Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay

Abstract

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

ddpcr: an R package and web application for analysis of droplet digital PCR data

“…We have applied ddpcr to data ( Dataset 1) from a novel ddPCR assay against somatic point mutations in the BRAF -V600 codon that was applied to FFPE specimens from a cohort of colorectal cancer (CRC) patients 8 . V600 mutations are observed in approximately 10% of colorectal tumours 9 and their detection in CRC patients helps determine disease prognosis and treatment regimen.…”

“…V600 mutation frequencies computed from automated ddpcr results were within 3% of those obtained by manual analysis of the ddPCR data by an experienced operator ( Supplementary Figure 4 and Supplementary Table 1). In addition, the BRAF -V600 status for each sample in the entire cohort was classified as mutant or wild-type by a certified pathologist using an immunohistochemical staining assay 8 . We obtained complete agreement between the pathologist’s binary classification of BRAF status and that determined using ddpcr .…”

Demographic trends and KRAS/BRAFV600E mutations in colorectal cancer patients of South China: A single‐site report

“…Besides, long‐term stored formalin‐fixed paraffin‐embedded (FFPE) tissues derived DNA is difficult to meet the requirements of Sanger sequencing . Various mutation detection techniques have been reported, with advantages and disadvantages concerning sensitivity, specificity, simplicity and cost …”

Analytic and clinical validity of thyroid nodule mutational profiling using droplet digital polymerase chain reaction

“…The most common mutation associated with thyroid cancer involves BRAF codon V600, followed by mutations in RAS [ 5 ]. The BRAF activating V600E mutation (BRAFV600E) is found in 29–83% of papillary thyroid cancers (PTC), and is associated with more aggressive disease [ 4 , 6 – 8 ]. A number of RAS mutations have been associated with thyroid cancer, with variable diagnostic utility [ 5 ].…”