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Optimizing NGS for NTRK  Gene Fusions

Identifying NTRK gene fusions with NGS

Many commercially available NGS kits can detect NTRK gene fusions. NGS enables sequencing of multiple nucleic acid targets in a rapid and massively parallel manner.1 This method is ideal because it addresses the unique multiple partner and variable structural properties of NTRK gene fusions.2

NGS can address the structural characteristics of NTRK gene fusions; thus, many commercially available kits for NTRK gene fusion detection are NGS-based2,3

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

References: 1. Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome Med. 2015;7(1):80. 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. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696.

RNA-based NGS methods are preferred for the detection of NTRK gene fusions1-3

  • RNA-based NGS sequences transcribed and spliced mature mRNAs (with introns already removed)3
  • Thus, RNA-based NGS avoids the difficulties associated with sequencing through challenging features of NTRK gene fusions and is able to identify in-frame fusion products2,3,5

General NGS

  • High sensitivity and specificity potential1,2
  • Multiplexing: simultaneously queries multiple potentially actionable targets4 (eg, ALK, ROS1, RET)
  • Detects both known and novel fusions, regardless of breakpoint or fusion partner (depending on the library prep method)2,5

RNA-Based NGS

  • Able to distinguish in-frame, transcribed gene fusions vs out-of-frame fusions2,5
  • Avoids difficulties of sequencing large intronic regions in the NTRK genes3

Planning for
your practice

Appropriately designed RNA-based NGS provides optimal method to detect NTRK gene fusions since no prior knowledge of fusion breakpoint and/or fusion partner is required and addresses the unique structural challenges present in NTRK gene fusions3

Watch Dr Andrew Turk discuss the considerations for using RNA- vs DNA-based NGS testing to detect NTRK gene fusions

Test Your Knowledge

Why are RNA-based NGS methods preferred to other methods for NTRK gene fusion detection?

ALK, anaplastic lymphoma kinase; DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; mRNA, messenger ribonucleic acid; RET, Ret proto-oncogene; RNA, ribonucleic acid; ROS1, ROS proto-oncogene 1.

References: 1. 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(7);25:513-523. 2. 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. 3. 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. 4. Farago AF, Azzoli CG. Beyond ALK and ROS1: RET, NTRK, EGFR and BRAF gene rearrangements in non-small cell lung cancer. Transl Lung Cancer Res. 2017;6(5):550-559. 5. Schram AM, Chang MT, Jonsson P, Drilon A. Nat Rev Clin Oncol. 2017;14(12):735-748.

There are 5 common NGS approaches for sequence data collection1-4

Choice of sequencing methodology is determined by the genomic regions and variant types of interest. Subsequently, this informs selection of NGS platform, NGS panel, bioinformatic analysis pipeline, and lab resources to support in-house verification and validation of NGS testing.3

Whole-genome sequencing

  • Sequences the majority of the genome, including coding (exons) and noncoding (introns) genomic regions5
  • Example: Illumina Nextera DNA Flex Library Prep Kit for whole-genome sequencing6,7

Whole-exome sequencing

  • Targets the coding (exon) region of an individual’s genome5
  • Example: Thermofisher Ion AmpliSeq™ Exome RDY Kit8

Whole-transcriptome sequencing

  • Targets mRNA transcripts. Enables determination of expression, quantity, and presence of alterations in the nucleic acid sequence of an individual’s genome5
  • Example: Illumina RNAseq Total Transcriptome Kit9,10

Targeted DNA/RNA sequencing

  • Targets selected genes of interest, focusing on those that are clinically meaningful and actionable (eg, EGFR, BRAF, KRAS, etc) for genomic alterations that include SNVs, indels, CNVs, and fusions5
  • Example: ThermoFisher Oncomine Comprehensive Assay11,12

Targeted hotspot sequencing

  • Targets frequently mutated “hotspot” regions of genes to detect well-known SNVs, indels, and gene fusions (if breakpoints and partners are known)13
  • Example: ThermoFisher Ion AmpliSeq™ Cancer Hotspot Panel v2 (does not include NTRK1, NTRK2, NTRK3)14-16

Planning for
your practice

Targeted RNA-based sequencing, or whole-exome/transcriptome sequencing are preferred, as they may be more suited to address some of the challenges in detecting NTRK gene fusions5

Watch Dr Alexander Lazar delineate the key distinctions among 5 common NGS approaches for sequence data collection

BRAF, v-raf murine sarcoma viral oncogene homolog B1; CNVs, copy number variations; DNA, deoxyribonucleic acid; EGFR, endothelial growth factor receptor; KRAS; Kirsten rat sarcoma viral oncogene homolog; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; mRNA, messenger ribonucleic acid; RNA, ribonucleic acid; SNV, single nucleotide variant.

