Detecting NTRK gene fusions

NTRK gene fusions can be detected with various testing methods^1,2

Next-generation sequencing (NGS)

• Multigene parallel sequencing assay¹
• Provides information about multiple biomarkers, including rare and common mutations, and can typically use less tissue^1,3
• Can detect fusions in all 3 NTRK genes, as well as other genetic alterations²
• Can detect NTRK fusion partner and position²
• Turnaround time for results may be longer than with IHC or FISH⁴

Immunohistochemistry (IHC)

• Uses specific antibodies to detect overexpression of the TRK fusion protein¹
• Can be used to screen patients for NRTK gene fusions, if included in panels¹
• Requires dedicated tissue and limits multiplexing^3,5
• Only detects the NTRK component of the fusion protein; molecular confirmation is necessary¹

Fluorescence in situ hybridization (FISH)

• Uses “break-apart” probes to show NTRK gene rearrangement¹
• Requires dedicated tissue^3,6
• Delivers rapid results⁴
• Cannot distinguish fusion variants⁴
• May be labor intensive, possibly more expensive for multiple assays¹

TRK=tropomyosin receptor kinase; NTRK=neurotrophic tyrosine receptor kinase.

Microsatellite instability (MSI) is an established biomarker in colorectal cancer, and an emerging biomarker for several other cancer types^7-9

MSI can increase the risk of developing certain cancers¹⁰

• Microsatellites are tandem repeats of nucleotides, usually a dinucleotide repeat of cytosine and adenine, which are prone to errors during DNA replication^10-12
• MSI is a hypermutable phenotype caused by the loss of DNA mismatch repair activity, usually caused by gene mutations^10,12
• MSI is detected in ≈15% of all colorectal cancers^10,13

Polymerase chain reaction (PCR) and next-generation sequencing (NGS) can test for MSI⁷

Tumors that undergo MSI testing can be classified as⁷:

For colon cancer, NCCN Guidelines consider IHC for mismatch repair (MMR) and DNA analysis for MSI to be different assays measuring the same biological effect ⁸

PCR-based MSI testing can be conducted in combination with MMR IHC¹³

• DNA from tumor samples can be tested for microsatellite instability using a panel consisting of mono- and di-nucleotide markers recommended by Bethesda/National Cancer Institute (BAT-25, BAT-26, D2S123, D5S346, D17S250)^7,12
  
• However, other markers are in use (eg, BAT-40, MONO-27, NR-21, NR-24)^7,12,14

NGS-based MSI testing enables massive parallel sequencing of MMR genes⁸

• The development of NGS enables more in-depth sequencing; however, it can potentially lead to identification of variants of unknown significance⁷
  • Mutations with unknown clinical significance may be detected that were at levels too low to identify with other methods⁷

Many different tumor types can be MSI-H, but prevalence is low¹²

Investigators identified over 386,000 microsatellite repeats in a recent study retrospectively analyzing 7,919 exomes and 1,000 whole genomes from The Cancer Genome Atlas (TCGA)¹²

• The goal of the study was to develop a profile of MSI mutation patterns across tumor types and understand the affected pathways as well as how they correlate with epigenomic features¹²
• Tumor-type specificity of frameshift MSI is evident for some well-known targets of MSI, such as ACVR2A (52% of MSI-H tumors) and TGFBR2 (44%) (enriched in both colon adenocarcinoma and stomach adenocarcinoma; P<0.05, one-tailed Fisher’s exact test) as well as RPL22 (31%), RNF43 (31%), MLL3 (27%), PRDM2 (21%), JAK1 (16%), and APC (3%)¹²

The graph shows the frequency of MSI-H samples from the 16 most prevalent tumor types in the dataset.

Prevalence of MSI-H tumors seen in a retrospective analysis of 7,919 matched tumor and somatic tissue pairs from the Cancer Genome Atlas^12*

*MSI-H predicted at a confidence level of 0.75.¹²
IHC=immunohistochemistry; MMR=mismatch repair; MSI=microsatellite instability; MSI-H=MSI-high; MSI-L=MSI-low; MSS=microsatellite stable; NCCN=National Comprehensive Cancer Network; NGS=next-generation sequencing; PCR=polymerase chain reaction.

Tumor mutational burden (TMB) is an investigational biomarker for response to cancer immunotherapy^15,16

TMB can be detected in solid tumors or blood by next-generation sequencing (NGS).^16-19

TMB has been determined using both broad and targeted panels and detected by mutational load in cfDNA samples and liquid biopsies.^17-20

TMB is the total number of mutations per coding area of a tumor genome¹⁵

TMB=tumor mutational burden.

The relationship between MSI and TMB is still being evaluated¹⁵

The relationship between TMB and microsatellite instability (MSI), both markers of genetic instability, was evaluated for 62,150 tumor samples.¹⁵

• A majority of tumors with microsatellite high (MSI-H) also showed high TMB (>20 mutations/Mb, 83%)¹⁵
• Only 16% of tumors that showed high TMB were also MSI-H¹⁵
• The co-occurrence of high TMB and MSI-H was dependent on the tumor type¹⁵

TMB levels vary across tumor type^12,15

• The figure shows whole genome sequencing results from 27 tumor types from 3,083 paired tumor and normal tissue samples²¹
• Mutation frequency can vary across cancer types, as well as across patients within a cancer type²¹
  • Pediatric and hematologic malignancies have the lowest frequencies, while tumors driven by environmental factors have higher mutation burdens²¹
    

Identification of mutational burden in 27 tumor types using whole genome sequencing²¹

Mb=megabases.
Republished with permission of Nature Publishing Group, from Lawrence MS, et al. Nature. 2013;499(7457):214-218; permission conveyed through Copyright Clearance Center, Inc.

Tumors with high TMB may potentially be more immunogenic^15,16,22

High levels of somatic mutations within tumor cells may result in expression of neoantigens that can induce a T cell response.²³

Increased mutation burden may be associated with a higher number of antigens, which may result in greater tumor immunogenicity.^9,12,23,24

Ongoing research is being conducted to evaluate TMB as a predictive biomarker for immunotherapy²⁴

The biologic rationale behind TMB’s potential as a biomarker is that tumors with high TMB are likely to accumulate neoantigens, making the tumors susceptible to activated immune cells.^9,24

cfDNA=circulating free DNA; NGS=next-generation sequencing; TMB=tumor mutational burden.