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Cancer Testing
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Let testing be your guide:

Educational Resources

NTRK Fusion and Cancer

Testing Algorithms

NTRK Fusion Detection

FAQ

 

Let testing be your guide.

 

Every cancer is unique. Let’s treat it that way.

 

Targeted therapies are being studied to advance treatment options for patients with actionable biomarkers – patients with gene fusions need high-quality molecular testing to realise these opportunities.1,2

 

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are emerging as actionable biomarkers and oncogenic drivers across a wide range of tumour types.1–6 NTRK fusion positive cancer currently has no known defining clinical or pathological features.1–3

 

Only high quality molecular testing such as next-generation sequencing (NGS) can confirm NTRK fusion positive cancer.1

 

Knowing a patient’s biomarker profile can open up new targeted treatment options.7–9 High-quality NGS helps you see beyond the tumour origin to know exactly which mutations are driving that cancer.10

 

Precision medicine combines different treatment options, including traditional cancer therapies and emerging targeted therapies, with the aim of achieving the best possible outcome for the patient.11

 

A number of diagnostic options are available for the identification of gene fusions. However, not all are equally reliable.1,12–17

 

Roche is committed to pioneering progress in precision medicine.18

 

1. Vaishnavi A, Le AT, Doebele RC. Cancer Discov 2015;5:25–34.

2. Lange AM, Lo HW. Cancers (Basel) 2018;10.

3. Amatu A, Sartore-Bianchi A, Siena S. ESMO Open 2016;1:e000023.

4. Khotskaya YB, et al. Pharmacol Ther 2017;173:58–66.

5. de Lartigue J. TRK inhibitors advance rapidly in “tumor-agnostic” paradigm. OncologyLive 2017;18. Available at: https://www.onclive.com/publications/oncology-live/2017/vol-18-no-15/trk-inhibitors-advance-rapidly-in-tumoragnostic-paradigm (Accessed July 2020).

6. Robbins HL, Hague A. Front Endocrinol (Lausanne) 2016;6:1–22.

7. Rozenblum AB, et al. J Thorac Oncol 2017;12:258–268.

8. Schwaederle M, Kurzrock R. Oncoscience 2015;2:779–780.

9. Mansinho A, et al. Expert Rev Anticancer Ther 2017;17:563–565.

10. Frampton GM, et al. Nat Biotechnol 2013;31:1023–1031.

11. Bode AM, Dong Z. NPJ Precis Oncol 2018;2:1.

12. Murphy DA, et al. Appl Immunohistochem Mol Morphol 2017;25:513–523.

13. Su D, et al. J Exp Clin Cancer Res 2017;36:1–12.

14. Abel HJ, Duncavage EJ. Cancer Genet 2013;206:432–440.

15. Hechtman JF, et al. Am J Surg Pathol 2016;41:1547–1551.

16. Aisner DL, et al. Arch Pathol Lab Med 2016;140:1206–1220.

17. Kumar-Sinha C, et al. Genome Med 2015;7:1–18.

18. Roche Media Release, 2018. Available at: www.roche.com/media/releases/med-cor-2018-06-19.htm (Accessed October 2020).

Educational Resources

infographic

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

 

infographic

Part 2: Pan-TRK IHC with VENTANA pan-TRK IHC (EPR17341) Assay
Dr. Bharathi Vennapusa, 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
     

infographic

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

infographic

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.

infographic

Validation of low prevalence biomarkers
Dr. Bharathi Vennapusa MD with Roche Medical and Scientific Affairs discusses sourcing samples, CAP recommendations and strategies for IHC validation of low prevalence biomarkers.

 

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NTRK: Testing: Practical Considerations for IHC Interpretation

NTRK Fusion and Cancer

 

NTRK Infographic

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

1. Vaishnavi A, Le AT, Doebele RC. Cancer Discov. 2015;5(1):25-34.

2. Murphy DA, Ely HA, Shoemaker R, et al. Appl Immunohistochem Mol Morphol. 2017;25(7):513-523.

3. Solomon JP, Benayed R, Hechtman JF, Ladanyi M. Ann Oncol. 2019;30(Suppl8):viii16-viii22.

4. Su D, Zhang D, Chen K, et al. J Exp Clin Cancer Res. 2017;36(1):121.

5. Du X, Shao Y, Qin HF, Tai YH, Gao HJ. Thorac Cancer. 2018;9(4):423-430.

6. Hechtman JF, Benayed R, Hyman DM, et al. Am J Surg Pathol. 2017;41(11):1547-1551.

7. Ali G, Bruno R, Savino M, et al. 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. 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.

