Publications by authors named "Valerie Taly"

72 Publications

Advances in multiplexed techniques for the detection and quantification of microRNAs.

Chem Soc Rev 2021 Feb 4. Epub 2021 Feb 4.

Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université de Paris, CNRS SNC5096, Equipe Labellisée Ligue Nationale Contre le Cancer, F-75006 Paris, France.

MicroRNA detection is currently a crucial analytical chemistry challenge: almost 2000 papers were referenced in PubMed in 2018 and 2019 for the keywords "miRNA detection method". MicroRNAs are potential biomarkers for multiple diseases including cancers, neurodegenerative and cardiovascular diseases. Since miRNAs are stably released in bodily fluids, they are of prime interest for the development of non-invasive diagnosis methods, such as liquid biopsies. Their detection is however challenging, as high levels of sensitivity, specificity and robustness are required. The analysis also needs to be quantitative, since the aim is to detect miRNA concentration changes. Moreover, a high multiplexing capability is also of crucial importance, since the clinical potential of miRNAs probably lays in our ability to perform parallel mapping of multiple miRNA concentrations and recognize typical disease signature from this profile. A plethora of biochemical innovative detection methods have been reported recently and some of them provide new solutions to the problem of sensitive multiplex detection. In this review, we propose to analyze in particular the new developments in multiplexed approaches to miRNA detection. The main aspects of these methods (including sensitivity and specificity) will be analyzed, with a particular focus on the demonstrated multiplexing capability and potential of each of these methods.
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http://dx.doi.org/10.1039/d0cs00609bDOI Listing
February 2021

Usefulness of Plasma SARS-CoV-2 RNA Quantification by Droplet-based Digital PCR to Monitor Treatment Against COVID-19 in a B-cell Lymphoma Patient.

Stem Cell Rev Rep 2021 02 5;17(1):296-299. Epub 2021 Jan 5.

Service de Microbiologie (Unité de virologie), Assistance Publique Hôpitaux de Paris-Centre (AP-HP.Centre), Hôpital Européen Georges Pompidou, Paris, France.

We report the case of an HIV-1-infected patient, treated with anti-CD20 monoclonal antibody for a B-cell lymphoma previously treated by autologous stem cell transplant. He suffered from chronic COVID19 and we monitored by plasma SARS-CoV-2 RNA by highly sensitive droplet-based digital PCR technology (ddPCR). Under tocilizumab therapy and despite a first clinical improvement biologically associated with decreasing inflammatory markers, a slight increase of SARS-CoV-2 RNAaemia quantified by ddPCR was highlighted, confirming the absence of viral efficacy of this treatment and predicting the subsequent observed deterioration. As expected, his complete recovery, finally achieved after COVID-19 convalescent plasmatherapy, strictly paralleled plasma SARS-CoV-2 RNA clearance. With these results, we confirmed the interest of SARS-CoV-2 RNAaemia monitoring by ddPCR in COVID-19 patients, particularly during treatment, and firstly showed that this new and specific biomarker could be helpful to select eligible patient for anti-IL6 receptors therapy considering the variable levels of efficacy recently observed with such therapy.
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http://dx.doi.org/10.1007/s12015-020-10107-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785125PMC
February 2021

Highly sensitive quantification of plasma SARS-CoV-2 RNA shelds light on its potential clinical value.

Clin Infect Dis 2020 Aug 17. Epub 2020 Aug 17.

Assistance Publique Hôpitaux de Paris-Centre (AP-HP.Centre), Service de Microbiologie (Unité de virologie), Hôpital Européen Georges Pompidou, Paris, France.

Background: Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARS-CoV-2 RNAaemia) in hospitalized COVID-19 patients.

Methods: Fifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ next-generation technology (Stilla Technologies, Villejuif, France).

Results: Overall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27.

Conclusion: SARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients.
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http://dx.doi.org/10.1093/cid/ciaa1196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454373PMC
August 2020

Author Correction: High throughput single cell counting in droplet-based microfluidics.

Sci Rep 2020 Jun 17;10(1):10061. Epub 2020 Jun 17.

INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer, Paris, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-66763-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299928PMC
June 2020

Plasma circulating tumor DNA in pancreatic adenocarcinoma for screening, diagnosis, prognosis, treatment and follow-up: A systematic review.

Cancer Treat Rev 2020 Jul 4;87:102028. Epub 2020 May 4.

