Tumor Mutational Burden and Comprehensive Cancer Profiling

TMB for Immunotherapy Response

Tumor Mutational & Cancer Profiling
  • Tumor mutational burden (TMB) is gaining tremendous interest in recent years as a biomarker particularly for predicting response to immuno-oncology (IO) therapies. 

  • TMB is measured as the total number of mutations per megabases within the coding region of a tumor genome. Tumors with high TMB are likely to harbor more proteins with mutations, which are presented as neoantigens that can be recognized by T cells. 

  • Patients with high TMB have been found to have more favorable response to checkpoint inhibitors, such as atezolizumab and pembrolizumab, than those with lower TMB.

Rapid Measurement of Tumor Mutational Burden

  • The gold standard for measuring TMB has been whole exome sequencing (WES). Recent studies have demonstrated that targeted NGS panels can be an effective and economical method for estimating TMB, provided that the panels contain at least 300 genes and cover more than 1 Mb of genomic content.

 

  • Amplicon-based targeted NGS panels require smaller DNA input amounts and provide a simpler and faster workflow, allowing sequencing to be carried out on the same day for rapid measurement of TMB. 

  • However, researchers should be mindful when selecting the appropriate technology for assay development since not all amplicon-based target enrichment technologies can interrogate hundreds of genes in a single assay. 

CleanPlex Streamlined Workflow

The CleanPlex TMB 500 Panel offers a simple and streamlined workflow. Starting from purified and quantitated DNA, the multiplex PCR-based protocol can be completed in just 6 hours, with 75 minutes of hands-on time, using a three-step workflow with minimal tube-to-tube transfers. Each step consists of a thermal cycling or incubation condition, followed by “with bead” purification using magnetic beads.

High TMB Correlation with Whole Exome Sequencing Data

  • Paragon Genomics has developed a high-performance CleanPlex TMB 500 Gene Panel that contains over 500 genes covered by approximately 27,000 amplicons. 

  • One custom version of the panel was tested by Berry Oncology (affiliated with Berry Genomics) to measure TMB in solid tumor FFPE samples. For each sample, the TMB was calculated, the types of mutations were documented, and the top mutated genes were identified. 

  • They compared the mutations identified using the TMB panel to those identified with WES for the same 103 samples and found a high degree of overlap between the two methods. They also found a high degree of correlation when they compared the TMB calculated using the two methods.

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