New treatment options for cancer patients with neurotrophic tyrosine kinase (NTRK) rearrangements are a tremendous success, demonstrating first-hand the importance of precision diagnostics. The FDA granted accelerated approval of Bayer's VITRAKVI (larotrectinib) for adult and pediatric solid tumors containing NTRK fusions1. The approval was one of the first tissue agnostic approvals based on genotyping results, and included patients with unresectable or metastatic cancer. For 12 cancer types, the overall response rate was 75% with 22% complete response and 53% partial response. In addition to VITRAKVI, Genentech's entrectinib has a "breakthrough" status and is being evaluated for the potential treatment of advanced or metastatic tumors that harbor NTRK or ROS1 gene rearrangements.
NTRK rearrangements are found in high frequency in some rare cases like secretory carcinoma of the breast and the salivary glands (ETV6-NTRK3) and congenital fibrosarcoma (TPM3-NTRK1, EML4-NTRK3, etc.) but for cancers overall, NTRK rearrangements are present in very low frequency (<0.5%). Okimoto and Bivona 2016)2 showed genetic profiling of various cancer subtypes and prevalence for basket trial concept.
In addition to low frequency, NTRKs 1,2,3 form fusions with numerous gene partners. There are over 40 fusion partners with clinical significance identified thus far. The partner gene may be ETV6, EML4, TPM4, CD74 or LMNA for example. Low frequency and a large number of fusion pairs together create a diagnostic challenge to select the most appropriate diagnostic technology that maximizes sensitivity and specificity at appropriate cost levels.
Detection of NTRK fusions
There are five approaches to detect NTRK fusions: IHC, FISH, RT-PCR, NGS RNA and NGS DNA. Each technology has its advantages and disadvantages thus making a rigorous and effective testing scheme important for today's constrained laboratory. Fortunately, two outstanding clinical research groups have studied the question and have just published their recommendations3,4. These two teams represent some of the top institutions worldwide: Centre Jean Perrin France, Seattle Children's Hospital Washington State, Columbia Hospital New York, University of Turin Italy, Candiolo Cancer Italy, Memorial Sloan Kettering New York, Mass General Boston, Caen Hospital France, University Medicine Berlin, University of Campania Italy, HM Sanchinarro Hospital Spain, and Institut Bustave Roussy France. The work of Marchio, et al 4 was sponsored by the European Society of Medical Oncology Switzerland. Let me highlight a few similarities and differences.
How do NTRK frequencies affect the detection approach?
The groups recommend a different approach depending on expected NTRK frequencies. If high-frequency NTRK is expected, Penault-Llorca, et al 3 recommend pan reactive IHC or FISH as the first test while Marchio et al. recommend only molecular approaches first e.g. FISH, RT-PCR or targeted RNA NGS. Since IHC sensitivity is only 45% - 75% compared to NGS, Penault-Llorca et al. recommend that all negative samples reflex to NGS. Marchio, et al 4 recommended FISH or RT-PCR for screening these cases as acceptable, principally due to turnaround and low costs. If the histological examination predicts low-frequency, both groups recommend NGS, but if NGS is not available both recommend pan IHC. In cases of expected low-frequency mutations, both groups recommend confirmation testing. Please check out the detailed testing schemes in each paper. These reviews are excellent sources for any lab setting up NTRK testing schemes. Each testing scheme does recommend NGS as the most comprehensive technology to ensure the detection of an NTRK alteration. Hence all roads lead to NGS.
For me, the most important conclusion is that it is critical for clinicians and cancer treatment centers to invest in NGS approaches now or at a minimum have easy access to clinical labs that do provide this testing. To achieve the benefits of individual testing and precision diagnostics, labs must have a robust NTRK fusions testing scheme that is comprehensive enough to detect all the potential breakpoints and fusion partners with high sensitivity and that is NGS.
Let me close by highlighting the NTRK reference material SeraCare has developed to assist labs and manufacturers during validation and QC. Table 1 lists the NTRK 1,2,3 fusion pairs we offer as either catalog multiplex products or offer unique mixes through customization.
Table 1 – NTRK fusion pairs in currently available at SeraCare
| Gene ID |
COSMIC ID |
| TPM3-NTRK1 |
COSF1329 |
| LMNA-NTRK1 |
COSF1654 |
| LMNA-NTRK1 |
NA |
| TFG-NTRK1 |
NA |
| TP53-NTRK1 |
COSF1655 |
| IRF2BP2-NTRK1 |
NA |
| SQSTM1-NTRK1 |
NA |
| NACC2-NTRK2 |
COSF1449 |
| AFAP1-NTRK2 |
NA |
| QKI-NTRK2 |
COSF1448 |
| TRIM24-NTRK2 |
NA |
| PAN3-NTRK2 |
NA |
| ETV6-NTRK3 |
COSF1537 |
| ETV6-NTRK3 |
COSF824 |
| ETV6-NTRK3 |
COSF1535 |
| BTBD1-NTRK3 |
NA |
| ETV6-NTRK3 |
COSF572 |
Additional fusion pairs are readily designed and custom prepared through an SOP process already in place at SeraCare. We offer product as RNA, FFPE blocks or as curl sections at 10 µm thickness. Each curl which yields ~400 ng of RNA or higher. These products are intended as a positive sample control reference material in translational research and/or cancer disease clinical research, sequencing library preparation, RNA targeted sequencing, fusion RNA detection, as well as bioinformatics pipeline optimization. See the schematic manufacturing workflow below (Figure 1).
Figure 1. Schematic for Production workflow for Seraseq® FFPE NTRK Fusion RNA Reference Material
For additional information, please check out a poster presentation on NTRK Reference Materials for Global Assay Standardization, delivered at AACR 2019 by my colleague, Katie Huang, where she focused on our NTRK collaborative work with Bayer Pharmaceuticals.
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