Université de Paris, Department of Hepatogastroenterology and GI Oncology, Georges Pompidou European Hospital, APHP Centre, Paris, France; Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Equipe labellisée Ligue Nationale contre le cancer, Paris, France. Electronic address:

While no biomarker is currently recommended for the management of pancreatic adenocarcinoma (PA), circulating tumor DNA (ctDNA) seems promising but little is known on how it may help to manage our patients in the near future. This systematic review of literature was designed to explore the current knowledge on ctDNA as a screening, diagnostic, prognostic, predictive and theranostic biomarker in the management of PA. We retrieved 62 full-text articles, 3 meta-analyses, 2 clinical trials, 1 abstract and 13 ongoing trials. Results were categorized into sections about screening, diagnosis, prognosis and follow-up of localized and advanced PA together with possible theranostics applications. Although its specificity is excellent, the current sensitivity of ctDNA remains a limitation especially in patients without metastatic disease. Therefore, this biomarker cannot be currently used as a screening or diagnostic tool. Increasing evidence suggests that ctDNA is a relevant candidate biomarker to assess minimal residual disease after radical surgery, but also a strong independent biomarker linked to a poor prognosis in advanced PA. Some recent data also indicates that ctDNA is an attractive biomarker for longitudinal follow-up and possibly early treatment adaptation. Its role in tumor profiling in advanced disease to decide targeted treatments remains to be explored. Altogether, ctDNA appears to be a reliable prognostic tool. Though promising results have been reported, further studies are still needed to define exactly how ctDNA can help physicians in the screening, diagnosis and treatment, as PA is expected to become a major cause of cancer-related deaths in the forthcoming decade.
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http://dx.doi.org/10.1016/j.ctrv.2020.102028DOI Listing
July 2020

"Decision for adjuvant treatment in stage II colon cancer based on circulating tumor DNA:The CIRCULATE-PRODIGE 70 trial".

Dig Liver Dis 2020 07 29;52(7):730-733. Epub 2020 May 29.

Sorbonne Université and Hôpital Saint Antoine, Paris, France.

Background: Adjuvant treatment for stage II colon cancer remains debated. Finding a tool to select patients at risk for disease recurrence may help the clinical decision. Circulating tumor DNA (ctDNA) has been reported recently as a potential predictive marker for disease recurrence. We thus aim to test its ability to better select stage II colon cancer patients for adjuvant therapy.

Methods: This national, phase III trial (NCT00002019-000935-15) conducted in more than 100 centers in France, plans to screen around 2640 patients in order to randomize (2:1; minimization method) 198 ctDNA positive patients. Patients aged 18 to 75 years with ECOG performance status ≤1 with R0 surgical resection of a pT3-T4aN0 colon or high rectum adenocarcinoma will be randomized within 63 days after curative-intent surgery, to adjuvant mFOLFOX6 (oxaliplatin 85 mg/m², leucovorin 400 mg/m², and 5-FU bolus 400 mg/m2 then 5FU Continuous infusion 2.4 g/m²) every two weeks for 12 cycles or observation. Patients will be followed for maximum 7 years. A gain of 17.5% in 3-yr disease free survival (DFS) is expected (42.5% in the experimental arm vs. 25% in the control arm; HR:0.62; α, 5% [two-sided log-rank test]; 1-β, 80%). Secondary endpoints include 2-yr DFS, overall survival, and toxicity. Recruitement began End of January 2020.
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http://dx.doi.org/10.1016/j.dld.2020.04.010DOI Listing
July 2020

Isothermal digital detection of microRNAs using background-free molecular circuit.

Sci Adv 2020 01 22;6(4):eaay5952. Epub 2020 Jan 22.

Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.
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http://dx.doi.org/10.1126/sciadv.aay5952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976291PMC
January 2020

[Development of molecular analysis by digital PCR for clinical practice: positioning, current applications and perspectives].

Ann Biol Clin (Paris) 2019 12;77(6):619-637

ID Solutions, Grabels, France, Service d'anatomie pathologique et neuropathologie, Assistance Publique Hôpitaux de Marseille, AP-HM, Marseille, France.

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.
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http://dx.doi.org/10.1684/abc.2019.1502DOI Listing
December 2019

Streamlined digital bioassays with a 3D printed sample changer.

Analyst 2020 Jan 26;145(2):572-581. Epub 2019 Nov 26.

Laboratoire Gulliver, UMR7083 CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.

