Identify NTRK fusion + cancers for all solid tumors


VENTANA pan-TRK (EPR17341) Assay

pan-TRK immunohistochemistry (IHC) has been shown to be a resource-efficient method to identify patients for genetic testing for the assessment of NTRK fusions.

pan-TRK IHC enables earlier detection of patients who potentially benefit from NGS testing.

  • Targeted therapies are being studied to advance treatment options for patients with actionable biomarkers—patients with gene fusions need high quality molecular testing to realize these opportunities.
  • To help patients with NTRK - fusion –positive cancer, we must first identify them.
  • Provides a resource-efficient method to identify patients for genetic testing confirmation.
  • Detects fusion proteins from all 3 TRK genes18
  • Enables earlier detection of patients who will potentially benefit from NGS

First pan-TRK IVD IHC assay

  • pan-TRK assay for use across neoplastic tissues.
  • pan-TRK immunohistochemistry (IHC) has been shown to be a resource-efficient method that may serve as an adjunct to genetic testing for the assessment of NTRK fusions.1

 

stain-1

Protein expression in head and neck squamous cell carcinoma with wild-type TRK (20x) 51

stain-2

Protein expression in secretory carcinoma of salivary gland with TRK fusion (20x) 52

stain-3

Protein expression in colorectal carcinoma with TRK fusion (20x) 52

Your assay choice matters

VENTANA pan-TRK (EPR17341) Assay is intended for the immunohistochemical detection of the C-terminal region of the tropomyosin receptor kinase (TRK) proteins A, B and C, which is known to be conserved across wild-type and fusion proteins, in formalin-fixed, paraffin-embedded (FFPE) neoplastic tissues stained with BenchMark IHC/ISH instruments. 

Note: This product should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information and proper controls. This antibody is intended for in vitro diagnostic (IVD) use. VENTANA pan-TRK (EPR17341) Assay has not been optimized to delineate between TRK wild-type and chimeric-fusion proteins

  • NTRK gene fusions drive cancer through aberrant signalling.1,2,9,10
  • NTRK gene fusions create an oncogenic fusion protein that activates a signalling cascade implicated in cell proliferation, survival, and angiogenesis.1,2
  • NTRK gene fusions may be mutually exclusive to other oncogenic drivers.2,11
  • Each NTRK gene can combine with multiple fusion partners; at least 25 distinct NTRK gene fusions have been identified to date.1-3,11
  • The human neurotrophic tyrosine kinase receptor genes NTRK1, NTRK2 and NTRK3, which encode the TrkA, TrkB and TrkC proteins, respectively, are oncogenes that can be activated and drive cancer progression when chromosomal rearrangements lead to their aberrant juxtaposition with other genes such as ETV6, EML4, LMNA and TPM3 (Figure 1).

https://roche63-h.assetsadobe2.com/is/image/content/dam/diagnostics/us/en/LandingPages/pan-trk/pan-TRK-tech-comparison.png?scl=1&fmt=png-alpha

 

Education Resources - Medical Affairs
 
“Down the Ocular” – Podcast
Listen to Dr. Bharathi Vennapusa discuss various topics related to NTRK gene fusion testing methodologies and recommendations for identifying patients with IHC testing prior to NGS confirmation.
 

Part 1: Biology and Testing methods for NTRK fusions
Dr. Denise Croix with Roche Diagnostics Medical and Scientific Affairs (MSA) provides an overview of the function and role pan-TRK IHC plays in testing methodology for NTRK gene fusions.

  • NTRK overview
  • Gene fusions
  • Prevalence
  • Methods of detection
     

Part 2: Pan-TRK IHC with VENTANA pan-TRK IHC (EPR17341) Assay
Dr. Bharathi Venapussa, a pathologist with Roche Diagnostics Medical and Scientific Affairs (MSA), discusses:.

  • Biology of NTRK fusions
  • IHC testing methodology
  • Staining patterns
  • Assay validation
  • Disease states
  • Challenges
     

Part 3: Pan-TRK testing algorithms
Dr. Katie Robertson with Roche Diagnostics Medical and Scientific Affairs (MSA) discusses multiple testing algorithms for the detection of NTRK fusion positive cancer.

