Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA

Abstract

subprojects C9 (R.F. and M.B.) and Z1 (M.B.); the German Federal Ministry of Education and Research within the framework of the e:Med research and funding concept CoNfirm FKZ 01ZX1708F (M.B.); and a Deutsche Gesellschaft für Hämatologie und Medizinische Onkologie e. V. (DGHO)eGesellschaft für Medizinische InnovationeHämatologie und Onkologie mbH (GMIHO) thesis fellowship (S.H.).Disclosures: None declared.

Longitudinal Analysis of Cell-free Mutated KRAS and CA 19-9 Predicts Survival Following Curative Resection of Pancreatic Cancer

“…We analyzed mutated KRAS in cell-free DNA with discriminatory ddPCR assays and integrated results with CA19-9 levels for association with relapse and survival endpoints. Numerous studies have unveiled the potential of the analysis of cell-free mutated tumor DNA as novel diagnostic [26] What takes our study apart is the use of discriminatory multi-target KRAS ddPCR assays [29] to directly identify KRAS SNVs without performing previous tumor NGS. These assays have higher sensitivity compared to many available NGS-based assays (Hussung et al).…”

“…We performed univariate and multivariate survival analyses (Supplemental Tables 1 and 2, Figure S1) for established clinicopathologic variables and found a trend towards inferior RFS but not OS for R1 resection (Figure S1 A,B), a signi cant inverse correlation between elevated CA19-9 in the rst sample collected after resection and RFS and OS (Figure S1 C,D) and signi cantly better OS for patients undergoing adjuvant chemotherapy ( Figure S1 E,F). [29]. At the postoperative stage, no molecular pathology data was available for any tumor.…”

“…The analysis of tumor-derived cell-free nucleic acids (ctDNA) extracted from the plasma and other body uids is a promising tool for molecular diagnostics and non-invasive monitoring of cancer patients [19] [28]. We recently described the development and validation of highly sensitive single-target and discriminatory multi-target KRAS ddPCR assays for the analysis of cfDNA [29]. These assays allow identi cation and quanti cation of mutated KRAS directly from circulation without previous knowledge of tumor KRAS mutational status, which is not routinely tested for resectable PDACs.…”

Longitudinal analysis of cell-free mutated KRAS and CA 19-9 predicts survival following curative resection of pancreatic cancer 

“…We analyzed mutated KRAS in cell-free DNA with discriminatory ddPCR assays and integrated results with CA 19-9 levels for association with relapse and survival endpoints. Numerous studies have unveiled the potential of the analysis of cell-free mutated tumor DNA as novel diagnostic [27] What takes our study apart is the use of discriminatory multi-target KRAS ddPCR assays [30] to directly identify KRAS SNVs without performing previous tumor NGS. These assays have higher sensitivity compared to many available NGS-based assays [29].…”

“…The absolute number of copies per milliliter of blood were calculated as follows: Copies/mL plasma =(copies per µL of reaction as per QuantaSoft analysis software version 1.7.4.0917) × (volume of ddPCR reaction) × ([volume eluted/volume of DNA used in reaction]/volume of plasma used for cfDNA extraction). Mutant allele frequency was calculated as: Mutant allele frequency = mutant copies/mL of plasma / (mutant copies/mL of plasma + wild−type copies/mL of plasma).Limit of detection (LOD) and limit of blank (LOB) of the individual assays have been previously described[30] .…”