 

NTRKs play an important role in healthy tissue

NTRK Small Infographic 1

 

  • The Neurotrophic tropomyosin receptor kinase (NTRK) receptor family is encoded by the three NTRK genes that code for three proteins1

  • In healthy tissue, the NTRK pathway is involved in the development and functioning of the nervous system as well as cell survival2,3

NTRK gene fusions create oncogenic proteins3

 

NTRK Small Infographic 2
 

  • Each of the three NTRK genes can combine with multiple fusion partners to create oncogenic proteins1–4

  • So far, 25 distinct fusions have been identified1

The oncogenic proteins drive cancer through aberrant signalling1,2,6–8

 

 

NTRK Small Infographic 3
 
  • The oncogenic chimera proteins activate a signalling cascade implicated in cell proliferation, survival and angiogenesis1,2,6–8

1. Vaishnavi A, Le AT, Doebele RC. Cancer Discov 2015;5:25–34.

2. Amatu A, Sartore-Bianchi A, Siena S. ESMO Open 2016;1:e000023.

3. Chong CR, et al. Clin Cancer Res 2017;23:204–213.

4. Lange AM, Lo HW. Cancers (Basel) 2018;10.

5. Stransky N, et al. Nat Commun 2014;5:4846.

6. Khotskaya YB, et al. Pharmacol Ther 2017;173:58–66.

7. de Lartigue J. TRK inhibitors advance rapidly in “tumor-agnostic” paradigm. OncologyLive 2017;18. Available at: https://www.onclive.com/publications/oncology-live/2017/vol-18-no-15/trk-inhibitors-advance-rapidly-in-tumoragnostic-paradigm (Accessed July 2020).

8. Robbins HL, Hague A. Front Endocrinol (Lausanne) 2016;6:1–22.

Testing Algorithms

 

NTRK fusion positive cancer currently has no known defining clinical or pathological features. Only high-quality molecular testing can confirm its presence.1 It is important to ensure that the diagnostic test covers NTRK 1, 2, 3 fusion genes and is validated with appropriate reference standards.1

 

ESMO recommendation: NTRK testing2

 

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.

 

NTRK Small Flowchart

1. Vaishnavi A, Le AT, Doebele RC. Cancer Discov 2015;5:25–34.

2. Marchio C. et al. Ann Oncol 2019;30:1417–1427.

 

NTRK Fusion Detection

Only sensitive and specific tests can reliably detect NTRK.1–3 There are multiple molecular tests available for detecting gene fusions, but some are more specific than others.1,2,4–11 

 

NTRK Fusion Detection Table

FFPE = Formalin-fixed and paraffin-embedded

NGS = Next-generation sequencing

IHC = Immunohistochemistry

FISH = Fluorescence in situ hybridisation

RT-PCR = Reverse transcriptase polymerase chain reaction

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Text References:

1. Su D, et al. J Exp Clin Cancer Res 2017;36:1–12.

2. Hechtman JF, et al. Am J Surg Pathol 2016;41:1547–1551.

3. Kumar-Sinha C, et al. Genome Med 2015;7:1–18.

4. Vaishnavi A, et al. Cancer Discov 2015;5:25–34.

5. Murphy DA, et al. Appl Immunohistochem Mol Morphol 2017;25:513–523.

6. Stack EC, et al. Methods 2014;70:46–58.

7. Knezevich SR, et al. Nat Genet 1998;18:184–187.

8. Naidoo J, Drilon A. Am J Hematol Oncol 2014;10:4–11.

9. International Association for the Study of Lung Cancer. IASLC Atlas of ALK and ROS1 Testing in Lung Cancer. Available at: https://www.iaslc.org/research-education/publications-resources-guidelines/iaslc-atlas-alk-and-ros1-testing-lung-cancer (Accessed November 2020).

10. Shan L, et al. PLoS One 2015;10:e0120422.

11. Bubendorf L, et al. Virchows Arch 2016;469:489–503.

 

Table References:


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. 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. 

