Tumor mutational burden (TMB) and Microsatellite Instability (MSI) are now established biomarkers guiding immunotherapy decisions across a growing list of solid tumors. This article outlines how TMB and MSI are defined and assessed in VarSome Clinical and how they are related to regulatory approvals and clinical utility across different cancer types. By summarising the latest guidelines, FDA indications, and landmark trials, we highlight where these biomarkers are already shaping treatment and where their role is still emerging.
Tumor Mutational Burden
TMB is the count of somatic, coding mutations per megabase of interrogated DNA (mut/Mb). A common clinical definition for TMB-high is ≥10mut/Mb. Mechanisms associated with TMB include loss or weakening of DNA repair pathways (e.g., mismatch repair loss, POLE mutations); exogenous mutations (UV, tobacco smoke, some chemotherapies); and novel mutations may result in neoantigen production, increasing the likelihood that T cells recognise the tumor once immune checkpoints are blocked.
Microsatellite instability
MSI describes somatic insertions or deletions that change the length of microsatellites (short tandem repeats) in tumor DNA when compared with matched normal DNA. These repetitive tracks are prone to slippage during replication. When mismatch repair (MMR) activity is lost or inactive, the slippage loops are not repaired, and the repeat units expand or contract, producing an MSI signature. Loss of MMR also allows replication errors to persist in SNVs and short indels throughout the genome. Consequently, most MSI high (MSI-H) tumors also meet TMB-high thresholds.
TMB Calculation with VarSome Clinical
VarSome Clinical estimates TMB in tumor-only, targeted analyses of WES data (≥ 31 Mb capture, non-WGS) or Agilent SureSelect Cancer CGP panels, provided the mean coverage reaches 50x. We report two metrics:
- TMB (all) - all coding variants, including synonymous changes.
- TMB (non-synonymous) - excludes synonymous calls.
To keep noise to a minimum, the pipeline calls variants with Sentieon TNHaplotyper2, flags likely germline sites with a panel of normals and public germline databases, then applies additional filters to remove any residual germline variants, resulting in a TMB value more reflective of the true somatic load.
Users can define custom thresholds, with defaults set to:
- TMB-high = TMB ≥ 10 mut/Mb
- TMB-low = TMB < 10 mut/Mb
TMB scores are capped at 40 mut/Mb for reporting purposes, even if the calculated value exceeds this limit.
For more information on how we calculate TMB status, please visit: http://docs.varsome.com/en/tmb
MSI Calculation with VarSome Clinical
Unlike traditional MSI detection methods that require matched normal samples, VarSome Clinical enables accurate MSI evaluation from tumor-only sequencing data. It supports somatic analyses starting from FASTQ with various assays, currently including:
- Whole Exome Sequencing (WES)
- Agilent SureSelect Cancer CGP Sequencing Panel
The tool outputs the MSI score (High or Not Detected) as the fraction of unstable sites (sites with evidence of indels or misalignment in microsatellite repeats) to qualifying sites (sites with adequate coverage analyzed for instability).
Estimation method
- Baseline
- Built from BAM files of normal tissue in 1532 TCGA cases (CRC, UCEC, STAD).
- Probability distributions calculated at many microsatellite loci.
- Optimised on 80 TCGA tumours with known MSI status.
- Cancer-specific cut-offs for colorectal cancer (CRC), uterine corpus endometrial carcinoma (UCEC), and stomach adenocarcinoma (STAD).
- Type-agnostic cut-off for all other cancers.
For more information on how we calculate MSI status, please visit: https://docs.varsome.com/en/msi
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