Current and Emerging Applications of Droplet Digital PCR in Oncology

Abstract

The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. This methodology has proven useful for the evaluation of archival tumor tissues, where poor DNA quality and limited sample availability are major obstacles for standard methods, providing less subjective and more automated quantitative results. However, most applications of ddPCR in cancer are focused on liquid biopsies (including cell-free DNA as well as circulating tumor cells) because these represent non-invasive alternatives to tissue biopsies that can more accurately reflect intratumoral heterogeneity and track the dynamic changes in tumor burden that occur in response to treatment at different times during follow-up. A broad spectrum of molecular markers have been interrogated in blood using ddPCR for diagnostic, predictive, and monitoring purposes in various malignancies. Emerging alternative approaches using other body fluids such as cerebrospinal fluid and urine are also currently being developed. This article aims to give a complete overview of ddPCR applications for molecular screening in oncology.

Phenotypic association of presence of a somatic GNAQ mutation with port‐wine stain distribution in capillary malformation

“…Use of the sensitive ddPCR method to quantify the abundance of cells with the GNAQ p.Arg183Gln mutation allowed for an accurate quantification of the target nucleic acid samples. The reliability of ddPCR in the detection and quantification of DNA from FFPE samples has been already reported in other fields . The accuracy of this technique was further supported by its nondetection of the somatic mutation in the 14 samples not affected by capillary malformation, with an allele frequency of ≤0.5%.…”

“…The reliability of ddPCR in the detection and quantification of DNA from FFPE samples has been already reported in other fields. [9][10][11] The accuracy of this technique was further supported by its nondetection of the somatic mutation in the 14 samples not affected by capillary malformation, with an allele frequency of ≤0.5%.…”

Multiple KRAS mutations in the non‐mucinous epithelial lining in the majority of mucinous cystic neoplasms of the pancreas

“…In previous studies, Sanger sequencing, 10,19,21 PCR-based restriction fragment length polymorphism, 9 whole-exome sequencing 12 and next-generation sequencing have been used to detect KRAS mutations; however, droplet digital PCR is one of the most sensitive methods for the detection of mutations. 14,23 In this study, multiple types of KRAS mutations were observed in 83% (10/12) of NMLE foci and 78% (7/9) of MLE foci, with low allele frequencies. These observations suggest the presence of significant genetic heterogeneity in the early tumorigenesis of MCNs.…”

“…This higher rate of positive cases is probably the result of the mutation detection methods used. In previous studies, Sanger sequencing, PCR‐based restriction fragment length polymorphism, whole‐exome sequencing and next‐generation sequencing have been used to detect KRAS mutations; however, droplet digital PCR is one of the most sensitive methods for the detection of mutations …”

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

“…In this context, droplet digital PCR (ddPCR) provides the opportunity to fill the void in inversion characterization by detecting linkage between amplicons located at both sides of the breakpoint repeats, making it possible to jump long genomic distances. The ddPCR TM technology has already been used to detect copy number variation (Boettger et al 2012), viral load (Strain et al 2013), low-frequency transcripts , rare mutations or cell free DNA (Olmedillas-López et al 2017;Camunas-Soler et al 2018) among other applications.…”