Comprehensive RNA-Based Testing and Selective Next-Generation TKIs Are Reshaping Frontline ROS1+ NSCLC Care

Although ROS1 gene fusions define only approximately 1% to 2% of non–small cell lung cancer (NSCLC) cases, identifying them has taken on outsized importance as next-generation tyrosine kinase inhibitors (TKIs) have transformed outcomes for this molecularly defined population.^1,2 During a recent OncLive Peer Exchange, a panel of thoracic oncology and pathology experts discussed how comprehensive testing strategies, updated efficacy and safety data from pivotal trials, and an evolving understanding of resistance mechanisms are shaping treatment selection and sequencing for patients with ROS1-positive NSCLC.

“We have to test all patients; no patient should be missed,” Ticiana Leal, MD, of the Winship Cancer Institute of Emory University in Atlanta, Georgia, said. “Once you identify a ROS1 fusion, that’s a game changer with the evolving paradigm and the new treatments we have available.”

The panel, moderated by Julia Rotow, MD, of the Dana-Farber Cancer Institute in Boston, Massachusetts, also included Estelamari Rodriguez, MD, MPH, of the University of Miami Health System in Florida, and Ignacio Wistuba, MD, of Moffitt Cancer Center in Tampa, Florida.

Why is comprehensive RNA-based testing essential for identifying patients with ROS1-positive NSCLC?

Consensus guidelines from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology recommend that ROS1 testing be performed on all patients with advanced lung adenocarcinoma, independent of clinical characteristics, such as age or smoking history.^3,4 The panelists reinforced that recommendation, but emphasized that testing modality matters as much as testing itself.

“These are fusions that can be missed with our traditional next-generation sequencing [NGS] DNA platforms,” Rodriguez said. “Testing needs to happen for all patients, and it needs to be comprehensive, including DNA and RNA, because we do miss patients with our earlier technology.” Wistuba explained the underlying limitation: DNA-based NGS can capture most clinically relevant ROS1 fusions, but large intronic sequences and rare fusion partners are more reliably detected with RNA-based approaches run on the same tissue specimen.

Wistuba added that ROS1 immunohistochemistry (IHC) can serve as a screening tool given its high sensitivity, but noted that specificity is closer to 80%, meaning positive results still require confirmatory NGS. He recommended that any pathology laboratory using a reflex testing approach include PD-L1, ALK, and ROS1 IHC ahead of comprehensive DNA/RNA NGS.

On circulating tumor DNA (ctDNA) testing, Rodriguez said that allele fraction does not typically factor into her treatment decisions once an actionable alteration is identified. However, Leal noted that the inverse scenario is informative: a negative result paired with low tumor fraction signals that the assay was likely inadequate, rather than truly negative. Rotow agreed, describing ctDNA as “more of a non-diagnostic test than a ruling-out assay,” given that most platforms remain DNA-based and cannot capture RNA-only detectable fusions.

Rotow said her working definition of comprehensive testing in advanced NSCLC includes DNA-based NGS, RNA-based NGS, and IHC for PD-L1, MET, and HER2, ideally obtained up front. Leal added that testing should be revisited at every progression time point, since panels evolve and prior results may not reflect current standards.

“It’s such a good point to call out that sometimes runs under the radar,” Rotow said of the risk that DNA-only testing poses specifically for ROS1 fusions in younger, light- or never-smoking patients.

How do efficacy, CNS activity, and resistance coverage differentiate available and emerging ROS1 TKIs?

The panel traced a clear generational arc in ROS1-directed therapy. An integrated analysis of entrectinib (Rozlytrek) trials, including the phase 1 ALKA-372-001 (EudraCT 2012-000148-88), phase 1/2a STARTRK-1 (NCT02097810), and phase 2 STARTRK-2 (NCT02568267) studies, reported an objective response rate (ORR) of 67.1% (95% CI, 59.3%-74.3%), a median duration of response (DOR) of 15.7 months (95% CI, 13.9-28.6), a median PFS of 15.7 months (95% CI, 11.0-21.1) in TKI-naive patients with ROS1-positive NSCLC (n = 161).⁵ In patients with baseline central nervous system (CNS) metastases (n = 46), the intracranial ORR was 52.2% (95% CI, 37.0%-67.1%).