  • Overview
  • Testing algorithms
    • ESMO recommendation
    • MSKCC recommendation
    • NCCN guidelines
       

Interpretation of VENTANA pan-TRK (EPR17341) Assay – Digital Case Review
Dr. Bharathi Vennapusa from Roche MSA reviews example cases stained with VENTANA pan-TRK (EPR17341) Assay using digital slides. Content and approvals of any product related information is current as of the date of publishing.

 

 

Detecting NTRK Gene Fusions

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) are oncogenic drivers across a wide range of pediatric and adult cancer types.1-3

• The tropomyosin receptor kinase (TRK) family contains 3 members—TRKA, TRKB, and TRKC— and these proteins are encoded by the genes NTRK1, NTRK2, and NTRK3, respectively.3

pan-TRK-infographic

NTRK fusion genes are emerging as actionable biomarkers
and oncogenic drivers across a wide range of tumor types

TRK fusion proteins have been identified in a wide range of commonly occurring tumors, such as lung cancer, thyroid cancer and sarcoma, but at low frequencies.1-7 In very rare tumors, such as infantile fibrosarcoma, secretory/juvenile breast cancer and mammary analogue secretory cancers (MASC, secretory carcinoma) of the salivary glands, TRK fusion proteins are likely to be the defining genetic feature.8-12

pan-TRK-infographic-2

 

 

Only testing can uncover NTRK fusion cancer.1

  • Advanced testing is needed to uncover NTRK fusion + cancer
  • Diagnostic testing should detect NTRK 1,2,3 fusion genes and be validated with appropriate reference standards.1 
  • Appendix and Cerebelum tissue can be used for controls to assess for the presence of NTRK fusion proteins through IHC.

ESMO recommendation: NTRK testing10

European Society for Medical Oncology (ESMO) have published recommendations outlining a strategy for NTRK gene-fusion testing. As part of this testing approach, the use of pan-TRK IHC to identify patients with solid tumors allows you to focus on those who may benefit from next-generation sequencing.

ESMO
Frequently Asked Questions

Frequently Asked Questions

General

General

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 FISH1,2,4,5

• 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 proteins2,3

• 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 assays2,6

• 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.

IHC

IHC

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

Validation

Validation

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

Guideline

Guideline

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. 

 

Testing Algorithms

Testing Algorithms

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

1. Vaishnavi A, Le AT, Doebele RC. TRKing down an old oncogene in a new era of targeted therapy. Cancer Discov. 2015;5(1):25-34.

2. Murphy DA, Ely HA, Shoemaker R, et al. Detecting gene rearrangements in patient populations through a 2-step diagnostic test comprised of rapid IHC enrichment followed by sensitive next-generation sequencing. Appl Immunohistochem Mol Morphol. 2017;25(7):513-523.

3. Solomon JP, Benayed R, Hechtman JF, Ladanyi M. Identifying patients with NTRK fusion cancer. Ann Oncol. 2019;30(Suppl8):viii16-viii22.

4. Su D, Zhang D, Chen K, et al. High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods. J Exp Clin Cancer Res. 2017;36(1):121.

5. Du X, Shao Y, Qin HF, Tai YH, Gao HJ. ALK-rearrangement in non-smallcell lung cancer (NSCLC). Thorac Cancer. 2018;9(4):423-430.

6. Hechtman JF, Benayed R, Hyman DM, et al. Pan-Trk immunohistochemistry is an efficient and reliable screen for the detection of NTRK fusions. Am J Surg Pathol. 2017;41(11):1547-1551.

7. Ali G, Bruno R, Savino M, et al. Analysis of fusion genes by NanoString system: a role in lung cytology? Arch Pathol Lab Med. 2018;142(4):480-489.

8. US Food and Drug Administration. List of cleared or approved companion diagnostic devices (in vitro and imaging tools). https://www.fda.gov/medical-devices/vitro-diagnostics/listcleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Accessed February 3, 2020.

