Tumor mutational burden (TMB) & microsatellite instability (MSI)

What is tumor mutational burden (TMB) and microsatellite instability (MSI)?

Tumor mutation burden (TMB) is the total number of mutations identified in the genome of a tumor [1]. It can be used as a biomarker for predicting the effectiveness of immunotherapy on some types of cancer. Tumors with high TMB (TMB-H) have been shown to have more ‘neo-antigens’ which coat the tumor cell that leads to the identification of tumor cells by the immune system. Therefore, it is thought that some TMB-H tumors are more responsive to immunotherapy though many factors, such as ethnicity, can influence response [2].

Another predictive cancer biomarker is microsatellite instability (MSI). MSI occurs when a short sequence of DNA that is repeated (a microsatellite or short tandem repeat) undergoes a mutation when it is copied [1]. MSI is the result of a malfunction in the DNA mismatch repair (MMR) system, which generates mutations in microsatellites. MSI is associated with colon, gastric, ovarian, skin, and a variety of other types of cancers [1]. MSI is classified into three categories: high, low, and stable (MSI-H, MSI-L, MSS, respectively). The frequency of MSI, can also be analyzed to predict response to immunotherapies [3].

Even though TMB and MSI are both distinct biomarkers used in cancer research, studies have shown that using both can provide more precise and comprehensive data for determining the efficacy of immunotherapies (e.g., immune checkpoint inhibitors (ICI)) [4].

TMB and MSI as predictive biomarkers for cancer research

Classic approaches for identifying MSI in samples consistent of methods such as PCR, capillary electrophoresis, and immunohistochemistry. These methods are well established and can provide key insights into the levels of MSI in samples, however relative to next generation sequencing (NGS) these approaches are very low throughput [5]. The ability to screen a large number of samples in parallel has encouraged researchers to incorporate NGS techniques into studies of TMB and MSI.

More specifically, researchers employ targeted next generation sequencing techniques for TMB and MSI biomarker research. There are multiple modalities for targeted NGS—Anchored Multiplex PCR chemistry (AMP™),  amplicon sequencing and hybridization capture. AMP is a target enrichment method that uses molecular barcode adapters and single, nested, gene-specific primers for amplification, permitting open-ended capture of DNA, RNA, and cfDNA fragments. Alternatively, amplicon sequencing relies on specially designed primers and PCR to amplify genomic regions of interest, while hybridization capture uses biotinylated oligonucleotide probes.

One common type of hybridization capture for cancer research is exome sequencing, where the area of interest is the protein-coding regions of the human genome (the exome). Exome sequencing can be very informative for determining TMB and MSI levels as it allows researchers to quantify multiple biomarkers and mutations in a single sequencing run [6,7]. Custom hybridization gene panels are another popular option used for quantifying TMB and MSI.

Archer VARIANTPlex™ panels, which rely on AMP chemistry, offer an alternative to hybridization capture methods for quantifying TMB and MSI in the form of small (Pan Solid Tumor) or medium (Complete Solid Tumor) targeted NGS panels.