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Updated: Aug 23, 2025
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Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
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Development and validation of blood tumor mutational burden reference standards
Eun-Ang Raiber-Moreau1, Guillem Portella2, Matthew G Butler3
1Precision Medicine and Biosamples, Diagnostic Development Unit, AstraZeneca, Cambridge, UK.
Genes, Chromosomes & Cancer
|November 3, 2022
Summary
We developed reference materials to standardize tumor mutational burden (TMB) testing in blood. These standards help ensure accurate and reproducible TMB measurements for immunotherapy selection across different labs and platforms.
Area of Science:
- Genomics
- Biomarker Development
- Cancer Research
Background:
- Tumor mutational burden (TMB) is a predictive biomarker for immunotherapy response.
- Variability in TMB measurement across different platforms and laboratories poses a challenge.
- Standardized reference materials are needed for accurate TMB assessment.
Purpose of the Study:
- To develop and characterize contrived blood TMB (bTMB) reference materials.
- To support calibration and alignment of bTMB assays across laboratories and platforms.
- To evaluate the feasibility and challenges of using bTMB reference standards.
Main Methods:
- Created reference materials by blending tumor cell line DNA with donor-matched lymphoblastoid cell line DNA.
- Fragmented and size-selected DNA to mimic circulating cell-free tumor DNA.
- Assessed bTMB scores using PredicineATLAS and GuardantOMNI next-generation sequencing assays.
- Applied bioinformatic filtration to address low-VAF artifact variants.
Main Results:
- Contrived bTMB reference samples mimicked patient cfDNA size profiles and mutational patterns.
- bTMB scores from reference samples aligned with expected values and showed good reproducibility.
- A bioinformatic filtration step was necessary to correct for artifact variants.
Conclusions:
- Demonstrated the feasibility of producing and using bTMB reference standards.
- These standards can aid in the calibration and validation of bTMB platforms.
- Reference materials can promote harmonization of bTMB testing between laboratories and assays.