9. Precision Oncology News. Japanese Regulator Approves Foundation Medicine CDx Assay for Roche’s Rozlytrek. https://www.precisiononcologynews.com/regulatory-news/japaneseregulator-approves-foundation-medicine-cdx-assay-roches-rozlytrek#.Xj2faBd7lTY. June 27, 2019. Accessed February 3, 2020.

10. Penault-Llorca F, Rudzinski ER, Sepulveda AR. Testing algorithm for identification of patients with TRK fusion cancer. J Clin Pathol. 2019;72(7):460-467.

11. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Non-Small Cell Lung Cancer. V.2.2020.© National Comprehensive Cancer Network, Inc 2019. All rights reserved. Published December 23, 2019. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

12. Roche (via PR NewsWire). Roche launches first IVD pan-TRK immunohistochemistry assay. Nov 27, 2018. https://www.prnewswire.com/news-releases/roche-launches-first-ivd-pan-trk-immunohistochemistryassay-300755647.html. Accessed February 3, 2020.

13. Roche Tissue Diagnostics. VENTANA pan-TRK (EPR17341) Assay: Package Insert 1017533EN Rev A. https://productlibrary.ventana.com/ventana_portal/executeSearch.do. Accessed February 7, 2020.

14. Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue – a review. Diagn Pathol. 2014;9:221.doi:10.1186/s13000-014-0221-9.

15. Xu T, Wang H, Huang X, et al. Gene fusion in malignant glioma: an emerging target for next-generation personalized treatment. Transl Oncol. 2018;11(3):609-618.

16. Cocco E, Scaltriti M, Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy. Nat Rev Clin Oncol. 2018;15(12):731-747.

17. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57-63. doi:10.1038/nrg2484.

18. Aguado C, Giménez-Capitán A, Karachaliou N, et al. Fusion gene and splice variant analyses in liquid biopsies of lung cancer patients. Transl Lung Cancer Res. 2016;5(5):525-531.

19. Foundation Medicine. FoundationOne® Liquid: Technical Specifications. https://www.foundationmedicine.com/genomic-testing/foundation-one-liquid. Accessed February 7, 2020.

20. Foundation Medicine. FoundationOne® CDx: Technical Information. https://www.foundationmedicine.com/genomic-testing/foundation-one-cdx. Accessed February 7, 2020.

21. Foundation Medicine. FoundationOne® Heme: Technical Specifications. https://www.foundationmedicine.com/genomic-testing/foundation-one-heme. Accessed February 7, 2020.

22. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23(3):550-576.

23. College of American Pathologists. Principles of analytic validation of immunohistochemical assays: summary of recommendations. 2015. www.cap.org. Accessed March 3, 2020.

24. SeraCare. SeraCare announces launch of expanded NTRK Reference Material Panel. October 22, 2018. https://www.seracare.com/about-seracare/press-releases/seracare-announces-launch-of-expanded-ntrk-reference-material-panel. Accessed February 3, 2020.

25. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Rectal Cancer. V.1.2020. ©National Comprehensive Cancer Network, Inc 2019. All rights reserved. Published December 19, 2019. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

26. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Cutaneous Melanoma. V.1.2020. © National Comprehensive Cancer Network, Inc 2019. All rights reserved. Published December 19, 2019. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

27. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Occult Primary (Cancer of Unknown Primary [CUP]). V.2.2020. © National Comprehensive Cancer Network, Inc 2020. All rights reserved. Published January 31, 2020. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

28. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Colon Cancer. V.1.2020. © National Comprehensive Cancer Network, Inc 2019. All rights reserved. Published December 19, 2019. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

29. Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Head and Neck Cancers. V.3.2019. © National Comprehensive Cancer Network, Inc 2019. All rights reserved. Published September 16, 2019. Accessed February 7, 2020. To view the most recent and complete version of the guideline, go online to www.NCCN.org.

30. Marchiò C, Scaltriti M, Ladanyi M, et al. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research. Ann Oncol. 2019;30(9):1417-1427.