Droplet-based microfluidics has permeated many areas of life sciences including biochemistry, biology and medicine. Water-in-oil droplets act as independent femto- to nano-liter reservoirs, enabling the parallelization of (bio)chemical reactions with a minimum sample input. Among the range of applications spanned by droplet microfluidics, digital detection of biomolecules, using Poissonian isolation of single molecules in compartments, has gained considerable attention due to the high accuracy, sensitivity and robustness of these methods. However, while the droplet throughput can be very high, the sample throughput of these methods is poor in comparison to well plate-based assays. This limitation comes from the necessity to convert independently each sample into a monodisperse emulsion. In this paper, we report a versatile device that performs the quick sequential partitioning of up to 15 samples using a single microfluidic chip. A 3D printed sample rotor is loaded with all samples and connected to a pressure source. Simple magnetic actuation is then used to inject the samples in the microfluidic chip without pressure disruption. This procedure generates monodisperse droplets with high sample-to-sample consistency. We also describe a fluorescent barcoding strategy that allows all samples to be collected, incubated, imaged and analyzed simultaneously, thus decreasing significantly the time of the assay. As an example of application, we perform a droplet digital PCR assay for the quantification of a DNA amplicon from 8 samples in less than 2 hours. We further validate our approach demonstrating the parallel quantification of 11 microRNAs from a human sample using an isothermal nucleic acid amplification chemistry. As an off-chip device, the sample changer can be connected to a variety of microfluidic geometries and therefore, used for a wide range of applications.
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http://dx.doi.org/10.1039/c9an01744eDOI Listing
January 2020

Emerging isothermal amplification technologies for microRNA biosensing: Applications to liquid biopsies.

Mol Aspects Med 2020 04 23;72:100832. Epub 2019 Nov 23.

Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne. Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006, Paris, France; INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, 45 rue des Saints-Pères, Paris, 75006, France; Equipe labellisée Ligue Nationale contre le cancer, France. Electronic address:

The potential of microRNAs (miRNAs) as biomarker candidates in clinical practice for diagnosis, prognosis and treatment response prediction, especially in liquid biopsies, has led to a tremendous demand for techniques that can detect these molecules rapidly and accurately. Hence, numerous achievements have been reported recently in miRNA research. In this review, we discuss the challenges associated with the emerging field of miRNA detection, which are linked to the intrinsic properties of miRNAs, advantages and drawbacks of the currently available technologies and their potential applications in clinical research. We summarize the most promising nucleic acid amplification techniques applied to the in vitro detection of miRNAs, with a particular emphasis on the state of the art for isothermal alternatives to RT-qPCR. We detail the sensitivity, specificity and quantitativity of these approaches, as well as their potential for multiplexing. We also review the different detection formats to which these chemistries have been adapted, including analog readouts such as real-time monitoring, digital counting based on single-molecule amplification in compartments, and surface-based strategies.
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http://dx.doi.org/10.1016/j.mam.2019.11.002DOI Listing
April 2020

HPV circulating tumoral DNA quantification by droplet-based digital PCR: A promising predictive and prognostic biomarker for HPV-associated oropharyngeal cancers.

Int J Cancer 2020 Aug 18;147(4):1222-1227. Epub 2019 Dec 18.

Laboratoire de Virologie, Hôpital Européen Georges Pompidou, and Assistance Publique - Hôpitaux de Paris, Paris, France.

We aimed to determine whether pretherapeutic assessment of HPV circulating tumoral DNA (HPV ctDNA) by droplet-based digital PCR (ddPCR) could constitute a predictive and prognostic biomarker for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). A mono-institutional prospective biomarker study on 66 patients with p16+/HPV16-positive oropharyngeal squamous cell carcinoma (OPSCC) was conducted in European Georges Pompidou Hospital, Paris, France. Blood samples were collected at the time of diagnosis before any treatment. Optimized digital PCR assays were used to quantify HPV16 ctDNA. Forty-seven (71%) patients showed a positive pretherapeutic HPV ctDNA at time of diagnosis. Interestingly, the quantity of HPV16 ctDNA at baseline, as assessed by ddPCR, was significantly correlated with the T/N/M status or OPSCC stages according to the 2018 new staging criteria for high-risk human papillomavirus (HR HPV) related OPSCC from American Joint Committee on Cancer (AJCC). Moreover, all recurrences and the majority (83%) of death reported events occurred in patients with positive HPV16 ctDNA at baseline. Finally, when posttreatment blood samples were available (n = 6), the kinetic of pretreatment/posttreatment HPV16 ctDNA was clearly associated with treatment success or failure. HPV ctDNA monitoring by ddPCR could constitute a useful and noninvasive dynamic biomarker to select HR HPV-related OPSCC patients eligible for potential treatment de-escalation and to monitor treatment response.
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http://dx.doi.org/10.1002/ijc.32804DOI Listing
August 2020

Vemurafenib for Refractory Multisystem Langerhans Cell Histiocytosis in Children: An International Observational Study.

