Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

Abstract

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

“…Detection of mutations from tumor biopsies and more recently from circulating tumor DNA (ctDNA) has proved to be of great importance in clinical oncology and cancer research settings (Wan et al, 2017). In recent years, several methods have been developed for the detection of low‐abundance mutations in tumors, including quantitative real‐time polymerase chain reaction (PCR), coamplification at lower denaturation temperature‐PCR (Milbury et al, 2011), digital PCR (Vogelstein & Kinzler, 1999; Zonta et al, 2016), and pyrosequencing (Herreros‐Villanueva, Chen, Yuan, Liu, & Er, 2014; Huggett & Whale, 2013; Tsiatis et al, 2010). Despite recent advances in diagnostic technology, several challenges still exist to accurately identify mutations in cancers, particularly in the presence of high backgrounds of wild‐type DNA, as is typical in early‐stage cancers or liquid biopsies.…”

Minimal residual disease in acute myelogenous leukemia

“…NPM1 mut assessment and schematic of dPCR technology are shown (Figure B). Outside of the AML, recent data also indicate similar results for multiplex detection of point mutations including EGFR mutations …”

Optofluidic Modular Blocks for On‐Demand and Open‐Source Prototyping of Microfluidic Systems

“…Microfluidics and related biotechnologies have made great advances over the past decades, realizing applications that were not possible before the development of microfluidics technology . For instance, the high‐throughput generation and analysis of nanoliter and picoliter droplets have enabled rapid diagnosis of low‐abundance biomarkers such as pathogens and molecular markers .…”

“…Microfluidics and related biotechnologies have made great advances over the past decades, realizing applications that were not possible before the development of microfluidics technology . For instance, the high‐throughput generation and analysis of nanoliter and picoliter droplets have enabled rapid diagnosis of low‐abundance biomarkers such as pathogens and molecular markers . Simulation of tissue or organ‐level physiology has been realized by generating physiologically relevant microenvironments in physical and chemical ways, thereby providing a potential alternative to animal testing for drug screening and toxicology studies .…”

“…We next applied the modular platform to on‐chip droplet generation and detection, which is an important application in the field of bio(chemical) analysis . For such an application, we developed a pumping block (part number 202) as a portable pressure source ( Figure ).…”