References: 1. Wang Q, Xia J, Jia P, Pao W, Zhao Z. Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives. Brief Bioinform. 2013;14(4):506-519. 2. Dong L, Wang W, Li A, et al. Curr Genomics. 2015;16(4):253-263. 3. 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. 4. Basho RK, Eterovic AK, Meric-Bernstam F. Clinical applications and limitations of next-generation sequencing. Am J Hematol Oncol. 2015;11(3):17-22. 5. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. 6. Illumina. Nextera DNA Flex Library Prep Kit. https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-dna-flex.html. Accessed March 27, 2019. 7. Illumina. TruSight HLA v2 Sequencing Panel. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-hla-v2-data-sheet-070-2016-002.pdf. Accessed March 27, 2019. 8. Thermo Fisher Scientific. Ion AmpliSeq™ Exome RDY Kit 4X2. https://www.thermofisher.com/order/catalog/product/A38264. Accessed March 27, 2019. 9. Illumina. Study gene expression using RNA sequencing. https://www.illumina.com/techniques/sequencing/rna-sequencing.html. Accessed March 27, 2019. 10. Illumina. A comprehensive picture of the transcriptome. https://www.illumina.com/techniques/sequencing/rna-sequencing/total-rna-seq.html. Accessed March 27, 2019. 11. Ion Torrent. Oncomine comprehensive assay v3 flyer. https://tools.thermofisher.com/content/sfs/brochures/oncomine-comprehensive-assay-v3-flyer.pdf. Accessed March 27, 2019. 12. Thermo Fisher Scientific. Ion Torrent. Oncomine comprehensive assays. https://www.thermofisher.com/us/en/home/clinical/preclinical-companion-diagnostic-development/oncomine-oncology/oncomine-cancer-research-panel-workflow.html. Accessed March 27, 2019. 13. Oliveira DM, Mirante T, Mignogna C, et al. Simultaneous identification of clinically relevant single nucleotide variants, copy number alterations and gene fusions in solid tumors by targeted next-generation sequencing. Oncotarget. 2018;9(32):22749-22768. 14. Thermo Fisher Scientific. Ion AmpliSeq cancer hotspot panel v2. https://www.thermofisher.com/order/catalog/product/4475346. Accessed March 27, 2019. 15. Thermo Fisher Scientific. Ion AmpliSeq cancer hotspot panel v2. https://assets.thermofisher.com/TFS-Assets/LSG/brochures/Ion-AmpliSeq-Cancer-Hotspot-Panel-Flyer.pdf. Accessed March 27, 2019. 16. Thermo Fisher Scientific. Ion Torrent. Ion AmpliSeq panels for focused next-generation sequencing flyer. https://assets.thermofisher.com/TFS-Assets/CSD/Flyers/flyer-ampliseq-ampliseq-hd.pdf. Accessed March 27, 2019.

Optimizing NGS workflow at every step is required to enable robust testing and reporting1

Nucleic acid extraction graphic

Preanalytic phase

1 Nucleic Acid Extraction: Sample

  • Using tissue from biopsy or blood sample, obtain purified DNA or RNA to be enriched and sequenced1

Analytic phase

2 Library Prep: Panel

  • Select genes/regions to sequence (from a few to hundreds)1
  • Perform target enrichment (via hybrid capture or PCR) to select and amplify regions of interest1
  • Perform clonal amplification (if required) to ensure sufficient material for successful sequencing1

3 Sequencing: DNA Sequencer

  • Determines the sequence of nucleotides in the nucleic acid sample tested1

Postanalytic phase: Bioinformatics

4 Analysis

  • Separates unique patients by barcode2
  • Aligns sequence reads to a reference sequence1
  • Filters noise, repeated sequences1
  • Identifies variations1
  • Determines what is potentially clinically meaningful vs usual human variation1

5 Annotation and Report Generation

Planning for
your practice

Understanding the stepwise approach outlined in this section can help streamline the NGS testing process to optimize conditions for NTRK gene fusion detection1

Explore the journey for RNA-based NGS testing at the Providence St. Joseph Health Molecular Genomics Laboratory

Test Your Knowledge

Which of the following are key steps in the NGS workflow?