J Clin Oncol 2019 11 12;37(31):2857-2865. Epub 2019 Sep 12.

Trousseau Hospital, Paris, France.

Purpose: Off-label use of vemurafenib (VMF) to treat mutation-positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated.

Patients And Methods: Fifty-four patients from 12 countries took VMF 20 mg/kg/d. They were classified according to risk organ involvement: liver, spleen, and/or blood cytopenia. The main evaluation criteria were adverse events (Common Terminology Criteria for Adverse Events [version 4.3]) and therapeutic responses according to Disease Activity Score.

Results: LCH extent was distributed as follows: 44 with positive and 10 with negative risk organ involvement. Median age at diagnosis was 0.9 years (range, 0.1 to 6.5 years). Median age at VMF initiation was 1.8 years (range, 0.18 to 14 years), with a median follow-up of 22 months (range, 4.3 to 57 months), whereas median treatment duration was 13.9 months (for 855 patient-months). At 8 weeks, 38 complete responses and 16 partial responses had been achieved, with the median Disease Activity Score decreasing from 7 at diagnosis to 0 ( < .001). Skin rash, the most frequent adverse event, affected 74% of patients. No secondary skin cancer was observed. Therapeutic plasma VMF concentrations (range, 10 to 20 mg/L) seemed to be safe and effective. VMF discontinuation for 30 patients led to 24 LCH reactivations. The blood allele load, assessed as circulating cell-free DNA, decreased after starting VMF but remained positive (median, 3.6% at diagnosis, and 1.6% during VMF treatment; < .001) and was associated with a higher risk of reactivation at VMF discontinuation. None of the various empirical therapies (hematopoietic stem-cell transplantation, cladribine and cytarabine, anti-MEK agent, vinblastine, etc) used for maintenance could eradicate the clone.

Conclusion: VMF seemed safe and effective in children with refractory -positive LCH. Additional studies are needed to find effective maintenance therapy approaches.
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http://dx.doi.org/10.1200/JCO.19.00456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823889PMC
November 2019

Plasma clearance of RAS mutation under therapeutic pressure is a rare event in metastatic colorectal cancer.

Int J Cancer 2020 Aug 12;147(4):1185-1189. Epub 2019 Oct 12.

Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006, Paris, France.

In metastatic colorectal cancer (mCRC), circulating tumor DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. Patients with a RAS mutated tumor included in the prospective PLACOL study were monitored for ctDNA. Analyses were based on optimized targeted next-generation sequencing and/or droplet-based digital polymerase chain reaction (ddPCR). For plasma samples without detectable mutations at progression disease, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. Among the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only two out of the eight patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only 2 samples among 36 were confirmed for clearance of RAS mutation in our series. In conclusion, this study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored.
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http://dx.doi.org/10.1002/ijc.32657DOI Listing
August 2020

Mutation Status in Circulating Tumor DNA from Patients with Metastatic Colorectal Cancer: Extended Mutation Analysis from the AGEO RASANC Study.

Cancers (Basel) 2019 Jul 17;11(7). Epub 2019 Jul 17.

Université Sorbonne Paris Cité, INSERM UMR-S1147 MEPPOT, CNRS SNC5014, Centre Universitaire des Saints-Pères, Equipe labellisée Ligue Nationale Contre le Cancer, 75006 Paris, France.

In patients with metastatic colorectal cancer (mCRC), and mutations are currently determined by tumor sample analysis. Here, we report mutation status analysis in paired tumor tissue and plasma samples of mCRC patients included in the AGEO RASANC prospective cohort study. Four hundred and twenty-five patients were enrolled. Plasma samples were analyzed by next-generation sequencing (NGS). When no mutation was identified, we used two methylated specific biomarkers (digital droplet PCR) to determine the presence or absence of circulating tumor DNA (ctDNA). Patients with conclusive ctDNA results were defined as those with at least one mutation or one methylated biomarker. The kappa coefficient and accuracy were 0.79 (95% CI: 0.67-0.91) and 97.3% (95% CI: 95.2-98.6%) between the status in plasma and tissue for patients with available paired samples ( = 405), and 0.89 (95% CI: 0.80-0.99) and 98.5% (95% CI: 96.4-99.5%) for those with conclusive ctDNA ( = 323). The absence of liver metastasis was the main factor associated to inconclusive ctDNA results. In patients with liver metastasis, the kappa coefficient was 0.91 (95% CI, 0.81-1.00) and accuracy was 98.6% (95% CI, 96.5-99.6%). We demonstrate satisfying concordance between tissue and plasma mutation detection, especially in patients with liver metastasis, arguing for plasma ctDNA testing for routine mutation analysis in these patients.
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http://dx.doi.org/10.3390/cancers11070998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679157PMC
July 2019

Liquid Biopsy: General Concepts.

