Detection of ROS1, RET, and ALK fusions in non-small cell lung cancer

ROS1 (c-ros oncogene 1), RET (rearranged during transfection), and ALK (anaplastic lymphoma kinase) are receptors of tyrosine kinases that play important roles in cell growth and differentiation. ROS1, RET or ALK fusion is formed by chromosomal rearrangements that lead to other genes fused with these three genes, causing development and progression of cancer. These genetic alterations have been identified in lung cancer, particularly non-small cell lung cancer (NSCLC). Although the fusions occur at a low-frequency, identification of these fusions proves critical for targeted therapy selection to improve patients’ outcomes.

ROS1 Fusion: A ROS1 fusion, found in a small subset (1-2%) of NSCLC cases, produces a fusion protein with constitutively activated kinase activity. Patients with ROS1-positive tumors may benefit from targeted therapies such as crizotinib, ceritinib, and entrectinib.
RET Fusion: RET fusion, found in approximately 1-2% of NSCLC cases, leads to a fusion protein with constitutive kinase activity. Selpercatinib and pralsetinib have shown efficacy in treating NSCLC patients with RET fusions.
ALK Fusion: ALK fusion, found in about 3-5% of NSCLC cases, also generates an oncogenic protein with constitutively activated kinase activity. Drugs such as crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib have been developed for target therapy on NSCLC patients with ALK fusions.

Detection of ROS1, RET and ALK Fusions using FISH, NGS and RT-qPCR

Fish (Fluorescence In Situ Hybridization), NGS (Next-Generation Sequencing), and RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) are most commonly used molecular techniques to detect ROS1, RET, and ALK fusions, with over 70% concordance of testing results between any two techniques. Here’s how each method works and its application in detecting these fusions:

FISH (Fluorescence In Situ Hybridization):
FISH is a traditional molecular cytogenetic technique that uses different fluorescent probes to detect specific DNA sequences within a fusion rearrangement through hybridization.It detects fusion qualitatively and is particularly useful when evaluating tumor heterogeneity using tissue or FFPE (formalin-fixed paraffin-embedded) samples.

NGS (Next-Generation Sequencing):
NGS is a high-throughput sequencing technology that sequences the fusion genes.
NGS can provide comprehensive genomic profiling with design, not just fusion gene alone. Gradually gaining more traction in clinical setting, NGS can work for fresh tissue, FFPE, as well as liquid biopsy samples.

RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction):
RT-qPCR is a sensitive and quicker molecular technique used to quantify the gene fusion RNA. It is especially detecting the fusions with known transcripts with high sensitivity and specificity.

Advantages and limitations of Fusion Detection TechniquesEach of these techniques has its advantages and limitations.

The following factors affect selection of the detection methods: sensitivity, specificity, cost, turnaround time, and available sample types, and laboratory instrument setting and expertise in the clinical laboratory. In clinical practice, it may be possible to use two methods to comprehensively evaluate gene fusions. For instance, NGS for initial testing and FISH for confirmation.

FISH:
Pros:
• High Sensitivity: highly sensitive, allowing direct visualization of fusion events.
• Specificity: highly specific for known fusion, minimizing false positives.
• Tumor Heterogeneity: provides tumor heterogeneity information in individual cells.
• Well-Established: gold-standard with standardized protocols and interpretation guidelines.
Cons:
• Labor-Intensive: requires specialized equipment and expertise.
• Limited Multiplexing: Limited to one or a few fusions per assay.
• Cost: Higher cost compared to RT-qPCR.
• Turnaround Time: longer turnaround time compared to RT-qPCR.

NGS:
Pros:
• Comprehensive Profiling: allows for simultaneous information in addition to gene fusions.
• High Throughput: detects multiple samples and targets in a single run.
• Sensitivity: high sensitivity for both known and novel fusion detection.
• Sample Flexibility: both tissues and liquid biopsy.
Cons:
• Cost: higher upfront cost including equipment and bioinformatics investment.
• Complexity: requires bioinformatics analysis expertise.
• Turnaround Time: much longer turnaround time compared to RT-qPCR.
• Detection Limitations: may miss novel fusion events that occurs at low-frequency.

RT-qPCR:
Pros:
• Sensitivity: highly sensitive for known fusions.
• Specificity: highly specific for known fusions.
• Quantification: allows for quantitation of fusion frequencies.
• Speed and cost: faster turnaround time and lower cost compared to both FISH and NGS.
• Sample flexibility: use sample types including tissues and liquid biopsies.
Cons:
• Limited Multiplexing: only detects one or a few fusion targets per assay if targets need to be identified.
• Detection Limitations: miss novel fusions.
• Design and optimization: is required before the assay is finalized.

QFusion™ ROS1, RET and ALK fusions RT-qPCR Products

DiaCarta has developed ROS1, RET and ALK fusions RT-qPCR kits to detect these fusions by leveraging our proprietary XNA technology to further improve sensitivity and specificity. All these fusion designs target the most common fusions found. These tests have a LoD of 50 copies of RNA when 50 ng of total RNA is used for the test.

> Download the Qfusion™ ALK gene fusion brochure

> Download the Qfusion™ ROS1 gene fusion brochure

> Download the Qfusion™ RET gene fusion brochure

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