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Detecting NTRK Gene Fusions

A variety of methods can detect NTRK gene fusions and fusion proteins1

Gene fusion detection capabilities vary by method2

Gene fusion detection capabilities and targets for NGS, IHC, FISH, and RT-PCR graphic3-7
Gene fusion detection capabilities and targets for NGS, IHC, FISH, and RT-PCR graphic3-7
Gene fusion detection capabilities and targets for NGS, IHC, FISH, and RT-PCR graphic3-7
Gene fusion detection capabilities and targets for NGS, IHC, FISH, and RT-PCR graphic3-7

NGS is a high-throughput technology capable of sequencing genomic strands in parallel7

  • Allows multiplexing, whereby it can simultaneously interrogate multiple potentially actionable targets in a single sample7,8
  • Detects fusions among any of the 3 NTRK genes (NTRK1, NTRK2, or NTRK3) and a gene partner (known or novel), regardless of breakpoint or fusion partner, depending on library prep method2
    • Hybrid-capture library prep is required to detect novel fusion partners9,10
    • Amplicon-based prep detects only known fusion partners, but may have lower nucleic acid–input requirements11
  • DNA- and RNA-based NGS methods exist; however, RNA-based methods are preferred, as they avoid the difficulties of sequencing large intronic regions in NTRK gene fusions2

IHC is a technique of visualizing proteins in histological tissue sections

  • Detects expression of the TRK protein, potentially driven by an NTRK gene fusion3,12
  • Lacks specificity to be a stand-alone assay; confirmatory NGS should be performed12,13
  • Pan-TRK IHC has ability to detect all 3 fusion proteins–TRKA, TRKB, and TRKC–in addition to wild-type TRK proteins14

FISH is a DNA-based technique that allows for the detection of translocations, amplifications, or deletions on intact chromosomes. Fluorescence associated with break-apart probes allows visualization of the gene fusion within the histological context of the sample4,15

  • Requires 3 sets of break-apart probes (ie, one for each NTRK gene)13
  • Uses specific probe sets for known fusions (eg, ETV6-NTRK3 )13
  • Can detect novel fusion partner if break-apart probes are used13

RT-PCR is targeted amplification of a predefined region of interest on an RNA template. Products are visualized using numerous downstream techniques, including gel electrophoresis or real-time quantitative PCR, that can be used to monitor the response of cancer cells to drugs16:

  • Highly sensitive methodology that already exists in most laboratories16
  • Detects fusion transcript with known partner—novel fusions are not identified13,17
  • Difficult to amplify across large introns16,17

Planning for
your practice

Several methods exist to detect NTRK gene fusions. RNA-based NGS methods may be ideal because they can identify the many known and novel fusion partners and variable structural properties of NTRK gene fusions8

Watch Dr Alexander Lazar explain the distinctions among the key testing methods for NTRK gene fusions

DNA, deoxyribonucleic acid; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid; RT-PCR, reverse-transcription polymerase chain reaction; TRK, tropomyosin receptor kinase.

