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Published on: March 14, 2019
Comparison of NTRK fusion detection methods in microsatellite-instability-high metastatic colorectal cancer
Suzanna J Schraa1, Ellen Stelloo2, Miangela M Laclé3
1Department of Medical Oncology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.
Abstract:
Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboring NTRK fusions, but the detection of NTRK fusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence of NTRK fusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5'/3' imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in which NTRK fusions are most prevalent (1-5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10 NTRK fusions were detected. In 33 IHC negative samples, no additional transcribed NTRK fusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection of NTRK fusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection, after enrichment with pan-TRK IHC, in routine clinical practice.
Insights
Detecting NTRK fusions in metastatic colorectal cancer (mCRC) is challenging. Quantitative RT-PCR (qRT-PCR), RNA-NGS, and FFPE-TLC show promise as effective diagnostic tools following pan-TRK immunohistochemistry (IHC) screening.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Tropomyosin receptor kinase (TRK) inhibitors are approved for NTRK fusion-positive metastatic solid tumors.
- Accurate detection of NTRK fusions is crucial but challenging, especially in metastatic colorectal cancer (mCRC).
- Current guidelines suggest pan-TRK immunohistochemistry (IHC) screening followed by RNA-based next-generation sequencing (NGS), but RNA extraction from FFPE tissues and cost are limitations.
Purpose of the Study:
- To evaluate the diagnostic performance of various assays for NTRK fusion detection in microsatellite-instability-high mCRC.
- To compare RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and quantitative RT-PCR (qRT-PCR) post-IHC screening.
- To identify suitable alternatives for routine clinical practice in NTRK fusion detection.
Main Methods:
- Assessed diagnostic performance of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR in 268 mCRC patients after pan-TRK IHC screening.
- Determined consensus results by reviewing all assay outcomes.
- Evaluated sensitivity, specificity, and robustness (interpretable results on the first run).
Main Results:
- Out of 16 IHC-positive tumors, 10 NTRK fusions were detected; no additional fusions were found in 33 IHC-negative samples.
- Sensitivity: qRT-PCR (100%), RNA-NGS (90%), FFPE-TLC (90%), FISH (78%). Specificity was 100% for all assays.
- Robustness: FFPE-TLC (100%), RNA-NGS (85%), FISH (70%), qRT-PCR (70%). FISH showed low sensitivity and robustness.
Conclusions:
- Pan-TRK IHC is a highly sensitive screening tool for NTRK fusions in mCRC.
- RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection in routine clinical practice after IHC.
- FISH is not recommended for NTRK fusion detection in mCRC due to suboptimal sensitivity and robustness.
