Detection of ROS1 rearrangement in lung cancer remains ‘complex’
No testing methodology demonstrated 100% sensitivity in the detection of ROS1 rearrangements or fusions among patients with non-small cell lung cancer, according to study results.
Thus, available tests may have inherent limitations that could fail to identify patients who could benefit from ROS1-directed therapy.
“The main point is just to be aware of the deficiencies in these assays and not to always trust a negative result from a single test,” Kurtis D. Davies, PhD, lead assay development scientist at Colorado Molecular Correlates Laboratory and instructor at University of Colorado Anschutz Medical Campus, said in a press release. “If you're suspicious that a patient could be ROS1 positive — maybe they’re a never-smoker without other known drivers such as EGFR, ALK, KRAS, BRAF — then it may be useful to try another kind of test.”
Preclinical and clinical data have shown cancer cells that harbored ROS1 fusions appeared sensitive to small molecule tyrosine kinase inhibitors with activity against ROS1, such as crizotinib (Xalkori, Pfizer).
ROS1 inhibition has improved efficacy and a more tolerable side effect profile than standard-of-care chemotherapy among patients with ROS1 rearrangement or fusion, which has created a need for accurate detection for clinical management.
Break-apart fluorescence in situ hybridization, or FISH, has served as the gold standard assay. However, immunohistochemistry and reverse-transcription polymerase chain reaction also are successfully used.
Still, assays remain limited in how they can be multiplexed, so that simultaneous evaluation of ROS1 and other gene fusions with these approaches is inefficient, according to the researchers.
Davies and colleagues compared the efficacy of the break-apart FISH assay with the efficacy of an RNA-based next-generation sequencing (NGS) approach and a DNA-based hybrid capture library approach followed by NGS, for the detection of ROS1 rearrangements or fusions in 23 clinical NSCLC samples. All samples were known to be ROS1 positive.
“The University of Colorado has been a big player in the clinical research of ROS1 lung cancer, and so we have a lot more ROS1-positive samples than almost anywhere else,” Davies said. “This allowed us to go back to our large bank of samples to test them in these three ways.”
Overall, none of the approaches demonstrated 100% sensitivity for the detection of ROS1 rearrangements or fusions.
Researchers reported false-negative findings for two of 20 samples on FISH, four of 18 samples on the DNA-based NGS assay, and three of 19 samples on the RNA-based NGS assay.
Of the false-negative FISH findings, one appeared to occur because of the inability of some FISH probes to detect rearrangements that result from small genomic deletions. A deletion on chromosome 6 in the sample left enough of the hybridization regions for the probes to bind. “Thus, despite being clearly positive for a GOPC-ROS1 fusion via both NGS-based assays, the sample appeared rearrangement negative by FISH,” the researchers wrote.
The second false-negative FISH result occurred for a complex staining pattern that had many atypical fusion doublets, but the percentage of cells with the typical split signals was below cutoff (15%).
For the DNA-based test, Davies attributed negatives to the assay’s inability to cover large areas of the gene.
“You have large swaths of unsequenced DNA and, if the ROS1 change is in one of those areas, it's possible to miss it,” Davies said.
For the RNA approach, RNA typically looks at what is manufactured from the DNA as opposed to taking a snapshot of altered ROS1 DNA, according to the press release.
For all three false-negative findings on RNA-based NGS cases, an important quality control metric was not met. These samples had poor-quality RNA and, thus, were deemed uninformative, instead of being true false-negative findings.
“The deficiency in assays based on RNA is that they depend on RNA quality, which can be bad in clinical samples,” Davies said. “If you take out the negatives due to RNA quality, which we don't really regard as negative, there were no false negatives with this kind of test.”
A mindful approach that may use an orthogonal assay following a negative ROS1 finding may be necessary if no other alterations are present, according to the researchers.
“ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results,” they wrote. – by Melinda Stevens
Disclosures: Davies reports no relevant financial disclosures. Please see the full study for all other authors’ relevant financial disclosures.