Abstract
Simple SummaryCtDNA analysis is a promising tool in liquid biopsy for the detection of tumor recurrence and progression, and is increasingly adopted into clinical practice. Still, guidelines for the accurate clinical interpretation of ctDNA analysis results are largely lacking, especially for tumor mutant variants detected at very low frequencies. Here, we show that cutoff determination for the detection and quantification of low-frequency mutant variants enables the accurate prediction of residual disease, tumor recurrence and progression, even before clinical evidence. CtDNA analysis using these cutoffs outperformed cfDNA and CEA level measurements. With these findings, we highlight the need to thoroughly validate each liquid biopsy assay and define the assay-specific limit of blanks (LOB) and limit of quantifications (LOQ) of BRAF p.V600E and KRAS p.G12/p.G13 assays for clinical interpretation. Our approach enables accurate clinical interpretation to support clinical decision making.Circulating tumor DNA (ctDNA) is a promising liquid biopsy (LB) marker to support clinical decisions in precision medicine. For implementation into routine clinical practice, clinicians need precise ctDNA level cutoffs for reporting residual disease and monitoring tumor burden changes during therapy. We clinically validated the limit of blank (LOB) and the limit of quantification (LOQ) of assays for the clinically most relevant somatic variants BRAF p.V600E and KRAS p.G12/p.G13 in colorectal cancer (CRC) in a study cohort encompassing a total of 212 plasma samples. We prove that residual disease detection using the LOB as a clinically verified cutoff for ctDNA positivity is in concordance with clinical evidence of metastasis or recurrence. We further show that tumor burden changes during chemotherapy and the course of disease are correctly predicted using the LOQ as a cutoff for quantitative ctDNA changes. The high potential of LB using ctDNA for accurately predicting the course of disease was proven by direct comparison to the routinely used carcinoembryonic antigen (CEA) as well as the circulating free DNA (cfDNA) concentration. Our results show that LB using validated ctDNA assays outperforms CEA and cfDNA for residual disease detection and the prediction of tumor burden changes.
Highlights
CfDNA is released from both tumor and normal cells into the circulation [1,2]
Precise cutoff definition of positive Circulating tumor DNA (ctDNA) status in a plasma sample is a prerequisite for the ctDNA-based detection of residual disease and recurrence, and was determined previously using well-characterized WT control reference materials according to CLSI
Disease recurrence could not be predicted around one month prior to clinical evidence in one patient, overall, these results indicate the potential of ctDNA analysis as a complementary marker for molecular residual disease (MRD) and recurrence detection
Summary
CfDNA is released from both tumor and normal cells into the circulation [1,2]. Besides ctDNA, other LB analytes including circulating tumor cells (CTCs) have been investigated for clinical application. CTCs allow the extraction of detailed information at the single cell level [6,7], numerous studies have shown that ctDNA profiles are highly concordant with the molecular profile of primary tumors and metastases [8,9], and are considered to have a higher likelihood to enter clinical application [10]. In certain clinical courses of patients with non-small cell lung cancer (NSCLC) and breast cancer, LB using ctDNA analysis is already recommended to guide therapeutic decisions [14,15], and is even covered by health insurance. LB is expected to enter the clinical routine for CRC patient management in the near future, once clinical utility is proven for the following applications: (1) residual disease detection after surgery in CRC to facilitate decision on adjuvant therapy, (2) recurrence monitoring and (3) real-time monitoring of treatment response during chemotherapy
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