Module 3: Pan-Cancer Biomarkers: TMB, MSI, VAF, CNV & Loss of Heterozygosity

1. Tumor Mutational Burden (TMB)

Tumor Mutational Burden (TMB) quantifies the total number of non-synonymous somatic mutations per megabase (Mb) of examined coding genome. Mechanistically, tumors harboring high mutational loads generate elevated frequencies of novel peptide sequences (neoantigens). These neoantigens are processed and presented on tumor cell surfaces via MHC-I complexes, provoking cytotoxic CD8+ T-cell antitumor immune responses.

$$\text{TMB (mut/Mb)} = \frac{\text{Count of Eligible Somatic Non-Synonymous Coding Variants}}{\text{Total Interrogated Coding Target Space (Mb)}}$$

2. Microsatellite Instability (MSI)

Microsatellites are short, repetitive nucleotide tandem repeat sequences (1–6 bp repeats, e.g. $(A)_n$ or $(CA)_n$) distributed throughout the genome. When the cellular DNA Mismatch Repair (MMR) machinery—encoded by MLH1, MSH2, MSH6, and PMS2—is inactivated through somatic mutation, promoter hypermethylation, or germline Lynch syndrome mutations, DNA polymerases slip across these repeat tracts during replication.

This failure to repair slippage creates length alterations across microsatellite loci, termed Microsatellite Instability-High (MSI-H). Computational algorithms (such as MSIsensor-pro and mSINGS) quantify the percentage of mutated microsatellite loci directly from targeted NGS reads:

$$\text{MSI Score (\%)} = \frac{\text{Unstable Microsatellite Sites}}{\text{Total Evaluated Microsatellite Sites}} \times 100\%$$

Tumors with $\text{MSI Score} \ge 20\% - 30\%$ are classified as MSI-H and are strongly hypersensitive to immune checkpoint blockade regardless of tissue of origin.

3. Variant Allele Fraction (VAF) & Tumor Purity Deconvolution

The Variant Allele Fraction (VAF) is the proportion of sequenced sequencing reads at a given genomic locus that support the mutant allele relative to total depth:

$$\text{VAF} = \frac{\text{Alternative (Mutant) Read Depth (AD)}}{\text{Total Read Depth (DP)}}$$

A variant's VAF is governed by three biological and technical parameters: Tumor Purity ($p$), Clonal Fraction ($c$), and Copy Number Status ($CN$). For a heterozygous somatic mutation in a diploid tumor with purity $p$:

$$\text{Expected VAF} = \frac{p \cdot c}{2(1 - p) + 2p} = \frac{p \cdot c}{2}$$

For example, if a biopsy specimen contains 40% tumor cells ($p = 0.40$), a clonal heterozygous driver mutation will present at $\text{VAF} = 0.40 / 2 = 0.20$ ($20\%$). Variants with VAF significantly below expected clonal levels represent subclonal sub-populations indicative of intratumor heterogeneity.

4. Copy Number Alterations (CNVs) and Loss of Heterozygosity (LOH)