References: 1. Vaishnavi A, Le AT, Doebele RC. Cancer Discov. 2015;5(1):25-34. 2. Schram AM, Chang MT, Jonsson P, Drilon A. Nat Rev Clin Oncol. 2017;14(12):735-748. 3. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media, LLC; 2014:3-11. 4. O’Connor C. Fluorescence in situ hybridization (FISH). Nat Educ. 2008;1(1):171. 5. Teixidó C, Giménez-Capitán A, Molina-Vila MÁ, et al. RNA analysis as a tool to determine clinically relevant gene fusions and splice variants. Arch Pathol Lab Med. 2018;142:474-479. 6. Kroneis T, Jonasson E, Andersson D, Dolatabadi S, Ståhlberg A. Global preamplification simplifies targeted mRNA quantification. Sci Rep. 2017;7:45219. 7. Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. 8. Kummar S, Lassen UN. Target Oncol. 2018;13(5):545-555. 9. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. 10. Basho RK, Eterovic AK, Meric-Bernstam F. Clinical applications and limitations of next-generation sequencing. Am J Hematol Oncol. 2015;11(3):17-22. 11. Vendrell JA, Taviaux S, Béganton B, et al. Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches. Sci Rep. 2017;7:1-11. 12. Fitzgibbons PL, Cooper K. Immunohistochemistry of biomarkers. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. Molecular Pathology Library Series. New York, NY: Springer Science+Business Media, LLC; 2009:133-137. 13. Church AJ, Calicchio ML, Nardi V, et al. Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy. Mod Pathol. 2018;31(3):463-473. 14. 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. 15. Halling KC, Wendel AJ. In situ hybridization; principles and applications. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. Molecular Pathology Library Series. New York, NY: Springer Science+Business Media, LLC; 2009:109-118. 16. Olsen JL. Polymerase chain reaction. In: Vohr H-W, ed. Encyclopedia of Immunotoxicology. Berlin, Germany: Springer-Verlag; 2016:715-720. 17. Argani P, Fritsch M, Kadkol SS, Schuster A, Beckwith JB, Perlman EJ. Detection of the ETV6-NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin-embedded tissue: application to challenging pediatric renal stromal tumors. Mod Pathol. 2000;13(1):29-36.

An algorithmic approach for NTRK gene fusion detection

Algorithmic approach for NTRK gene fusion detection graphic

*If histology typical, then confirmation by NGS recommended.

CMN, congenital mesoblastic nephroma; FISH, fluorescence in situ hybridization; IFS, infantile fibrosarcoma; IHC, immunohistochemistry; MASC, mammary analogue secretory carcinoma; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; NTRK, neurotrophic tyrosine receptor kinase; SBC, secretory breast cancer; TRK, tropomyosin receptor kinase.

Reference: 1. Penault-Llorca F, Rudzinski ER, Sepulveda AR. Testing algorithm for identification of patients with TRK fusion cancer. J Clin Pathol. 2019 May 09. doi: 10.1136/jclinpath-2018-205679 [Epub ahead of print]

Key considerations for detecting NTRK gene fusions

  • RNA-based NGS is preferred to DNA-based NGS, as it enables distinction of in-frame, transcribed gene fusions vs out-of-frame fusions and avoids sequencing large intronic regions associated with NTRK gene fusions1,2
  • Not all commercially available NGS panels comprehensively cover NTRK1, NTRK2, and NTRK3 gene fusions3,4

Advantages

  • Multiple targets simultaneously analyzed1,5,6
  • Availability of commercial kits that cover gene fusions5
  • RNA-based NGS only identifies actively transcribed chimeric fusions, which is particularly advantageous in unstable cancers and has a faster sequencing process compared to DNA-based NGS1,7,8

Challenges

  • Depending on input template sample, handling protocols could be complex1,5
  • Detection capabilities vary depending on enrichment method (hybrid capture vs amplicon based)1
  • Longer turnaround time1,5,9,10
  • Requires complex bioinformatics1,10

Planning for
your practice

A variety of commercially available NGS kits can be used to efficiently detect fusions across NTRK1, NTRK2, and NTRK3 genes. Sample requirements (DNA vs RNA) and bioinformatic requirements vary among these kits and associated enrichment methods with these kits impact each kit’s detection capabilities11

Testing workflow overview5,12

1 Nucleic acid extraction (from various sample types)

2

Simultaneous amplification of regions of interest (hybrid capture– vs amplicon-based target enrichment)

3

Massively parallel sequencing (instrument reads massive amounts of DNA fragments simultaneously)

4 Sequence analysis (complex bioinformatic analysis)

  • Computer software aligns, filters noise, and compares sequence to reference genome

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid; TRK, tropomyosin receptor kinase.