3. Horak P, Fröhling S, Glimm H. Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls. ESMO Open. 2016;1(5):e000094. 

4. Hovelson DH, McDaniel AS, Cani AK, et al. Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors. Neoplasia. 2015;17(4):385-399. 

5. Chen H, Luthra R, Goswami RS, Singh RR, Roy-Chowdhuri S. Analysis of pre-analytic factors affecting the success of clinical next-generation sequencing of solid organ malignancies. Cancers (Basel). 2015;7(3):1699-1715. 

6. 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. 

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

8. Kummar S, Lassen UN. TRK inhibition: a new tumor-agnostic treatment strategy. Target Oncol. 2018;13(5):545-556. 

9. Jensen E. Technical review: in situ hybridization. Anat Rec (Hoboken). 2014;297(8):1349-1353. 

10. Wolff DJ, Bagg A, Cooley LD, et al. Guidance for fluorescence in situ hybridization testing in hematologic disorders. J Mol Diagn. 2007;9(2):134-143.

11. 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. 

12. 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.

13. Srinivasan M, Sedmark D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161(6):1961-1971. 

14. 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. 

15. Liu ML, Jeong J, Ambannavar R, et al. Rt-PCR gene expression profiling of RNA from paraffin-embedded tissues prepared using a range of different fixatives and conditions. Methods Mol Biol. 2011;724:205-237. 

16. Cleveland Clinic Laboratories. CC-SIGN® NTRK Plus Gene Fusion NGS Panel. https:// clevelandcliniclabs.com/2019/01/31/new-cc-sign-ntrk-gene-analysis/. Accessed January 31, 2020. 

17. University of Washington, Department of Laboratory Medicine. UW OncoPlex Cancer Gene Panel. http://tests.labmed.washington.edu/UW-OncoPlex. Accessed January 31, 2020. 

18. NeoGenomics. NTRK NGS Fusion Profile. https://neogenomics.com/ test-menu/ntrk-ngs-fusion-profile. Accessed January 31, 2020. 

19. Thermo Fisher Scientific. Oncomine NTRK Testing Flyer. 2018. Available for download at https://www.oncomine. com/ngs-for-pathologists. Accessed February 7, 2020. 

20. Molecular MD. NGS Lung-Thyroid Fusion Cancer Panel. December 16, 2016. www.molecularmd.com. Accessed February 7, 2020. 

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

22. 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. 

23. Stack EC, Wang C, Roman KA, Hoyt CC. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 2014;70(1):46-58. 

24. Doebele RC, Davis LE, Vaishanavi A, et al. An oncogenic NTRK fusion in a patient with soft-tissue sarcoma with response to the tropomyosin-related kinase inhibitor loxo-101. Cancer Discov. 2015;5(10):1049-1057. 

25. College of American Pathologists. Anatomic Pathology Checklist. July 28, 2015. www.cap.org. Accessed February 7, 2020. 

26. NeoGenomics. Pan-TRK. https://neogenomics.com/test-menu/pan-trk. Accessed January 31, 2020. 

27. Hung YP, Fletcher CDM, Hornick JL. Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics. Histopathology. 2018;73(4):634-644. 

28. Croce S, Hostein I, Longacre TA, et al. Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms. Mod Pathol. 2019;32(7):1008-1022. 

29. Rudzinski ER, Lockwood CM, Stohr BA, et al. Pan-Trk immunohistochemistry identifies NTRK rearrangements in pediatric mesenchymal tumors. Am J Surg Pathol. 2018;42(7):927-935. 

30. Wang L, Busam KJ, Benayed R, et al. Identification of NTRK3 fusions in childhood melanocytic neoplasms. J Mol Diagn. 2017;19(3):387-396. 

31. Tognon C, Knezevich SR, Huntsman D, et al. Expression of the ETV6- NTRK3 gene fusion as a primary event in human secretory breast carcinoma. Cancer Cell. 2002;2(5):367-376. 

32. Luk PP, Selinger CI, Mahar A, Cooper WA. Biomarkers for ALK and ROS1 in lung cancer: immunohistochemistry and fluorescent in situ hybridization. Arch Pathol Lab Med. 2018;142(8):922-928. 