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; RNA, ribonucleic acid.

References: 1. 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. 2. Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med. 2015;139(4):481-493.

Detection of genomic alterations using NGS is significantly affected by quality of starting sample material1

It is critical for the anatomical pathologist to assess tumor samples under a microscope before the sample can be accepted for NGS testing. During this time, it’s also important that the sample is enriched by macro- or microdissection techniques, even when the relative proportion of tumor nuclei is prevalent at high frequency.1

Key considerations for sample handling

  • Heterogeneity
    • Ensuring sufficient tumor cell content compared to normal cells1
    • Differing subclone populations may result in different mutation combinations
  • Size of sample1
    • Biopsying technique may limit amount of DNA/RNA available1
  • Preprocessing
    • Formalin fixation can create random artificial mutations that may result in false positives when using a highly sensitive technique like NGS1

Planning for
your practice

Preanalytical review of the sample and tumor enrichment by microdissection helps ensure sufficient tumor for analysis and increases sensitivity for the gene alteration1

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; RNA, ribonucleic acid.

Reference: 1. 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.

Library prep is a critical step in the NGS process1

Library prep generates a pool of sequencing targets representing areas of interest ready for sequencing on an appropriate NGS platform. Target enrichment is a key component of this step. During target enrichment, DNA or RNA isolated from a sample is enriched for regions of interest and formatted for sequencing on the chosen platform.1

Choice of target enrichment method during library prep is a key consideration when detecting NTRK gene fusions2,3

Two potential methods for target enrichment are amplification and hybridization. Both methods have unique attributes that can influence their ability to optimally detect various genomic alterations.1

Amplification vs Hybridization-based Capture
Amplification Hybridization-Based Capture
Variants Detected
  • Best suited for detecting known variants of interest (SNVs or indels)4
  • Can include known, specific gene fusions (eg, EML4-ALK) and CNVs1
  • Potential for broad detection of all actionable mutations (CNVs, SNVs, indels, and fusions) depending on assay design, potentially including novel gene fusion partners1
Benefits
  • Commonly used for small panels; ideal for SNVs, indels, and known fusions1
  • Requires low input of nucleic acid1
  • Can detect larger number of genes and genomic regions1
  • Sensitivity for detecting fusions with novel fusion partners1
Drawbacks
  • Requires complex amplicon design to sufficiently cover all genomic alterations including gene fusions1
  • Requires more input of nucleic acid than amplification-based methods1

Planning for
your practice

Hybridization-based capture approach may be better than amplicon-based approach as this method can detect a broad range of NTRK gene fusions (known and novel)1-3,5

Clonal amplification generates required copies of templates of interest for accurate sequencing1,6

Depending on NGS platform, clonal amplification may be required to ensure sufficient material for successful sequencing.1

Clonal amplification approach with Thermo Fisher Ion Personal Genome Machine, Thermo Fisher Proton, or Qiagen GeneReader6-8

Clonal amplification approach of emulsion, on-bead amplification, and final product graphic

Clonal amplification approach with Illumina MiSeq or Illumina NextSeq6,9

Clonal amplification approach of template binding step, bridge PCR amplification, and cluster generation graphic

No clonal amplification needed with PacBio RS 1/II, Oxford nanopore, or GridlON6,10,11

Circular DNA graphic Circular DNA graphic and Hairpin DNA graphic

Current NGS technologies employ different sequencing methods6

Sequencing by synthesis using serial DNTP flow (Thermofisher Scientific): Semiconductor sequencing6

1 Template-enriched beads are carefully arrayed into a microtitre plate chip6

2

Each of the 4 DNA nucleotide species is added iteratively to ensure only 1 dNTP is responsible for the signal6

3

As each base is incorporated, a small unit change in pH is detected by an integrated CMOS–ISFET sensor device6

4

The pH change detected by the sensor is proportional to the number of nucleotides detected6

5

After signal detection and recording, unincorporated bases are washed away and the next one is added to repeat the process6

Sequencing by synthesis using REVERSIBLE TERMINATORS (ILLUMINA AND QIAGEN)6

1

Primers, DNA polymerase, and modified nucleotides added to template-enriched clusters (Illumina) or beads (Qiagen) on flow cell6