References: 1. Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. 2. Schram AM, Chang MT, Jonsson P, Drilon A. Nat Rev Clin Oncol. 2017;14(12):735-748. 3. QIAGEN. GeneRead™ QIAact Lung All-in-One UMI Assay [product profile]; 2017. https://amp.qiagen.com/2017/wp-content/uploads/sites/2/2017/11/PROM-11408-001_1108382_PP_GeneRead_QIAact_LungAiO_0917_WW.pdf. Accessed March 3, 2019. 4. QIAGEN. GeneRead™ QIAact Lung RNA Fusion UMI Panel Handbook. April 2018. https://QIAGEN.+GeneRead%E2%84%A2+QIAact+Lung+RNA+Fusion+UMI+Panel+Handbook&oq=QIAGEN.+GeneRead%E2%84%A2+QIAact+Lung+RNA+Fusion+UMI+Panel+Handbook&aqs=chrome..69i57.2540j0j4&sourceid=chrome&ie=UTF-8. Accessed March 3, 2019. 5. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing–based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. 6. Abel H, Pfeifer J, Duncavage E. Translocation detection using next-generation sequencing. In: Kulkarni S, Pfeifer J, eds. Clinical Genomics. New York, NY: Elsevier/Academic Press; 2015:151-164. 7. Li MM, Datto M, Duncavage EJ, et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017;19(1):4-23. 8. Teixidó C, Giménez-Capitán MA, Molina-Vila MÁ, et al. RNA analysis as a tool to determine clinically relevant gene fusions and splice variants. Arch Pathol Lab Med. 2018;142:474-479. 9. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media, LLC; 2014:3-11. 10. 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. 11. Kummar S, Lassen UN. Target Oncol. 2018;13(5):545-556. 12. Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome Med. 2015;7(1):80.

Key considerations for detecting TRK fusion proteins

  • Pan-TRK IHC can detect all 3 fusion proteins–TRKA,TRKB, and TRKC–in addition to wild-type TRK proteins1
  • Strong positive staining for TRK protein is not always due to the presence of an NTRK gene fusion and can have a negative impact on the specificity of the assay performance2
  • Some cancers are associated with high physiological expression of wild-type TRK protein, and are not the result of a gene fusion, thus limiting the usefulness of IHC methodology2,3

Advantages

  • Rapid and inexpensive1,4
  • Well established and widely available within clinical laboratories to support the diagnostic evaluation of cancer1,2

Challenges

  • Typically limited to investigating only one analyte at a time in the clinical setting5
  • Requires experienced and qualified pathologists to correctly evaluate the extent or degree of intensity of the staining to determine whether the test is positive2
  • Lack of standardization is common in preanalytical, analytical, and postanalytical aspects of testing2
  • Low specificity for TRK fusion proteins3

Planning for
your practice

Because IHC measures protein expression, results should be confirmed using NGS to ensure abnormal expression is associated with a fusion protein vs wild-type TRK protein2

Testing workflow overview6

1 Tissue fixed and embedded within paraffin block

  • Only 10% NBF is recommended
  • Fix immediately; delayed time to fixation may result in tissue degradation

2 Sections cut and placed on a microscope slide

  • Cut sections approximately 3-6 μm in thickness
  • Mount on positively charged slides

3 Sections labeled with antibody

4 Sections stained and visualized

  • Immediate staining is recommended, as antigenicity may diminish over time
  • Consult assay for instruction for recommended staining protocol

Using pan-TRK IHC as a screening tool

Pan-TRK IHC detects TRKA, TRKB, and TRKC, but lacks specificity to serve as stand-alone test6

  • IHC detects both wild-type and fusion proteins, thus protein expression may not be directly correlated to the presence of an NTRK gene fusion
  • After a positive TRK IHC test, confirm NTRK gene fusions via a validated molecular test (eg, NGS)

Commercially available assays and antibodies can detect TRK proteins: TRKA, TRKB, TRKC

MANUFACTURER Antibody/Assay Classification/specific
Roche Tissue Diagnostics/Ventana VENTANA pan-TRK (EPR17341) Assay IVD/CE-IVD
Assay detects C-terminal region of TRKA, TRKB, TRKC6
Abcam Anti-TRKA+ TRKB+TRKC [EPR18413] Research use only
Synthetic peptide within Human TRKC amino acid to the C-terminus7
Abcam Anti-TRKA+ TRKB+TRKC [EPR17341] Research use only
Synthetic peptide within Human TRKC amino acid to the C-terminus8
Cell Signaling Technology TRK (pan) (A7H6R) Rabbit mAb Research use only
Rabbit mAb detects endogenous levels of TRKA, TRKB, and TRKC9