33. Empire Genomics. NTRK3 Break Apart FISH Probe. https://www.empiregenomics.com/fish-probes/hd-probes/NTRK3+Break+Apart+FISH+Probe. Accessed January 31, 2020. 

34. Tatematsu T, Sasaki H, Shimizu S, et al. Investigation of neurotrophic tyrosine kinase receptor 1 fusions and neurotrophic tyrosine kinase receptor family expression in non-small-cell lung cancer and sensitivity to AZD7451 in vitro. Mol Clin Oncol. 2014;2(5):725-730.

 

FAQ

1. What kinds of testing methods can be used to detect NTRK gene fusions? 

NTRK gene fusions can be detected using various testing methods, including next-generation sequencing (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 

 

2. How does each testing method work and what are the advantages and limitations of each? 

• NGS relies on deoxyribonucleic acid (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, the 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 the expression of the TRK component of the fusion protein. An antibody directed against a conserved region may detect fusion or wild-type proteins derived of the TRKA, TRKB, or TRKC proteins.2,3

• Like IHC, FISH probes cannot distinguish fusion variants. FISH assays can often be laborintensive 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 variants.7

 

3. Is there a companion diagnostic assay for selecting NTRK fusion-positive eligible patients? 

To date, NGS tests are in development for this use, but only one is currently approved as a companion diagnostic for the detection of NTRK rearrangements.8 Japan’s Ministry of Health, Labour, and Welfare has granted additional approval of Foundation Medicine’s FoundationOne® CDx Cancer Genomic Profile as a companion diagnostic for entrectinib (ROZLYTREK®), marketed in Japan by Chugai.9

1. What is the role of NGS vs IHC in NTRK testing strategy? 

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 tumours 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 tumours 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

 

2. How is IHC different from NGS? 

IHC is a protein biomarker test that utilizes specific antibodies to detect expression of the TRK component of the fusion protein.2 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 three NTRK genes, as well as the NTRK gene fusion partner and position. However, turnaround time for results with NGS may be longer than with IHC. IHC mainly detects gene expression at the protein level. It doesn’t detect the fusion itself.1,2,4-6,11

 

3. Does IHC miss any NTRK fusions? 

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 

 

4. Does IHC detect NTRK fusions? 

The VENTANA® pan-TRK (EPR17341) assay is a CE-IVD analytic assay intended for the immunohistochemical detection of the C-terminal region of the TRKA, TRKB and TRKC proteins, 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

 

5. What is the sensitivity of IHC?

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

 

6. Is there a scoring algorithm or criteria to determine positivity of IHC?

No, there is no scoring algorithm or criteria to determine IHC positivity. The VENTANA® pan-TRK (EPR17341) assay is a CE-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 chimeric fusion proteins.12,14 

 

1. When should I use RNA- vs DNA-based NGS? 

NGS detects DNA or RNA at nucleotide, exon, and whole-genome levels.RNA-based NGS sequences a small portion of the genome that is transcribed, and, thus, may have a lower cost. In RNA-based NGS, the introns are spliced out of the RNA, which removes the technical limitations of intronic coverage. In addition, detection of RNA-level fusions provides direct evidence that they are functionally transcribed, and analysis of the spliced sequence can determine whether the protein would be translated and in-frame. Fusion transcripts can also be detected with high confidence in the RNA of low tumour purity samples because gene fusions are often highly expressed in the tissue. However, this method relies heavily on the quality and length of the RNA. DNA-based sequencing may provide a more comprehensive characterization of genetic alterations in the genome, but this technique is typically more expensive and has a longer turnaround time.3,10,15-17

 

2. Can I use liquid biopsy to test for NTRK gene fusions?

Liquid biopsy may serve patients with suboptimal or a lack of available tissue samples.18

 

3. Does FoundationOne® liquid report NTRK fusions? 

No, the current version of FoundationOne® liquid does not detect NTRK1, NTRK2, or NTRK3 fusions.19

 

4. What is the difference between FoundationOne® Heme and FoundationOne® CDx?

Foundation Medicine Inc’s FoundationOne® CDx test detects genetic abnormalities in 324 genes as well as genomic signatures, including microsatellite instability (MSI) and tumor mutational burden (TMB). It is FDA-approved as well as CE-marked, and can assist in the identification of patients who may be eligible for treatment with targeted therapies in accordance with the approved therapeutic product labeling. FoundationOne® Heme is validated to detect genomic alterations in more than 400 cancer-related genes. In addition to DNA sequencing, FoundationOne® Heme employs RNA sequencing across more than 265 genes to capture a broad range of gene fusions, common drivers of hematologic malignancies, and sarcomas.8,20,21 We are not promoting or endorsing these products. If more information is needed, we can refer you to the diagnostic manufacturer and their information (eg, websites). 