2 Nucleotide addition

  • All 4 uniquely fluorophore-labeled, terminally blocked nucleotides added and hybridize to complementary base. Each cluster incorporates a different base (Illumina)6 or
  • All 4 uniquely fluorophore-labeled, terminally blocked and unlabeled, blocked nucleotides added and hybridize to complementary base. Each bead incorporates a different base (Qiagen)6

3 Imaging

  • Slides are imaged with 2 or 4 laser channels. Each cluster emits color corresponding to base incorporated during cycle (Illumina)6 or
  • Slides are imaged with 4 laser channels. Each bead emits color corresponding to base incorporated during cycle (Qiagen)6

4 Cleavage

  • Fluorophores cleaved and washed away. A new cycle begins with the addition of new nucleotides6

NGS platform considerations12-18

MANUFACTURER METHOD INSTRUMENT RUN TIME READ LENGTH OUTPUT (Gb)
Illumina Bridge PCR
Chemistry: hybridization
Sequence by synthesis
Paired end sequencing
MiSeq v2
MiSeq v3
NextSeq
HiSeq
~39 hours
~56 hours
≤35 hours
6 days
2x250
2x300
2x150
2x125
7.5-8.5
13.2-15
≥90
450-500
ThermoFisher Emulsion PCR
Semiconductor sequencing
Single end sequencing
Ion PGM (CE/IVD)
Ion Proton
Ion S5
5 hours
2.5 hours
12 hours
400
200
400
0.6-1
15
1.2-2
Qiagen Emulsion PCR
Sequencing by synthesis
Single end sequencing
GeneReader 30 hours 134 1

Planning for
your practice

To ensure a testing method is most appropriate for your existing laboratory workflow, considerations for choosing an NGS platform and a commercial kit should be made together

Watch Dr Michelle Shiller compare target enrichment techniques for NTRK gene fusion detection

Test Your Knowledge

Fill in the blank.
____________ requires more input of nucleic acid than amplification-based methods.

CNV, copy number variations; DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; RNA, ribonucleic acid; SNV, single nucleotide variant; ssDNA, single-strand DNA.

References: 1. 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. 2. 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. 3. Kummar S, Lassen UN. Target Oncol. 2018;13(5):545-556. 4. 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):1-11. 5. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. 6. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genetics. 2016;17:333-351. 7. Platform manufacturer website: ThermoFisher Scientific. https://www.thermofisher.com/us/en/home/applications-techniques.html. Accessed February 27, 2019. 8. Platform manufacturer website: Qiagen. https://www.qiagen.com/us/products/ngs/ngs-life-sciences/dna-amplicon-sequencing/. 9. Platform manufacturer website: Illumina. https://www.illumina.com/techniques/sequencing/dna-sequencing.html. Accessed February 27, 2019. 10. Platform manufacturer website: PacBio. https://www.pacb.com/applications/rna-sequencing/. Accessed February 27, 2019. 11. Platform manufacturer website: Oxford Nanopore. https://nanoporetech.com/applications/basic-genome-research. Accessed February 27, 2019. 12. Platform manufacturer website: Illumina. Specifications for the MiSeq system. Cluster generation and sequencing. https://www.illumina.com/systems/sequencing-platforms/miseq/specifications.html. Accessed February 26, 2019. 13. Platform manufacturer website: Illumina. System specifications for NextSeq 550Dx. Performance parameters. https://www.illumina.com/systems/sequencing-platforms/nextseq-dx/specifications.html?langsel=/us/. Accessed February 26, 2019. 14. Platform manufacturer website: Illumine. Performance specifications for the HiSeq 2500 system. High output run mode. https://www.illumina.com/systems/sequencing-platforms/hiseq-2500/specifications.html. Accessed February 26, 2019. 15. Platform manufacturer website: ThermoFisher Scientific. Ion PGM™ Sequencer Specifications/Ion GM System specifications. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-pgm-system-for-next-generation-sequencing/ion-pgm-system-specifications.html. Accessed February 26, 2019. 16. Platform manufacturer website: ThermoFisher Scientific. Ion Proton™ Sequencer Specification. Ion Proton System performance specifications with Ion PI Chip. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-proton-system-for-next-generation-sequencing/ion-proton-system-specifications.html. Accessed February 26, 2019. 17. Platform manufacturer website: ThermoFisher Scientific. Ion GeneStudio S5 Spec. Flexibility in throughput and workflow for a broad range of NGS applications. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-s5-ngs-targeted-sequencing/ion-s5-specifications.html. Accessed February 26, 2019. 18. Platform manufacturer website: DeciBio. With the GeneReader launch Qiagen offers a true end-to-end clinical NGS solution.https://www.decibio.com/2015/11/06/with-the-genereader-launch-qiagen-offers-a-true-end-to-end-clinical-ngs-solution/. Accessed February 26, 2019.

