What is Tumor Mutational Burden (TMB)?
Tumor mutational burden (TMB) is a quantitative measure of the total number of somatic mutations present in a tumor genome, most commonly expressed as the number of mutations per megabase (mut/Mb) of DNA sequenced. 1
Definition and Measurement
TMB denotes the relative number of somatic coding mutations harbored by tumor cells, typically calculated from next-generation sequencing (NGS) data of tumor tissue or circulating cell-free DNA (blood TMB). 1, 2
The measurement specifically counts somatic mutations—genetic alterations acquired by cancer cells that are not present in normal germline DNA. 2
TMB values vary dramatically across cancer types, ranging from approximately 1 mutation/Mb in hematologic malignancies to over 10 mutations/Mb in solid tumors with hypermutant phenotypes. 1
Clinical Significance and FDA Approval
The FDA has approved pembrolizumab for patients with unresectable or metastatic TMB-high (TMB-H) solid tumors, defined as ≥10 mutations/megabase by an FDA-approved test, who have progressed following prior treatment and have no satisfactory alternative treatment options. 3
This approval was based on the phase II KEYNOTE-158 trial, where patients with TMB-H tumors treated with pembrolizumab achieved an objective response rate of 29% compared to 6% in non-TMB-H tumors. 1
TMB has demonstrated predictive value for response to immune checkpoint inhibitors (particularly PD-1/PD-L1 inhibitors) across multiple cancer types, both retrospectively and prospectively. 1
The relationship between TMB and immunotherapy response stems from the principle that higher mutational burden generates more tumor neoantigens (TNAs), which can be recognized by the immune system and enhance response to checkpoint blockade. 1
Testing Methodologies
Whole exome sequencing (WES) is considered the gold standard for TMB assessment, providing comprehensive measurement across the entire coding genome. 4
Targeted gene panels of various sizes (typically 0.5-1.5 Mb) are increasingly used in clinical practice due to lower cost and faster turnaround, though they sample smaller genomic space. 1, 4
Blood-based TMB (bTMB) can be measured from circulating cell-free DNA, though it requires higher tumor content (typically ≥5% tumor fraction for accurate assessment) and has lower limits of detection compared to tissue-based testing. 1
Important Clinical Limitations and Pitfalls
TMB is an unstable metric with significant variability in measurement and interpretation, and its predictive value should not be overstated—it functions at best as a limited surrogate biomarker of immunotherapy response. 5
TMB showed predictive value for immunotherapy response in melanoma and non-small cell lung cancer, but not in renal cell carcinoma, highlighting tumor-type specific differences. 5
In colorectal cancer specifically, the NCCN Panel does not currently recommend TMB biomarker testing outside of clinical trials, as limited data showed insufficient clinical activity (disease control rate 31%, objective response rate only 11% in TMB-H metastatic colorectal cancer treated with pembrolizumab). 1
Low circulating tumor DNA fractions significantly limit accuracy of TMB calling from blood samples, particularly when tumor content is below 1-2%. 1
Lack of standardization across different testing platforms, bioinformatic pipelines, and panel sizes creates challenges in comparing TMB values and establishing universal predictive cutoffs. 4
TMB assessment requires careful filtration to exclude low variant allele frequency artifact variants and potential clonal hematopoiesis (CHIP)-related mutations that originate from blood cells rather than tumor. 1, 6
Relationship to Other Biomarkers
The relationship between TMB, microsatellite instability (MSI), and PD-L1 expression is complex and tumor-type dependent. 1, 7
Simultaneous presence of TMB-high, MSI-high, and PD-L1 expression occurred in only 2.9% of all cancers analyzed, with higher percentages in colorectal (12.8%) and esophagogastric cancers. 1
MSI-high tumors frequently demonstrate high TMB due to defective DNA mismatch repair, but TMB-high status can occur independently of MSI status. 1
NGS platforms that couple TMB analysis with MSI determination may represent a decisive tool for selecting patients for immunotherapy, particularly in cancers not belonging to the Lynch syndrome spectrum. 1