Acta Cytol 2019 15;63(6):449-455. Epub 2019 May 15.

INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer, Paris, France,

Liquid biopsy provides the opportunity of detecting, analyzing and monitoring cancer in various body effluents such as blood or urine instead of a fragment of cancer tissue. It is composed of different biological matrices such as circulating tumor cells (CTCs), cell free nucleic acids, exosomes or tumors "educated platelets." In addition to representing a non- or minimally invasive procedure, it should represent a better view of tumor heterogeneity and allows for real-time monitoring of cancer evolution. Recent technological and molecular advances, greatly facilitated by the use of microfluidics in many cases, have permitted large progresses both in our ability to purify and analyze liquid biopsy components. In particular, the great developments of droplet-based digital PCR and the various optimizations of next generation sequencing technologies are central to the several validations of CTC-free DNA as a strong cancer biomarker. However, complete adoption of liquid biopsy in clinics will require pursuing recent efforts in the standardization of procedures both on the pre-analytical and analytical aspects.
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http://dx.doi.org/10.1159/000499337DOI Listing
October 2019

High-throughput multiplexed fluorescence-activated droplet sorting.

Microsyst Nanoeng 2018 22;4:33. Epub 2018 Oct 22.

1INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer, Paris, France.

Fluorescence-activated droplet sorting (FADS) is one of the most important features provided by droplet-based microfluidics. However, to date, it does not allow to compete with the high-throughput multiplexed sorting capabilities offered by flow cytometery. Here, we demonstrate the use of a dielectrophoretic-based FADS, allowing to sort up to five different droplet populations simultaneously. Our system provides means to select droplets of different phenotypes in a single experimental run to separate initially heterogeneous populations. Our experimental results are rationalized with the help of a numerical model of the actuation of droplets in electric fields providing guidelines for the prediction of sorting designs for upscaled or downscaled microsystems.
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http://dx.doi.org/10.1038/s41378-018-0033-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220162PMC
October 2018

Coins in microfluidics: From mere scale objects to font of inspiration for microchannel circuits.

Biomicrofluidics 2019 Mar 9;13(2):024106. Epub 2019 Apr 9.

Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, I-44121 Ferrara, Italy.

The fabrication of microfluidic chips remains a complex and expensive process requiring specific equipment and protocols, often if not always limited to the most privileged laboratories. As an alternative to the most sophisticated methods, the present paper describes the fabrication of microfluidic chips by an approach that uses coins as positive master for the rapid production of multigeometry chips. All steps of chip production were carried out using inexpensive approaches by low-cost chemicals and equipment. The chips were validated by different "classic" microfluidic tasks, such as hydrodynamic focusing, droplets generation, micromixing, and on-chip cell culture. The use of coins is not only an efficient method for rapid prototyping but also represents an inspiring possibility for the design of new microfluidic chips. Finally, coin-inspired chips could represent a laboratory experiment doable at a high school level.
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http://dx.doi.org/10.1063/1.5086535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456355PMC
March 2019

Gelatin-Coated Microfluidic Channels for 3D Microtissue Formation: On-Chip Production and Characterization.

Micromachines (Basel) 2019 Apr 19;10(4). Epub 2019 Apr 19.

INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe Labellisée Ligue Nationale Contre le Cancer, 75005 Paris, France.

Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.
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http://dx.doi.org/10.3390/mi10040265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523541PMC
April 2019

HPV-circulating tumoural DNA by droplet-based digital polymerase chain reaction, a new molecular tool for early detection of HPV metastatic anal cancer? A case report.

Eur J Cancer 2019 05 22;112:34-37. Epub 2019 Mar 22.

Laboratoire de virologie, Hôpital Européen Georges Pompidou, Assistance Publique - Hôpitaux de Paris, France; Faculté de Médecine Paris Descartes, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; INSERM U970, PARCC, Hôpital Européen Georges Pompidou, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.ejca.2019.02.012DOI Listing
May 2019

Incidence and risk factors for clinical neurodegenerative Langerhans cell histiocytosis: a longitudinal cohort study.