Bioinformatics: Analyzing, interpreting, and reporting NGS data

Multiple bioinformatic tools available from sequencing providers and third-party vendors are evaluated, selected, and then integrated to create a validated bioinformatic process for NGS that allows for the discovery and identification of gene fusions.1

Bioinformatics workflow: Key processes for accurate gene fusion detection1

  • Sequencing data: Platform-specific DNA sequences determined and demultiplexed to produce pool of patient specific-sequence reads1
  • Alignment: Patient-specific sequence reads assembled or mapped by comparing to a control (reference) DNA genome sequence1
  • Variant calling: DNA base pairs in the reads or segments of the reads different from the reference are noted as variants1
  • Variant annotation: Variants are compared against public or proprietary databases to determine biological relevance1
  • Variant interpretation: Relevant variants compared against public or proprietary databases to determine and add clinical context to the annotation1
  • Sequencing report: Provides key findings and actionable steps to end-user (ie, triage patients to a clinical trial or prescribe a targeted therapy)1

Planning for
your practice

Choosing the appropriate bioinformatics tools directly impacts the diagnostic utility of sequencing by NGS. For this reason, sequencing data should be analyzed with bioinformatics processes that are optimized for the identification of NTRK gene fusions2

Experimental and bioinformatic considerations for carrying out RNA-based NGS

Number of reads

  • Total number of RNA transcript sequence reads should be optimized for system under study to support gene fusion detection3

Length of reads

  • Paired-end sequencing and longer sequence reads provide data that accumulate overlapping coverage with higher specificity to fused genes during alignment3
  • This can be problematic with FFPE samples that generally contain fragmented DNA/RNA3

Filtering of reads

  • Reads originating from highly expressed genes that are unlikely to be involved in gene fusions, such as ribosomal RNA, must be anticipated and filtered out appropriately3

Reads ratio

  • Low ratio of chimeric to wild type reads in the vicinity of the fusion boundary may indicate spurious mismapping of reads3

How to detect gene fusions

  • Highly promising fusion-sequence predictions evident if sequences in the read strongly align to sequences in 1 of the genes that constitute the gene fusion3
  • Gene fusions usually follow splicing patterns of wild-type genes; thus, their detection at exon boundaries that match those of the wild-type gene(s) is another powerful indicator of gene fusion sequence identification3

Planning for
your practice

Chosen library type should be supportive of laboratory sample batching considerations and be able to comprehensively detect gene fusions (known/novel)3

NGS test reporting

A clear and concise report is crucial to ensure immediate clinical use to the ordering physician4

The report should contain all the information required for the ordering clinician to know what was tested and the results obtained from the test. Incomplete or unclear representation of the data can lead to clinical errors and incorrect patient management.4

The report should contain all the information required for the ordering clinician to know what was tested and the results obtained from the test. Incomplete or unclear representation of the data can lead to clinical errors and incorrect patient management.4

  • All clinically critical information should be clear and concise and displayed prominently at the beginning of the report, to increase the likelihood that it will be seen and understood
  • Using graphs, charts, and tables may help increase clarity of the report4
  • Recommendations should be made where possible when based on clinical evidence and appropriately cited4
  • Reports should not be limited to positive findings. Pertinent negatives are equally important in clinical decisions and, thus, should also be reported4
  • Report any additional preanalytical, analytical, or postanalytical testing variables, as these can provide context for some test results and influence the result’s clinical interpretation4

In addition, details about the testing methodology should be presented at the bottom of the report, including description of the method, assay performance characteristics, and critical quality metrics for the assay run. Unless the entire gene is tested, specific gene loci, exons, or hot spots tested should be listed in the final report.4

Review a sample test report5

NGS sample test report image
Detected genomic alterations should be classified under a 4-tiered system4
Tier I

Variants of strong clinical significance

  • Therapies approved by the FDA, and professional guidelines, or on well-powered clinical studies with expert consensus
  • Diagnostic or prognostic biomarker in specific tumor types, based on professional guidelines or well-powered clinical studies with expert consensus
Tier II

