VENTANA pan-TRK (EPR17341) Assay
 

NTRK gene fusions can be detected using various testing methods, including NGS, immunohistochemistry (IHC), which detects protein expression of both fusion and wild-type; fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR).^1-3

• NGS relies on DNA or ribonucleic acid (RNA) sequencing technologies that are capable of processing multiple DNA or RNA sequences in parallel, and provides the most comprehensive view of several biomarkers. NGS can detect fusions in all 3 NTRK genes (must have NTRK 1, 2, 3 gene-fusion coverage), as well as the NTRK gene fusion partner and position.

However, turnaround time for results with NGS may be longer than with IHC or FISH^1,2,4,5

• IHC is a protein biomarker test that utilizes specific antibodies to detect expression of the TRK component of the wild type and fusion protein. An antibody directed against a conserved region may detect fusion or wild-type proteins derived from TRKA, TRKB, or TRKC proteins^2,3

• Like IHC, FISH break-apart probes can distinguish fusion variants. FISH assays can often be labor intensive and more expensive than IHC when performing multiple assays^2,6

• RT-PCR requires multiple primer sets for each gene, since the location of the gene rearrangement is not known. It is very reliable for known fusion variants but requires multiple reactions with specific primers for known variants and can miss detection of unknown/untested variants⁷

On October 23, 2020, the Food and Drug Administration approved the next-generation sequencing (NGS)-based FoundationOne CDx test (Foundation Medicine, Inc.) as a companion diagnostic to identify fusions in neurotrophic receptor tyrosine kinase (NTRK) genes, NTRK1, NTRK2, and NTRK3, in DNA isolated from tumor tissue specimens from patients with solid tumors eligible for treatment with larotrectinib (VITRAKVI, Bayer Healthcare Pharmaceuticals, Inc.).

^{*This document doesn’t contain any information on ROS1.}

Per ESMO, in solid tumours where gene fusions are common but the frequency of NTRK gene
fusions is lower, an NGS panel that includes NTRK fusions is recommended. For tumors with a
very low frequency of NTRK gene fusions but where molecular screening is common, inclusion of
NTRK genes in routine NGS analysis is recommended. For tumors with a low frequency of NTRK
fusions, where NGS is not available or is not routinely performed for a histotype, pan-TRK IHC
should be performed for screening with NGS confirmation of positive IHC results.¹⁰

IHC is a protein biomarker test that utilizes specific antibodies to detect the expression of the antigen/protein.² In case of pan-TRK IHC assay, our assay detects the c-terminal region of TRK A, B, and C proteins in wild type as well as fusion proteins. NGS relies on DNA or RNA sequencing technologies that are capable of processing multiple DNA or RNA sequences in parallel and provides the most comprehensive view of several biomarkers, including rare and common alterations. NGS can detect fusions in all 3 NTRK genes, as well as the NTRK gene fusion partner and position. However, the turnaround time for results with NGS may be longer than with IHC. IHC mainly detects gene expression at the protein level and pan-TRK IHC assay is not optimized to differentiate the wild type and fusion TRK protein expression. ^1,2,4-6,11

In order to detect all TRK proteins, the antibody must detect TRKA, TRKB, and TRKC proteins. Some clones are less sensitive than others and there are reports that the sensitivity of IHC for some NTRK3 fusions is lower than for NTRK1 and NTRK2.³

The VENTANA^® pan-TRK (EPR17341) assay is a class 1 analytic assay intended for the immunohistochemical detection of the C-terminal region of the TRK proteins A, B, and C, which is known to be conserved across wild-type (WT) and chimeric fusion proteins. As the VENTANA^® pan-TRK (EPR17341) assay can’t differentiate between WT and NTRK fusions, it is important to note that WT TRK proteins are not expected in non-neural tissues, and this expression may indicate a fusion that should be confirmed with other methods.^6,12,13

Pan-TRK antibodies have been reported to have 95% to 100% sensitivity and up to 100% specificity.^2,6

No, there is no scoring algorithm or criteria to determine IHC positivity. The VENTANA^® pan-TRK (EPR17341) assay is a IVD analytic assay intended for the immunohistochemical detection of the C-terminal region of the TRK proteins A, B, and C, which is known to be conserved across WT and fusion proteins.^12,14

Laboratory directors should use studies performed by the laboratory or reported in published or other reliable sources to validate new assays. The College of American Pathologists recommends that laboratory medical directors determine that fewer than 20 validation cases are sufficient for a specific marker (eg, rare antigen). When that determination is made, the rationale must be documented.^22,23

IHC, examples of positive control tissues for the Ventana^® pan-TRK (EPR17341) assay include cerebellum and appendix.^13,24

The National Comprehensive Cancer Network (NCCN) guidelines for treatment by cancer type include NTRK gene-fusion testing.

• Non−Small Cell Lung Cancer (NSCLC) v3.2019 / Category 2A: Recommends NTRK gene-fusion testing as part of broad molecular profiling in patients with advanced or metastatic adenocarcinoma, large-cell, or NSCLC not otherwise specified, based on recent data.¹¹

As of August 2019, some additional NCCN panel guidelines that consider NTRK gene-fusion testing as an option include the following:

• Rectal Cancer v2.2019 / Category 2A: Recommends NTRK gene-fusion testing in patients with metastatic colorectal cancer.²⁵
• Cutaneous Melanoma v2.2019 / Category 2A: Recommends NTRK gene-fusion testing as an emerging molecular technology for cutaneous melanoma diagnosis and prognostication.²⁶
• Occult Primary v2.2019 / Category 2A: Per physician discretion, TRK protein testing may be considered as part of broad IHC testing (a positive test should then be confirmed with NGS).²⁷

Molecular Testing

• Colon Cancer v2.2019 / Category 2A: Recommends NTRK gene-fusion testing in patients with metastatic colorectal cancer.²⁸
• Head and Neck Cancers v2.2019 Salivary Gland Tumors / Category 2A: Recommends NTRK gene-fusion status should be checked for secretory carcinoma.²⁹

Although tumor testing has been focused primarily on the use of FFPE tissues, laboratories often accept other specimen types, particularly cytopathology preparations not processed by FFPE methods. 

Experts who were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group have published their recommendations. There are other recommendations published, such as one from Dr Penault-Llorca.^10,30