Repotrectinib built on that CNS signal in the phase 1/2 TRIDENT-1 trial (NCT03093116). In the TKI-naive cohort (n = 71), the confirmed ORR (cORR) was 79% (95% CI, 68%-88%), the median DOR was 34.1 months (95% CI, 25.6-not estimable [NE]), and the median PFS was 35.7 months (95% CI, 27.4-NE); the intracranial ORR reached 89% (95% CI, 52%-100%) among patients with measurable baseline brain metastases who had not previously received a ROS1 TKI (n = 9).⁶

“We are seeing that these drugs can work in the brain,” Rodriguez said.

Taletrectinib represented a further step in selectivity. In a pooled analysis of the phase 2 TRUST-I (NCT04395677) and TRUST-II (NCT04919811) trials (n = 273), TKI-naive patients (n = 160) achieved a cORR of 88.8% (95% CI, 82.8%-93.2%) and an intracranial cORR of 76.5% (95% CI, 50.1%-93.2%); the median DOR was 44.2 months (95% CI, 30.4-not reached [NR]), and the median PFS was 45.6 months (95% CI, 29.0-NR). TKI-pretreated patients (n = 113) achieved a cORR of 55.8% (95% CI, 46.1%-65.1%), including a 61.5% (95% CI, 31.6%-86.1%) response rate among those with the ROS1 G2032R resistance mutation (n = 8/13).⁷

“That’s my go-to for first line,” Leal said of taletrectinib. “When you look across trials, with the caveats of cross-trial comparisons, you see the longest PFS of over 45 months.”

Zidesamtinib (Jideytro), a TRK-sparing ROS1 inhibitor, was approved by the FDA in July 2026 for the treatment of adult patients with previously treated ROS1-positive NSCLC, based on phase 1/2 ARROS-1 trial (NCT05118789) data showing an ORR of 44% (95% CI, 34%-53%) among 117 TKI-pretreated patients.⁸

On resistance biology, Wistuba noted that the ROS1 G2032R solvent-front mutation remains the most frequent on-target resistance mechanism.⁹ Rodriguez said the newest agents already cover ROS1 G2032R mutations, arguing for starting with the most potent, most selective TKI rather than risking resistance that a first-generation agent would leave unaddressed. Leal added that emerging data with zidesamtinib suggest activity even after taletrectinib, an early signal for future sequencing strategies.

The panel agreed that intracranial activity in the 60%-to-80% range is now a baseline expectation rather than a differentiator among newer agents, given that ROS1-positive tumors have a strong predilection for CNS involvement at both diagnosis and progression.

“A lot of times, we can treat the brain with the ROS1-targeted therapy,” Leal said, noting that this can spare patients from radiation therapy and its associated late toxicities.

What do current safety data show with TKIs in lung cancer, and how should treatment be sequenced going forward?

Toxicity profiles have shifted substantially alongside the move toward more selective agents. Rodriguez recalled that crizotinib and entrectinib were associated with visual field changes and liver function test abnormalities, whereas repotrectinib’s off-target TRK inhibition drives a distinct pattern of neurologic toxicity. In TRIDENT-1, treatment-emergent dizziness occurred in 62% of patients and was mostly low grade.⁶ Leal noted that dose holds and reductions have been shown to preserve treatment efficacy in patients who have already responded but experience toxicities.

Taletrectinib’s safety profile is centered on gastrointestinal (GI) and transaminase-related adverse effects (AEs), rather than neurotoxicity. In the pooled TRUST-I and TRUST-II analysis, among 352 patients treated with taletrectinib at 600 mg once daily, the most frequent treatment-emergent AEs were GI events (88%) and elevated aspartate aminotransferase (72%) and alanine aminotransferase (68%) levels, most grade 1. Neurologic events were infrequent (dizziness, 21%; dysgeusia, 15%) and mostly grade 1, and patients in this arm had a 6.5% discontinuation rate due to treatment-emergent AEs.⁷

“Most of [these toxicities] don’t really interfere with quality of life [QOL],” Rodriguez said, adding that proactive antiemetic and antidiarrheal counseling before treatment initiation, along with pharmacist-led education sessions, helps patients stay on therapy through the first month of dose adjustment.

