Fanny Garlan

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker

Purpose: Despite recent therapeutic advances, prognosis of patients with pancreatic adenocarcinoma remains poor. Analyses from tumor tissues present limitations; identification of informative marker from blood might be a promising alternative. The aim of this study was to assess the feasibility and the prognostic value of circulating tumor DNA (ctDNA) in pancreatic adenocarcinoma. Experimental Design: From 2011 to 2015, blood samples were prospectively collected from all consecutive patients with pancreatic adenocarcinoma treated in our center. Identification of ctDNA was done with next-generation sequencing targeted on referenced mutations in pancreatic adenocarcinoma and with picoliter droplet digital PCR. Results: A total of 135 patients with resectable (n = 31; 23%), locally advanced (n = 36; 27%), or metastatic (n = 68; 50%) pancreatic adenocarcinoma were included. In patients with advanced pancreatic adenocarcinoma (n = 104), 48% (n = 50) had ctDNA detectable with a median mutation allelic frequency (MAF) of 6.1%. The presence of ctDNA was strongly correlated with poor overall survival (OS; 6.5 vs. 19.0 months; P < 0.001) in univariate and multivariate analyses (HR = 1.96; P = 0.007). To evaluate the impact of ctDNA level, patients were grouped according to MAF tertiles: OS were 18.9, 7.8, and 4.9 months (P < 0.001). Among patients who had curative intent resection (n = 31), 6 had ctDNA detectable after surgery, with an MAF of 4.4%. The presence of ctDNA was associated with a shorter disease-free survival (4.6 vs.17.6 months; P = 0.03) and shorter OS (19.3 vs. 32.2 months; P = 0.027). Conclusions: ctDNA is an independent prognostic marker in advanced pancreatic adenocarcinoma. Furthermore, it arises as an indicator of shorter disease-free survival in resected patients when detected after surgery. Clin Cancer Res; 23(1); 116–23. ©2016 AACR.

Early Evaluation of Circulating Tumor DNA as Marker of Therapeutic Efficacy in Metastatic Colorectal Cancer Patients (PLACOL Study)

Purpose: Markers of chemotherapy efficacy in metastatic colorectal cancer (mCRC) are essential for optimization of treatment strategies. We evaluated the applicability of early changes in circulating tumor DNA (ctDNA) as a marker of therapeutic efficacy. Experimental Design: This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C0), second (C1) and/or third (C2) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation (KRAS, BRAF, TP53) or hypermethylation (WIF1, NPY). CT scans were centrally assessed using RECIST v1.1 criteria. Multivariate analyses were adjusted on age, gender, ECOG performance status (PS), metastatic synchronicity, and treatment line. Results: Eighty-two patients with mCRC treated in first- (82.9%) or second- (17.1%) line chemotherapy were included. Patients with a high (>10 ng/mL) versus low (≤0.1 ng/mL) ctDNA concentration at C0 had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5–12.6; P < 0.0001). By analyzing the evolution of the ctDNA concentration between C0 and C2 or C1 (C2or1), we classified the patients in two groups (named “good” or “bad ctDNA responders”). In multivariate analysis, patients belonging to the group called “good ctDNA responder” (n = 58) versus “bad ctDNA responder” (n = 15) had a better objective response rate (P < 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09–0.40; P < 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11–0.57; P < 0.001). Conclusions: This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC. Clin Cancer Res; 23(18); 5416–25. ©2017 AACR.

Chapter Three – Droplet-Based Digital PCR: Application in Cancer Research

The efficient characterization of genetic and epigenetic alterations in oncology, virology, or prenatal diagnostics requires highly sensitive and specific high-throughput approaches. Nevertheless, with the use of conventional methods, sensitivity and specificity were largely limited. By partitioning individual target molecules within distinct compartments, digital PCR (dPCR) could overcome these limitations and detect very rare sequences with unprecedented precision and sensitivity. In dPCR, the sample is diluted such that each individual partition will contain no more than one target sequence. Following the assay reaction, the dPCR process provides an absolute value and analyzable quantitative data. The recent coupling of dPCR with microfluidic systems in commercial platforms should lead to an essential tool for the management of patients with cancer, especially adapted to the analysis of precious samples. Applications in cancer research range from the analysis of tumor heterogeneity to that of a range of body fluids. Droplet-based dPCR is indeed particularly appropriate for the emerging field of liquid biopsy analysis. In this review, following an overview of the development in dPCR technology and different strategies based on the use of microcompartments, we will focus particularly on the applications and latest development of microfluidic droplet-based dPCR in oncology.

