS1-fusions, KRAS mutations, EML4-ALK translocations, and EGFR mutations. The median PFS of patients with EGFR mutated, and ALK rearranged NSCLC were 3.7 months (95% CI, 2.2–5.2) and 5.4 months (95% CI, 2.7–8.2), respectively. When compared to other oncogene-driven lung malignancies, patients with ROS1-rearranged NSCLC had a higher ORR (57.9%), and a longer median PFS of 7.5 months (95% CI, 0.6–14.3) months; P = 0.003.
There is no direct comparison between the efficacy of TKI and chemotherapy for ROS1-fusion positive NSCLC. The clinical outcome of crizotinib (30 patients) has been compared to that of platinum-pemetrexed in 47 patients in a retrospective analysis in the first line setting. The use of crizotinib led to better responses and PFS [ORR of 86.7% versus 44.7%, median PFS of 18.4 (95% CI = 6.4-30.3) versus 8.6 months (95% CI = 6.9 – 10.3), P < 0.001]. The median OS in the crizotinib group was not reached; it was 28.4 months (95% CI = 20.7–36.0) in the chemotherapy group, although the difference was not significantly different (P = 0.176).
Immunotherapy in ROS1-fusion positive lung cancer
It is now well recognized that the use of immune checkpoint inhibitors results in poor outcomes in NSCLC with driver mutations. An international retrospective study in 551 patients, seven of whom had ROS1-rearranged NSCLC, looked at the effectiveness of immunotherapy in oncogenically driven lung malignancies. Anti-PD1 antibodies were mostly used to treat ROS1-fusion positive tumors following at least one line of treatment. The best response was progressive disease in all but one patient with ROS1-fusion positive NSCLC. There are insufficient data to support the use of chemo-immunotherapy in ROS1-rearranged cancers.
Resistance mechanism to crizotinib in ROS1-fusion positive NSCLC
The two important proposed mechanisms of resistance to crizotinib in ROS1-fusion positive NSCLC include bypass signaling activation and changes in the therapeutic targets [Figure 2]. Crizotinib-resistance has been attributed to point mutations in the ROS1 gene, which include D2033N, G2032R, L2026M, L2155S, and S1986F/Y. In addition, bypass signaling activation has been shown in ~45% of crizotinib-resistant NSCLC cases which have developed resistance to crizotinib. By reducing the dependency on ROS1 activity, activation of the EGFR, or KIT pathways have been demonstrated to impart crizotinib-resistance. In such cases, an EGFR inhibitor like gefitinib or a KIT inhibitor like ponatinib may be useful. Last but not the least, crizotinib-resistant cells showed enhanced MAPK signaling as well as amplified HER2 and TP53, suggesting that bypass mechanisms were at work.
Treatment post progression on ROS1 targeted TKI
The development of resistance to first line ROS1 TKIs is inevitable and is a serious concern. To determine the resistance mechanisms, collecting circulating cell-free DNA and/or tissue biopsy samples is recommended. The ROS1 G2032R mutation status generally decides further therapy. Lorlatinib can be considered in the second line in patients not harboring the G2032R mutation. Patients who progress on a first line TKI and harbor G2032R can be offered chemotherapy or can be enrolled in a clinical trial.
Lorlatinib is recommended in cases with asymptomatic, CNS-only progression while in cases of symptomatic, CNS-only progression, lorlatinib should be preceded by local therapy. Oligoprogression may be managed with local therapy and continuation of the TKI. Figure 3 summarizes the suggested treatment algorithm in patients with ROS1-positive lung cancer.
Several trials have demonstrated the efficacy of ROS1 TKIs. Patients with ROS1-positive NSCLC are also likely to be more responsive to pemetrexed-based chemotherapy, as demonstrated in several studies. As the oral TKI arsenal directed at ROS1-fusion positive NSCLC continues to grow, sequencing of TKIs has become increasingly difficult. Several factors, including safety, efficacy, CNS activity, and resistance mechanisms, must be taken into account while determining treatment. Currently, only crizotinib and entrectinib are approved for the first-line treatment for ROS1-positive NSCLC. Lorlatinib can be considered in the second line setting (off-label) in patients without G2032R mutation.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initials will not be published and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
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