Rotow noted that reported experience with zidesamtinib to date has been notable primarily for peripheral edema, without significant TRK-mediated neurotoxicity.

Looking ahead, the panelists cited several open questions related to understanding resistance mechanisms to newer-generation TKIs, extending ROS1-directed therapy into earlier-stage and consolidation settings, managing leptomeningeal disease, and improving RNA-based liquid biopsy technology. Wistuba also raised the potential future role of computational pathology as a ROS1 screening aid.

Asked to define success in treating ROS1-positive NSCLC, Leal pointed to durable disease control that translates into meaningful survival outcomes, delayed development of brain metastases, and QOL outcomes that allow patients to “live for years and lead lives that are a new normal.”

Rodriguez emphasized that testing remains the foundation, saying “You won’t find the mutations you’re not looking for.”

Rotow closed by echoing that message for community oncologists, emphasizing that “We have a lot of exciting advances for patients with ROS1-positive lung cancer... but only if we know about the target.”

Julia Rotow, MD, is the clinical director of the Lowe Center for Thoracic Oncology, the director of clinical research, and a physician at Dana-Farber Cancer Institute; as well as an assistant professor of medicine at Harvard Medical School, in Boston, Massachusetts.

Ticiana Leal, MD, is a professor and director of the Thoracic Oncology Program in the Department of Hematology and Medical Oncology at the Emory University School of Medicine, as well as the Medicial Director of the Clinical Trials Office at the Winship Cancer Institute of Emory University in Atlanta, Georgia.

Estelamari Rodriguez, MD, MPH, is the associate director of Community Outreach - Thoracic Oncology at the University of Miami Health System in Florida.

Ignacio Wistuba, MD, is the research department chair, program lead, and the chair of translational pathology at Moffitt Cancer Center in Tampa, Florida.

References

1. Desilets A, Repetto M, Yang S, Drilon A. Targeting ROS1 rearrangements in non–small cell lung cancer: current insights and future directions. Cancer. 2025;131(suppl 1):e35784. doi:10.1002/cncr.35784
2. Drilon A, Jenkins C, Iyer S, Schoenfeld A, Keddy C, Davare MA. ROS1-dependent cancers - biology, diagnostics and therapeutics. Nat Rev Clin Oncol. 2021;18(1):35-55. doi:10.1038/s41571-020-0408-9
3. Lindeman NI, Cagle PT, Aisner DL, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med. 2018;142(3):321-346. doi:10.5858/arpa.2017-0388-CP
4. Kalemkerian GP, Narula N, Kennedy EB, et al. Molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement summary of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology clinical practice guideline update. JCO Oncol Pract. 2018;14(5):323-327. doi:10.1200/JOP.18.00035
5. Dziadziuszko R, Krebs MG, De Braud F, et al. Updated integrated analysis of the efficacy and safety of entrectinib in locally advanced or metastatic ROS1 fusion-positive non-small-cell lung cancer. J Clin Oncol. 2021;39(11):1253-1263. doi:10.1200/JCO.20.03025
6. Drilon A, Camidge DR, Lin JJ, et al. Repotrectinib in ROS1 fusion-positive non-small-cell lung cancer. N Engl J Med. 2024;390(2):118-131. doi:10.1056/NEJMoa2302299
7. Pérol M, Li W, Pennell NA, et al. Taletrectinib in ROS1+ non–small cell lung cancer: TRUST. J Clin Oncol. 2025;43(suppl 16):1920-1929. doi:10.1200/JCO-25-00275
8. FDA approves zidesamtinib for previously treated ROS1-positive non-small cell lung cancer. FDA. Updated July 24, 2026. Accessed August 27, 2026. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-zidesamtinib-ros1-positive-non-small-cell-lung-cancer
9. Drilon A, Camidge DR, Lin JJ, et al. Repotrectinib in ROS1 fusion-positive non-small-cell lung cancer. N Engl J Med. 2024;390(2):118-131. doi:10.1056/NEJMoa2302299