Tumor mutational burden (TMB) is one of the most promising biomarkers for the clinical understanding of patient response to immune check point inhibitors. Current clinical application of TMB has focused on the use of whole exome sequencing (WES), however, targeted NGS assays appear to be a much better fit for meeting needs of timely turn-around of patient test results and broader applicability beyond a few send-out clinical laboratories.
Accurate and consistent TMB measurement by targeted NGS panels requires ground-truth controls with a range of TMB scores that support analytical validation of these assays to measure TMBs and the concomitant bio-informatics analysis pipelines for generating the patient TMB scores. SeraCare has partnered with key industry stakeholders to design, develop, and commercialize a range of human cell line-derived TMB reference standards that can be used to validate targeted NGS panels designed for TMB measurements and the concomitant TMB scoring informatics pipeline, allowing precise TMB measurements.
We have developed new TMB reference standards from matched tumor-normal human cell lines in both purified gDNA and FFPE formats. These cell lines are primarily derived from diseased patients and their matched peripheral blood lymphoblastoid normal cell lines. High molecular weight DNAs were extracted and annotated for their TMB scores by whole exome sequencing.
Seraseq® TMB Reference Standards and TMB Scores1
|TMB Reference Standards
||TMB Scores (gDNA)*
||TMB Scores (FFPE)*
|Seraseq® TMB Mix Score 7
||7.2 ± 0.2
||7.15 ± 0.4
|Seraseq® TMB Mix Score 9
||9.5 ± 0.4
||7.52 ± 1.3
|Seraseq® TMB Score 13
|| 12.6 ± 0.02
||12.1 ± 0.3
|Seraseq® TMB Mix Score 20
||20.1 ± 0.2
||18.59 ± 0.5
|Seraseq® TMB Mix Score 26
||25.8 ± 0.5
||22.8 ± 3.6
*Whole Exome Sequencing (WES)-derived TMB scores (replicates).
Consortia-Developed TMB Reference Standards
- LGC SeraCare partnered with the following organizations in the development and evaluation of the gDNA and FFPE TMB reference standards:
- Memorial Sloan Kettering Cancer Center (MSKCC)
- Illumina Inc.
- Neo New Oncology GmbH
- Roche Sequencing
- The Broad Institute
- Sema4 Genomics
- Intermountain Precision Genomics
- Augusta University, Medical College of Georgia
- LGC SeraCare was selected to provide TMB reference materials for an IQN Path TMB Pilot Project2 as well as to the Friends of Cancer Research TMB Harmonization Project,3 focused on harmonizing and standardizing TMB scores between targeted NGS panels and WES-derived measurements. 16 laboratories in the FoCR consortium evaluated the Seraseq gDNA TMB reference samples by WES and targeted NGS panels, and results presented at the 2019 ASCO and 2020 AACR Meetings4,5.
The Seraseq TMB reference standards are now available for immediate purchase. We can also build-to-suit or customize reference standards for your tissue or blood-based TMB projects. Please contact us at 800-676-1881 to discuss your specific needs.
1. TMB scores were determined by WES using Agilent SureSelectXT2 Human All Exon V6 +COSMIC target enrichment kit and sequenced on an Illumina NovaSeqTM 6000. Sequence reads were aligned to the hg38 genome assembly with BWA-MEM and variants were called with Mutect2. Variants were filtered using criteria described by the Friends of Cancer Research TMB Harmonization Working Group (see Reference #2).
2.Press release announcing the selection of SeraCare as provider of FFPE TMB reference controls for IQN Path’s TMB EQA Pilot Program, see: https://www.seracare.com/about-seracare/press-releases/iqn-path-selects-seracare-life-sciences-for-first-ever-tmb-external-quality-assessment-program/
3. Press Release on Friends of Cancer Research’s announcement of Phase 2 of the TMB Harmonization Project, see: https://www.focr.org/news/friends-cancer-research-announces-launch-phase-ii-tmb-harmonization-project
4. Diana M. Merino, Laura M. Yee, Lisa M. McShane, Mathew G. Butler, Vincent Funari, Mathew D. Hellmann, Ruchi Chaudhary, Shu-Jen Chen, Wandjuh Chen, Jeffrey M. Conroy, David Fabrizio, Laura E. MacConaill, Aparna Pallavajjala, Arnaud Papin, Mark Sausen, Victor J. Weigman, Mingchao Xie, Ahmet Zehir, Chen Zhao, and P. Mickey Williams, TMB Standardization by Alignment to Reference Standards: Phase 2 of the Friends of Cancer Research TMB Harmonization Project, Poster #268, 2019 ASCO Meeting, Chicago, USA; https://meetinglibrary.asco.org/record/172797/abstract
5. Diana Merino Vega, Alignment of TMB measured on clinical samples: Phase 2B of the Friends of Cancer Research TMB Harmonization Project, 2020 AACR Virtual Meeting, April 2020. https://www.focr.org/sites/default/files/AACR_2020_TMB.pdf