Pan-TRK staining varies in intensity and percentage across tumor types3

  • Pan-TRK IHC may be helpful in tumor types with low prevalence of wild-type expression (such as CRC and papillary thyroid) where it can serve as a screening tool to identify NTRK gene fusion-harboring tumors3,6
  • However, for tumors with high prevalence of wild-type TRK protein expression, IHC is less helpful, as staining intensity and percentage for fusion proteins cannot be distinguished from wild-type expression6

PAN-TRK STAINING ACROSS TUMOR TYPES10,*

Ventana Pan-TRK [EPR17341] Assay.

TRK fusion-positive MASC IHC staining image TRK fusion-positive colorectal carcinoma IHC staining image Wild-type TRK expression in HNSCC staining image Wild-type TRK expression in salivary tumor staining image

*Fusion status based upon next-generation sequencing reported from external laboratory-developed test using Oncomine™ Focus Assay.

Planning for
your practice

Pan-TRK IHC can provide a low-cost method to help you screen your patients for TRK protein expression; however, results should be confirmed with NGS to ensure expression is associated with a fusion protein1,4

Watch Dr Erin Rudzinski explain key considerations for using pan-TRK IHC as screening tool for TRK protein expression

Test Your Knowledge

Fill in the blank.
Pan-TRK IHC can provide a ______ to help you screen your patients for TRK protein expression.

HNSCC, head and neck squamous cell carcinoma; IHC, immunohistochemistry; MASC, mammary analogue secretory carcinoma; NBF, neutral buffered formalin; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; NTRK, neurotrophic tyrosine receptor kinase; TRK, tropomyosin receptor kinase.

References: 1. 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. 2. Fitzgibbons PL, Cooper K. Immunohistochemistry of biomarkers. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. Molecular Pathology Library Series. New York, NY: Springer Science+Business Media, LLC; 2009:133-137. 3. Feng J, Ebata K, Hansen F, et al. TRK wild-type and fusion protein expression in solid tumors: characterization by immunohistochemistry and in situ hybridization. Poster presented at: MAP 2018—Molecular Analysis for Personalised Therapy. European Society for Medical Oncology Congress (ESMO); September 15, 2018; Paris, France. 4. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media, LLC; 2014:3-11. 5. Halling KC, Wendel AJ. In situ hybridization: principles and applications. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. Molecular Pathology Library Series. New York, NY: Springer Science+Business Media, LLC;2009:118-127. 6. VENTANA pan-TRK (EPR17341) Assay [product information]. Mannheim, Germany: Roche Diagnostics GmbH: 2018. 7. Abcam [product data sheet]. Anti-TrkA+TrkB+TrkC antibody [EPR18413] ab189903. https://www.abcam.com/pan-trk-antibody-epr18413-ab189903. Accessed February 27, 2019. 8. Abcam [product data sheet]. Anti-TrkA+TrkB+TrkC antibody [EPR17341] ab181560. https://www.abcam.com/pan-trk-antibody-epr17341-ab181560.html. Accessed February 27, 2019. 9. Cell Signaling Technology [product data sheet]. Trk (pan) (A7H6R) Rabbit mAb. https://media.cellsignal.com/pdf/92991.pdf. Accessed February 27, 2019. 10. Ventana Medical System, Inc. VENTANA pan-TRK (EPR17341) Assay: your assay choice matters [brochure]. Tucson, AZ: 2018.