 

5. Does NGS miss any NTRK fusions? 

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. One disadvantage of DNA-NGS is that DNA-NGS is limited by intron size.1,3,6

1. The prevalence of NTRK rearrangements and expression is low across most tissues. How should I validate the assays? 

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

 

2. What is the control tissue? 

For NGS and RT-PCR, commercial sources exist, such as the Seraseq® FFPE Tumour Fusion RNA Reference Material. Seraseq® is manufactured for use as positive reference standards in molecular assay testing (PCR or NGS) of 15 NTRK gene fusions in adult and pediatric cancer patients. For IHC, examples of positive control tissues for the Ventana® pan-TRK (EPR17341) assay include cerebellum and appendix.13,24

1. Are pediatric patients eligible to be tested using all methodologies?

Yes, pediatric patients are eligible to be tested using all methodologies.10

1. Are there disease state guidelines that recommend NTRK gene fusion testing? 

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

Non−Small Cell Lung Cancer (NSCLC) v6.2022 / Category 2A: The NCCN Panel recommends testing for NTRK1/2/3 gene fusions in all patients with metastatic nonsquamous NSCLC or NSCLC not otherweise specified based on clinical trial data. NTRK1/2/3 gene fusion testing should be considered patients with metastatic squamous cell carcinoma.11

As of December 2022, some additional NCCN panel guidelines that consider NTRK gene-fusion testing as an option include the following: 

  • Rectal Cancer v3.2022 / Category 2A: Recommends NTRK gene-fusion testing in patients with wild-type KRAS, NRAS, and BRAF or with dMMR.25

  • Cutaneous Melanoma v3.2022 / Category 2A: Recommends NTRK gene-fusion testing as an emerging molecular technology for cutaneous melanoma diagnosis and prognostication.26 

  • Occult Primary v2.2023 / 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

  • Colon Cancer v2.2022 / Category 2A: Recommends NTRK gene-fusion testing in patients with wild-type KRAS, NRAS, and BRAF or with dMMR.28

  • Head and Neck Cancers v2.2019 Salivary Gland Tumors / Category 2A: Recommends NTRK gene-fusion status should be checked for salivary ductal carcinomas and adenocarcinomas.29

 

2. Are there specific guidelines for specimen acquisition and management for NTRK gene-fusion testing? 

Although tumour 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. Although testing on cell blocks is not included in the FDA approval for multiple companion diagnostic assays, testing on these specimen types is highly recommended when it is the only or best material available.11

 

1. What is the ESMO algorithm?

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 

 

2. Are there testing algorithms for different tumour types?

From the ESMO working group, it was recommended that in tumours where NTRK fusions are highly recurrent, FISH, RT-PCR, or NGS-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK 1, 2, 3 fusions are uncommon, either frontline sequencing (preferentially RNA-sequencing) or screening by IHC followed by sequencing of positive cases should be pursued.According to Dr Penault-Llorca’s findings in tumours with a high frequency of NTRK gene fusion events, FISH is recommended, with pan-TRK IHC as an alternative if FISH is unavailable. Confirmation by targeted NGS in those cases with positive pan-TRK IHC can be conducted concurrently with treatment considerations. In solid tumours where gene fusions are common, but the frequency of NTRK gene fusions is lower (5%–25%), an NGS panel that includes NTRK fusions is recommended as the preferred test for patients. For tumours with a very low frequency of NTRK gene fusions (<5%), but where molecular screening is common, inclusion of NTRK genes in routine NGS analysis is recommended. For tumours with a low frequency of NTRK fusions, where NGS is not available or is not routinely performed, pan-TRK IHC should be performed for screening with NGS confirmation of positive IHC results.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. 

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