Proof of Principle for Mutations Monitoring Using Picoliter-Droplet Digital PCR on DNA and Living Cells: Application to Myelodysplastic Syndromes
Authors: Guermouche Hélène , Nizard Philippe , Lu Heng , Decraene Charles , Nectoux Juliette , Laurent-Puig Pierre , Proudhon Charlotte , Fontenay Michaela , Taly Valérie , Kosmider Olivier 
Published: October 2019

Abstract
Introduction
Myelodysplastic syndromes (MDS) are phenotypically and genotypically heterogeneous diseases with several driver mutations, which are closely related to patient prognosis. Dynamic evolution of mutations reflects the selection of subclones during disease evolution until transformation in secondary acute myeloid leukemia. Thanks to increasing knowledge in gene functions, target drugs are now available in therapeutic. However, questions remain on the impact of such treatments on malignant cells. We have previously investigated the effects of lenalidomide on clonal evolution, by monitoring variant allele frequencies (VAF) using next generation sequencing (NGS) in non-del5q MDS patients (Chesnais et al, Blood 2015). Here, we present a rapid and ultra-sensitive method using picoliter-droplet digital PCR for mutation detection in MDS with ring sideroblasts (RS).
Materials and Methods
Bone marrow aspirates were obtained from MDS patients included at diagnosis in a multicentric observational trial (PHRC MDS-04, NCT02619565). Three cell lines (HL60, OCI-AML3, UKE-1) were also used to establish the specificity and the sensitivity of assays. Both frozen living cells and extracted DNA were used. Selected samples were screened for mutations in 39 genes by an NGS approach using a Personal Genome Machine® (PGM, ThermoFisher Scientific, Waltham, MA, USA). Primers and probes were designed for Taqman assays based on allelic discrimination of recurrent mutations found in DNMT3A, SF3B1, JAK2 and NRAS genes. For the detection of SF3B1 p.K700E mutation, 3 locked nucleic acids were notably added to the probes to improve specificity. Picoliter-droplet digital PCR was performed on RainDrop® Digital PCR System (RainDance™ Technologies).
Results
Allelic discrimination assays were validated on genomic DNA extracted from cell lines and patient samples harboring or not targeted mutations using the RainDance system. About 5.106 droplets were generated using RainDrop Source. Wild-type (WT) DNA was tested in order to assess false positive signals for each design, characterized by λFP (mean number of false positive signals), limit of blanck (LOB) and limit of detection (LOD) for all experiments. The limit of blanck (LOB) defined here the highest number of droplets corresponding to apparent droplets containing mutated amplicons while testing wild type DNA. The limit of detection (LOD) was the lower number of droplets which can be distinguish from LOB while testing DNA with very low concentration of mutant genome.
All the designed assays were also strongly approved for linearity using mixtures of mutated and WT DNA from cell lines (0.01% to 100% mutated allele frequency). Specificity, linearity and sensibility of the selected assays were validated on genomic DNA. Later on, we investigate genomic DNA of 3 MDS patients with RS and harboring JAK2 and SF3B1 mutations. For these patients, we obtained comparable results using both NGS and picoliter-droplet digital PCR in term of mutant allele burden quantification. Moreover, a triplex assay allowing mutant allele discrimination in JAK2 and SF3B1 genes was established on these patients.
Further analyses were conducted on living cells harboring JAK2 or NRAS mutations. This approach was first conducted using a "home made" microfluidic system based on the detection of fluorescent probes in living cells encapsulated into agarose beeds. We obtained specific fluorescent signals corresponding to the genotypes. In parallel, an alternative method based on the QX100™Droplet Digital™PCR system (Biorad) also demonstrated the feasibility of allelic discrimination in living cells. Experiments based on frozen cells of MDS patients are currently under investigation.
Conclusion
This study is the first application of multi-target digital PCR used to detect and quantify somatic mutations recurrently found in MDS. Analyses of the clonal architecture determined on living cells and its evolution upon treatment in MDS patients with RS by this approach will help us to investigate the monitoring of the therapeutic response. Our study supports a proof of principle for further large-scale analyses of MDS patients at diagnosis and follow-up.

Disclosures
No relevant conflicts of interest to declare.