Key considerations for detecting NTRK gene fusions

  • Does not establish if rearrangement results in an oncogenic fusion1
  • Diverse range of potential NTRK gene fusion partners associated with the 3 genes (NTRK1, NTRK2, and NTRK3 ) will make FISH a labor-intensive and inefficient option1
  • Limited biopsy material may present a barrier to detecting all relevant NTRK gene fusions if looking for the novel fusion partner2

Advantages

  • Detects alterations that occur only within a small subset of cells being analyzed1
  • Inexpensive process3
  • FISH as a methodology is well established and widely available within clinical laboratories for copy number variation and known translocation/gene fusions1

Challenges

  • Only detects rearrangements in the genes being evaluated1
  • Break-apart FISH probes may find novel fusion partners, but lack specificity1,4
  • FISH assays that detect a known fusion partner will miss other variants and novel partners1
  • Labor intensive and requires experienced and qualified pathologists1

Planning for
your practice

FISH does not establish if a rearrangement results in a functional fusion, thus results should be interpreted with caution. Due to the range of potential NTRK gene fusion partners, this will become highly labor intensive, as individual reactions for each NTRK gene will be required1

Testing workflow overview2

1 Cells fixed

2 Cells placed on a microscope slide

3 Break-apart/dual-colored probe hybridized to the cells (overnight incubation)

4 Analysis using fluorescence microscope

Watch Drs Jessica Davis and Erin Rudzinski break down the different considerations for NTRK gene fusion detection using FISH testing

FISH, fluorescence in situ hybridization; NTRK, neutrophic tyrosine receptor kinase.

References: 1. Church AJ, Calicchio ML, Nardi V, et al. Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy. Mod Pathol. 2018;31(3):463-473. 2. Halling KC, Wendel AJ. In situ hybridization: principles and applications. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. Molecular Pathology Library Series. New York, NY: Springer Science+Business Media, LLC; 2009:109-118. 3. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media, LLC; 2014:3-11. 4. Cheng L, Zhang S, Wang L, MacLennan GT, Davidson DD. Fluorescence in situ hybridization in surgical pathology: principles and applications. J Path Clin Res. 2017;3(2):73-99.

Key considerations for detecting NTRK gene fusions

  • Methodology already associated with detection of specific NTRK gene fusions, eg, ETV6-NTRK3  1-3
  • Generally requires knowledge of fusion partner and difficult to amplify across large introns4,5

Advantages

  • Rapid and sensitive test4
  • Assay can be multiplexed to cover a range of mutations within a single reaction4
  • Well established within molecular genetic laboratories for quantitatively monitoring therapy response4

Challenges

  • Assay probes have to be designed for each specific fusion combination1,5
  • Potential presence of PCR inhibitors in the biologic sample4
  • RT-PCR is susceptible to false positives due to RNA contamination4

Planning for
your practice

RT-PCR is already used to detect specific fusion combinations associated with certain cancer indications; however, it cannot be used for the detection of novel fusion partners1

Testing workflow overview4,6

1 RNA extraction and ctDNA synthesis (from various sample types and platforms)

2 RT-PCR reaction (counts normal and mutant copies amplified in real time, ie, as the PCR reaction takes place)

ctDNA, chloroplast deoxyribonucleic acid; NTRK, neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; RNA, ribonucleic acid; RT-PCR, reverse-transcription polymerase chain reaction.

References: 1. Church AJ, Calicchio ML, Nardi V, et al. Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy. Mod Pathol. 2018;31(3):463-473. 2. 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. 3. Vaishnavi A, Le AT, Doebele RC. Cancer Discov. 2015;5:25-34. 4. Olsen JL. Polymerase chain reaction. In: Vohr H-W, ed. Encyclopedia of Immunotoxicology. Berlin, Germany: Springer-Verlag; 2016:715-720. 5. Argani P, Fritsch M, Kadkol SS, Schuster A, Beckwith JB, Perlman EJ. Detection of the ETV6-NTRK3 chimeric RNA of infantile fibrosarcoma/cellular congenital mesoblastic nephroma in paraffin-embedded tissue: application to challenging pediatric renal stromal tumors. Mod Pathol. 2000;13(1):29-36. 6. Peter M, Gilbert E, Delattre O. A multiplex real-time PCR assay for the detection of gene fusions observed in solid tumors. Lab Invest. 2001;81:905-912.