Variants of potential clinical significance

  • Therapies based on FDA-approved therapies for different tumor types or investigational therapies
  • Based on multiple small studies with some consensus
  • Preclinical trials or a few case reports without consensus
Tier III

Variants of unknown clinical significance

  • None or no convincing evidence to determine clinical/biological significance
Tier IV

Variants of known insignificance

  • No existing published evidence of cancer association
  • Observed at significant allele frequencies in the general population or a specific subpopulation
  • Tier I–III alterations must be reported in descending order of clinical importance4
  • Clinically useful interpretations should be provided for tier I and II variants to inform disease management decisions4
  • Interpretations for tier III variants should be brief, bearing in mind the goal to keep critical information clear and concise4
  • Tier IV alterations are benign or likely benign; therefore, it is recommended that they not be reported4

Treatment decisions are multifaceted and often based on several factors that are unknown to the molecular pathologist. Therefore, recommendations in an NGS test report should be relevant to the patient’s cancer diagnosis and contain language clarifying that the recommendations are based on data available to the laboratory, but that additional factors need to be considered to create a treatment plan for each individual.4

Nomenclature

Detected genomic alterations should be annotated and reported by the HUGO Gene Nomenclature Committee. Gene fusions should be reported listing both gene partners separated by a dash. For example, a gene fusion between the NTRK1 gene and the partner gene TPM3 should be reported as TPM3-NTRK1 4

Use of standard nomenclature does not outweigh the goal of clear communication. Use colloquial nomenclature in addition to standard nomenclature, as needed, to clearly communicate results to the clinical provider.4

Other reporting elements

The report should also contain any information that can help provide a comparison with other results from the patient over time or provide additional context for the reported results, including4

  • Genomic coordinates
  • Genome build
  • Transcript reference sequence
  • Pertinent negatives
  • Sequencing coverage cutoff for the NGS assay used
  • Genes and/or hotspots that did not meet the sequencing coverage criteria should be declared as failed

Role of the Multidisciplinary Molecular Tumor Board

With rapid advancements in sequencing technology, implementing multidisciplinary molecular tumor board may be crucial to help translate genomic alteration findings into evidence-based recommendations.6 Diverse perspectives provide a multidisciplinary evaluation of the patient, as well as ensure expert interpretation of complex genomic data, especially when using whole-exome or whole-genome sequencing.6,7 Thus, the molecular tumor board should invite collaboration from multiple disciplines, including clinicians with varying specialties (eg, oncologists, hematologists, pathologists, geneticists, molecular biologists, and bioinformaticians).6,7

Molecular tumor boards can provide education, facilitate interpretation, and implementation of precision medicine by6

  • Improving clinician’s understanding of assay strengths, limitations, and results
  • Increasing clinician’s confidence in and efficiency of utilizing molecular diagnostics
  • Providing information about laboratory updates, analysis software, and challenges in data interpretation and utilization

Planning for
your practice

Clear and actionable communication is paramount when reporting NGS results to the clinical provider4

Test Your Knowledge

Select all that apply.
Which of the following are key bioinformatics processes for accurate gene fusion detection?

cDNA, complementary DNA; DNA, deoxyribonucleic acid; FDA, US Food and Drug Administration; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid.

References: 1. Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med. 2015;139(4):481-493. 2. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. 3. Conesa A, Madrigal P, Tarazona S, et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17(13):1-19. 4. 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. 5. Amatu A, Sartore-Bianchi A, Siena S. ESMO Open. 2016;1:e000023. 6. van der Velden DL, van Herpen CML, van Laarhoven HWM, et al. Molecular Tumor Boards: current practice and future needs. Ann Oncol. 2017;28(12):3070-3075. 7. Knepper TC, Bell GC, Hicks JK, et al. Key lessons learned from Moffitt’s molecular tumor board: the Clinical Genomics Action Committee experience. Oncologist. 2017;22(2):144-151.

