Common tests for ROS1 translocations in lung cancer

Published Mon Oct 15, 2018


For drugs that target specific gene mutations, such as crizotinib, it is critical to know a patient's mutation status to ensure they are receiving the most effective treatment; therefore, knowing the ROS1 mutation status of a patient with lung cancer is essential. A new article published in the Journal of Thoracic Oncology highlights the limitations in common lab tests used to determine ROS1 status in patients with lung cancer. In the study, researchers from the University of Colorado Cancer Center discovered unexpected false-negatives in their samples.

The researchers tested three methods for determining ROS1 status: fluorescence in situ hybridization (FISH), a DNA-based targeted hybrid capture sequencing test and Archer®'s FusionPlex® Solid Tumor assay, an RNA-based next-generation sequencing (NGS) test. The FISH test provided a false negative result for 2 of 20 samples known to be ROS1-positive. The DNA-based hybrid capture test was negative for 4 of 18 positive samples, and FusionPlex Solid Tumor was negative for 3 of 19 positive samples.

The study found that each test has limitations that led to the false-negatives. FISH assays can have difficulty identifying fusions that occur between partners that are in close genomic proximity. When this is the case, the deleted region between the two genes may be too small to detect. The DNA-based hybrid capture assay failed to cover regions of ROS1 introns where fusions can occur, which is an inheritant challenge associated with detecting fusions using DNA input. Finally, a false-negative was only reported when using FusionPlex Solid Tumor when the sample was of low quality, suggesting that the Archer RNA-based assay is limited only by the quality of the input material, making this method the most reliable.

"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,"

Dr. Kurtis Davies describing the results of the FusionPlex Solid Tumor assay

Importantly, Dr. Kurtis Davies, the first author of the study, noted that the quality of the RNA sample is known based on the results of the test; therefore, a secondary analysis can be run to verify the results of low quality samples. For more details, read the press release here>>

Dr. Lynette Sholl from Brigham and Women's Hospital and Harvard Medical School also discusses similar challenges associated with detecting fusions in the editorial Recognizing the Challenges of Oncogene Fusion Detection: A Critical Step toward Optimal Selection of Lung Cancer Patients for Targeted Therapies. Here she highlights the findings from the Davies et al. study above. Dr. Sholl points out the benefits of using an RNA-based anchored mulitplex PCR assay for fusion detection and goes on to discuss the importance of screening for ROS1 in lung cancer patients, even if it requires employing complimentary assays for optimal results.

"It is reasonable to conclude that for laboratories relying on comprehensive hybrid capture sequencing for fusion detection, alternative techniques, such as RNA transcript detection or immunohistochemistry to detect protein overexpression may be essential to providing a confident interpretation of a rearrangement event."

Dr. Lynette M. Sholl

Archer FusionPlex assays generate target-enriched cDNA libraries from RNA to characterize gene fusions, SNVs, indels and detect expression levels by next-generation sequencing (NGS), including ROS1 mutations. Archer's Anchored Multiplex PCR (AMP™) chemistry utilizes open-ended targeted amplification to identify both known and novel gene fusions, and was purpose-built for low-input and degraded samples such as FFPE tissue.

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For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.