A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

Abstract

Investigating the mutations driving non Hodgkin lymphomas and developing plasma-based assays for tumour detection and monitoring) (R.D.M.); a post-doctoral fellowship from the Michael Smith Foundation for Health Research (H.-L.W.); and a Bio-Rad Droplet Innovation Award (M.A.). M.A., M.C., and K.B. contributed equally to this work. Disclosures: A part of this study was also funded by a Droplet Innovation Award given to M.A. by Bio-Rad.

Identification of a Clinical Cutoff Value for Multiplex KRASG12/G13 Mutation Detection in Colorectal Adenocarcinoma Patients Using Digital Droplet PCR, and Comparison with Sanger Sequencing and PNA Clamping Assay

“…The ddPCR platform is an advanced digital PCR technology that has been used to detect and quantify target DNA or RNA in tissue or blood samples. ddPCR is a very sensitive method that can detect as little as 0.01% mutant DNA [ 4 ]. Because ddPCR provides an absolute number of fluorescent droplets, clinical applications of the ddPCR platform require delineation between positive (mutant) and negative (wild-type) KRAS G12/G13 mutation status, which is critical for therapeutic decision-making [ 11 , 12 ].…”

“…Because ddPCR provides an absolute number of fluorescent droplets, clinical applications of the ddPCR platform require delineation between positive (mutant) and negative (wild-type) KRAS G12/G13 mutation status, which is critical for therapeutic decision-making [ 11 , 12 ]. Previous studies reported various cutoffs for KRAS G12/G13 determined by ddPCR; Dong et al [ 13 ] set 0.02 to 0.56% cutoffs for multiple KRAS G12/G13 mutation site based on detection limit on their experiments of mixing mutant KRAS G12/G13 DNA to wild-type DNA; Vanova et al [ 14 ] determined an arbitrary 0.6% cutoff; Alcaide et al [ 4 ] set a MAF cutoff of 1%, which was a threshold above background gray-zone noisy; and Laurent-Puig et al [ 15 ] suggested a 1% threshold, which was a clinically relevant cutoff to discriminate a patient’s prognosis.…”

“…In 2017, a total of 50,260 CRC patients died in the United States [ 2 , 3 ]. Following the emergence of monoclonal antibodies against epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), cytotoxic chemotherapy plus targeted monoclonal antibody has been recommended as the standard treatment for metastatic CRC around the world [ 4 ]. Anti-EGFR monoclonal antibody is recommended for RAS–wild or left colon cancers, and VEGF antibody is recommended for RAS-mutant or right colon cancers.…”

“…By partitioning DNA into a large number of droplets, ddPCR can provide absolute quantification and detect target DNA in a much higher background of non-target (usually wild-type) DNA. Due to its technological advantages, ddPCR has been adopted for testing of KRAS mutation in patient samples [ 4 ]. However, the clinical performance of ddPCR-based KRAS mutation detection in CRC has not been carefully evaluated.…”

Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR

“…To solve this problem, we are currently developing a novel multiplex analysis method that identifies major KRAS and other gene mutations using 2D-spatial information regarding fluorescence intensity in dPCR. The dPCR system we used can distinguish two fluorescent colors 27 ; however, a novel dPCR platform would be capable of simultaneously detecting multi-color dyes. Such a new tool might further enhance the utility of the assay during multiplex analysis, potentially allowing the detection of driver mutations across multiple genomic regions 28 .…”