A variety of factors need to be considered when deciding which commercial kit should be implemented within a lab setting

Assay considerations to support the decision-making process when choosing a commercial kit

  • Platform availability and cost1
  • Manufacturer technical support1
  • Assay requirements, ie, number of genes and the extent of gene coverage1

Laboratory-specific considerations to ensure implementation of efficient workflows

  • In-house technical expertise1
  • Expected testing volume and scalability1
  • Required result turnaround time1

Commercially available NGS kits2-26

Vendor Kit Name Sample # of Genes Included Library Prep Method DNA/RNA NTRK 1, 2,
AND 3
Unknown Fusion Partners Integrated Bioinformatics Recommended Sequencer
Illumina®
TruSight™
Oncology 500
FFPE 523 Hybrid Capture DNA/RNA Y Y Variant Calling + Interpretation/ Reporting
(powered by PierianDx)
NextSeq®
TruSight®
Tumor 170
FFPE 170 Hybrid Capture DNA/RNA Y Y Variant Calling + Interpretation/ Reporting
(powered by PierianDx)
NextSeq®/
HiSeq
TruSight™ RNA Fusion Panel FFPE, bone
marrow
507 Hybrid Capture RNA Y Y Variant Calling MiniSeq™/
MiSeq™/
NextSeq®
TruSight®
RNA Pan-Cancer Panel
FFPE 1385 Hybrid Capture RNA Y Y Variant Calling MiniSeq™/
MiSeq™/
NextSeq®
Thermo Fisher
Ion Torrent™
Oncomine™
Focus Assay
FFPE 52 Amplicon DNA/RNA Y N Ion Reporter/
Oncomine™ Report
Ion PGM®
Dx System
Ion Torrent™
Oncomine™
Comprehensive
Assay v3
FFPE 161 Amplicon DNA/RNA Y N Ion Reporter/
Oncomine™
Report
Ion Chef™ and
Ion S5™
Systems
Ion Torrent™
Oncomine™
Childhood
Cancer
Research Assay
FFPE, blood
and bone
marrow
203 Amplicon DNA/RNA Y N Ion Reporter/
Oncomine™
Report
Ion GeneStudio™
S5/Ion Chef™
Archer DX
Archer®
FusionPlex®
Solid Tumor Kit
Fresh frozen
or FFPE
53 Anchored
Multiplex PCR
RNA Y Y Archer
Analysis
Illumina®/
Ion Torrent™
Archer®
FusionPlex®
Oncology
Research Kit
Fresh frozen
or FFPE
74 Anchored
Multiplex PCR
RNA Y Y Archer
Analysis
Illumina®/
Ion Torrent™
Archer®
FusionPlex®
Sarcoma Kit
Fresh frozen
or FFPE
26 Anchored
Multiplex PCR
RNA NTRK3
Only
Y Archer
Analysis
Illumina®/
Ion Torrent™
Archer®
FusionPlex®
CTL Kit
Fresh frozen
or FFPE
36 Anchored
Multiplex PCR
RNA Y Y Archer
Analysis
Illumina®/
Ion Torrent™
Archer®
FusionPlex®
Lung Kit
Fresh frozen
or FFPE
14 Anchored
Multiplex PCR
RNA Y Y Archer
Analysis
Illumina®/
Ion Torrent™
Qiagen
GeneRead
QIAact Lung
DNA UMI
Panel
FFPE/
liquid
biopsy
19 UMI/PCR DNA NTRK1
Only
N Qiagen
Clinical
Insight
GeneReader
GeneRead
QIAact Lung
RNA Fusion UMI
Panel
FFPE/
liquid
biopsy
19 UMI/PCR RNA NTRK1
Only
N Qiagen
Clinical
Insight
GeneReader

Information current as of February 2019.
Contact the laboratory vendors for more information. This list may not represent all tests for NTRK1, NTRK2, and NTRK3 gene fusions.
All kits listed above are for research use only (RUO).

Review important considerations for selecting a commercial NGS kit with Dr Jessica Davis

DNA, deoxyribonucleic acid; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; RNA, ribonucleic acid; NTRK, neurotrophic tyrosine receptor kinase.

