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Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden

 Kwang Seob Lee  ;  Jieun Seo  ;  Choong-Kun Lee  ;  Saeam Shin  ;  Zisun Choi  ;  Seungki Min  ;  Jun Hyuek Yang  ;  Woo Sun Kwon  ;  Woobin Yun  ;  Mi Ri Park  ;  Jong Rak Choi  ;  Hyun Cheol Chung  ;  Seung-Tae Lee  ;  Sun Young Rha 
 CLINICAL CHEMISTRY, Vol.68(12) : 1519-1528, 2022-12 
Journal Title
Issue Date
Biomarkers, Tumor / genetics ; Circulating Tumor DNA* / genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Liquid Biopsy ; Mutation
circulating tumor DNA ; liquid biopsy ; next-generation sequencing ; predictive biomarker ; targeted gene panel ; tumor mutational burden
Background: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation.

Methods: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines.

Results: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring.

Conclusions: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.
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1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > Yonsei Biomedical Research Center (연세의생명연구원) > 1. Journal Papers
Yonsei Authors
Kwon, Woo Sun(권우선) ORCID logo https://orcid.org/0000-0003-0268-5624
Rha, Sun Young(라선영) ORCID logo https://orcid.org/0000-0002-2512-4531
Shin, Saeam(신새암) ORCID logo https://orcid.org/0000-0003-1501-3923
Lee, Seung-Tae(이승태) ORCID logo https://orcid.org/0000-0003-1047-1415
Lee, Choong-kun(이충근) ORCID logo https://orcid.org/0000-0001-5151-5096
Chung, Hyun Cheol(정현철) ORCID logo https://orcid.org/0000-0002-0920-9471
Choi, Jong Rak(최종락) ORCID logo https://orcid.org/0000-0002-0608-2989
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