Droplet-Based Digital PCR: Application in Cancer Research

The efficient characterization of genetic and epigenetic alterations in oncology, virology, or prenatal diagnostics requires highly sensitive and specific high-throughput approaches. Nevertheless, with the use of conventional methods, sensitivity and specificity were largely limited. By partitioning individual target molecules within distinct compartments, digital PCR (dPCR) could overcome these limitations and detect very rare sequences with unprecedented precision and sensitivity. In dPCR, the sample is diluted such that each individual partition will contain no more than one target sequence. Following the assay reaction, the dPCR process provides an absolute value and analyzable quantitative data. The recent coupling of dPCR with microfluidic systems in commercial platforms should lead to an essential tool for the management of patients with cancer, especially adapted to the analysis of precious samples. Applications in cancer research range from the analysis of tumor heterogeneity to that of a range of body fluids. Droplet-based dPCR is indeed particularly appropriate for the emerging field of liquid biopsy analysis. In this review, following an overview of the development in dPCR technology and different strategies based on the use of microcompartments, we will focus particularly on the applications and latest development of microfluidic droplet-based dPCR in oncology.

[Digital PCR compartmentalization I. Single-molecule detection of rare mutations].

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients.

PCR digitale en micro-compartiments - I. Détection sensible de séquences d’acides nucléiques rares

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients.

BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/μL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.

Droplet-Based Digital PCR

The efficient characterization of genetic and epigenetic alterations in oncology, virology, or prenatal diagnostics requires highly sensitive and specific high-throughput approaches. Nevertheless, with the use of conventional methods, sensitivity and specificity were largely limited. By partitioning individual target molecules within distinct compartments, digital PCR (dPCR) could overcome these limitations and detect very rare sequences with unprecedented precision and sensitivity. In dPCR, the sample is diluted such that each individual partition will contain no more than one target sequence. Following the assay reaction, the dPCR process provides an absolute value and analyzable quantitative data. The recent coupling of dPCR with microfluidic systems in commercial platforms should lead to an essential tool for the management of patients with cancer, especially adapted to the analysis of precious samples. Applications in cancer research range from the analysis of tumor heterogeneity to that of a range of body fluids. Droplet-based dPCR is indeed particularly appropriate for the emerging field of liquid biopsy analysis. In this review, following an overview of the development in dPCR technology and different strategies based on the use of microcompartments, we will focus particularly on the applications and latest development of microfluidic droplet-based dPCR in oncology.

Hypermethylated circulating DNA detection using picoliter droplet-based PCR in colorectal cancer.

622 Background: Circulating tumor DNA (ctDNA) is thoroughly investigated as a surrogate biomarker of tumor follow-up, in different cancer types, such as colorectal cancer (CRC). Droplet-based digital PCR (ddPCR) is a highly sensitive and also quantitative method for detection of very low amount of ctDNA. Since many different genes can be mutated within a specific tumor type and also wide mutation spectrum can occur within a specific gene, procedures for ctDNA monitoring can be time consuming and need to be improved for a routinely use. To overcome these drawbacks, we characterized the methylation status of 3 genes frequently hypermethylated in CRC to identify universal markers for tumor follow-up. Methods: The characterization of the methylated status of the WIF, NPY and PENK genes in the tumor DNA was performed in 56 CRC of different stages and 45 corresponding plasma samples using droplet-based dPCR, after DNA bisulfite conversion. A two-panels assay (with albumin as a reference) was developed. Methylat...