References: 1. Jennings LJ, Arcila ME, Croless 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. 2. Illumina. TruSight Oncology 500 DNA/RNA Kit. https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500.html. Accessed February 27, 2019. 3. Illumina. TruSight Tumor 170 Datasheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-tumor-170-data-sheet-1170-2016-017.pdf. Accessed February 27, 2019. 4. Illumina. TruSight RNA fusion panel. https://www.illumina.com/products/by-type/clinical-research-products/trusight-rna-pan-cancer.html. Accessed February 27, 2019. 5. Illumina. NextSeq 550 data sheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/nextseq-550-system-spec-sheet-770-2013-053.pdf. Accessed March 1, 2019. 6. Illumina. TruSight RNA Pan-Cancer Panel. https://www.illumina.com/products/by-type/clinical-research-products/trusight-rna-pan-cancer.html. Accessed March 1, 2019. 7. Illumina. TruSight RNA Pan-Cancer Panel Datasheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-rna-pan-cancer-data-sheet-1170-2015-004.pdf. Accessed February 28, 2019. 8. lllumina. TruSight RNA Pan-Cancer Sequencing Panel Gene List. https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_pan_cancer.xlsx. Accessed February 27, 2019. 9. ThermoFisher. Oncomine Focus Assay, 318 Solution. https://www.thermofisher.com/order/catalog/product/A28548. Accessed February 28, 2019. 10. ThermoFisher. Oncomine Focus Assay White Paper. https://tools.thermofisher.com/content/sfs/brochures/oncomine-focus-assay-performance-white-paper.pdf. Accessed February 28, 2019. 11. ThermoFisher. Oncomine Comprehensive Assay v3 flyer. https://assets.thermofisher.com/TFS-Assets/LSG/brochures/oncomine-comprehensive-assay-v3-flyer.pdf. Accessed February 28, 2019. 12. ThermoFisher. Oncomine Childhood Cancer Research Assay. https://www.thermofisher.com/us/en/home/clinical/preclinical-companion-diagnostic-development/oncomine-oncology/oncomine-childhood-research-assay.html. Accessed February 27, 2019. 13. ThermoFisher Cancer Genomics Research Brochure. https://tools.thermofisher.com/content/sfs/brochures/cancer-genomics-research-brochure.pdf. Accessed February 27, 2019. 14. ArcherDX. Archer FusionPlex NGS Assays. http://archerdx.com/fusionplex-assays/. Accessed February 28, 2019. 15. ArcherDX. Archer FusionPlex Solid Tumor Kit. http://archerdx.com/fusionplex-assays/solid-tumor. Accessed February 28, 2019. 16. ArcherDX. Archer FusionPlex Solid Tumor Kit Product Insert. http://info.archerdx.com/acton/attachment/17223/f-05ae/1/-/-/l-0014/l-0014:2353/LA179-Product-Insert-FusionPlex-Solid-Tumor.pdf?nc=1. Accessed March 2, 2019. 17. ArcherDX. Archer FusionPlex Oncology Research Kit. http://archerdx.com/fusionplex-assays/oncology-research. Accessed March 4, 2019. 18. ArcherDx. Archer FusionPlex Oncology Research Kit Product Insert. http://info.archerdx.com/acton/attachment/17223/f-05b0/1/-/-/l-0014/l-0014:2356/LA181-Product-Insert-FusionPlex-Oncology-Research.pdf?nc=1. Accessed February 28, 2019. 19. Archer FusionPlex Sarcoma Kit. https://archerdx.com/fusionplex-assays/sarcoma. Accessed March 4, 2019. 20. Archer FusionPlex Sarcoma Kit Product insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA168.B%20Product%20Insert,%20FusionPlex%20Sarcoma.pdf. Accessed February 28, 2019. 21. Archer FusionPlex CTL Kit. https://archerdx.com/fusionplex-assays/ctl-rna. Accessed February 28, 2019. 22. Archer FusionPlex CTL Kit. Product Insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA180.B%20Product%20Insert,%20FusionPlex%20CTL.pdf. Accessed February 28, 2019. 23. Archer FusionPlex Lung Kit. Product Insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA671.B%20Product%20Insert,%20FusionPlex%C2%AE%20Lung,%20SK0133.pdf. Accessed March 2, 2019. 24. Archer FusionPlex Lung Kit. Protocol. http://cdn2.hubspot.net/hubfs/4445440/Protocols/LA135.F%20Protocol,% 20Archer%20FusionPlex%20Kits%20for%20Illumina.pdf. Accessed March 2, 2019. 25. Qiagen. GeneRead QIAact Lung DNA UMI Panel Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=94ab92d2-1918-4388-989b-4cefa8eed203&lang=en. Accessed March 4, 2019. 26. Qiagen. GeneRead QIAact Lung RNA Fusion UMI Panel Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=1a71d98a-c45c-44fa-b4af-874cd1d2b61f&lang=en. Accessed March 4, 2019.

NTRK, neurotrophic tyrosine receptor kinase.