Br J Haematol 2018 11 12;183(4):608-617. Epub 2018 Nov 12.

French Reference Centre for Langerhans Cell Histiocytosis, Trousseau Hospital, Paris, France.

Neurodegenerative (ND) complications in Langerhans cell histiocytosis (LCH) are a late-onset but dramatic sequelae for which incidence and risk factors are not well defined. Based on a national prospective registry of paediatric LCH patients, we determined the incidence rate of clinical ND LCH (cND-LCH) and analysed risk factors, taking into account disease extent and molecular characteristics. Among 1897 LCH patients, 36 (1·9%) were diagnosed with a cND-LCH. The 10-year cumulative incidence of cND-LCH was 4·1%. cND-LCH typically affected patients previously treated for a multisystem, risk organ-negative LCH, represented in 69·4% of cND-LCH cases. Pituitary gland, skin and base skull/orbit bone lesions were more frequent (P < 0·001) in cND-LCH patients compared to those without cND-LCH (respectively 86·1% vs. 12·2%, 75·0% vs. 34·2%, and 63·9% vs. 28·4%). The 'cND susceptible patients' (n = 671) i.e., children who had experienced LCH disease with pituitary or skull base or orbit bone involvement, had a 10-year cND risk of 7·8% vs. 0% for patients who did not meet these criteria. Finally, BRAF status added important information among these cND susceptible patients, with the 10-year cND risk of 33·1% if a BRAF mutation was present compared to 2·9% if it was absent (P = 0·002).
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http://dx.doi.org/10.1111/bjh.15577DOI Listing
November 2018

Development of digital PCR molecular tests for clinical practice: principles, practical implementation and recommendations.

Ann Biol Clin (Paris) 2018 Oct;76(5):505-523

Assistance publique - Hôpitaux de Marseille, Service d'anatomie pathologique et neuropathologie, Marseille, France, ID Solutions, Grabels, France.

Digital PCR (dPCR) is a 3rd generation technology that complements traditional end-point PCR and real-time PCR. It was developed to overcome certain limitations of conventional amplification techniques, in particular for the detection of small amounts of nucleic acids and/or rare variants. This technology is in a full swing because of its high sensitivity and major applications in various domains such as oncology, transplantation or non-invasive prenatal testing. Consequently, PCRd also has great interest in many areas of medical biology, particularly for clinical applications aiming at detecting and quantifying specific genetic or epigenetic alterations of nucleic acids, even with specimens containing very low concentration of the nucleic acids of interest (e.g. liquid biopsies). However, this technique requires a good training of users and compliance with certain precautions. A lack in such a knowledge can lead to many errors in the conduct of the experiment and the interpretation of the results. In this review, we present the context in which this technology has emerged by describing in particular its principle and the main factors that can influence the quality of the analysis. Then, we propose a number of practical recommendations for the implementation of a test based on dPCR in clinical laboratories with an eye on quality requirements.
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http://dx.doi.org/10.1684/abc.2018.1372DOI Listing
October 2018

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

Clin Chem 2018 09 14;64(9):1296-1307. Epub 2018 Jun 14.

Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;

Background: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.

Methods: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer ( c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement.

Results: Concentration values for samples of G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively).

Conclusions: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.
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http://dx.doi.org/10.1373/clinchem.2017.285478DOI Listing
September 2018

Role of circulating tumor DNA in the management of patients with colorectal cancer.

Clin Res Hepatol Gastroenterol 2018 10 5;42(5):396-402. Epub 2018 Apr 5.

INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer, Paris, France; Department of Gastroenterology and Digestive Oncology, European Georges Pompidou Hospital, AP-HP, Paris Descartes University, Paris, France. Electronic address:

Colorectal cancer is a major health burden with a prognosis that has been improved with the progresses in diagnosis and the advance of chemotherapy and personalized medicine. However, because of intra-tumor heterogeneity, clonal evolution and selection, tumors often develop resistance to treatments. "Liquid biopsy" is a minimally invasive method, based on analysis of tumor-specific material in peripheral blood samples of patients. Analysis of tumor specific genetic or epigenetic alterations in cell-free circulating nucleic acids may reflect the molecular heterogeneity of the underlying disease process and serial testing could allow to monitor its temporal genomic changing without using re-biopsy. In this review, we focused on the role of circulating tumor DNA (ctDNA) as a biomarker in the management of patients with colorectal cancer at early and advanced stages. Through recent studies, we described its promising clinical applications for diagnosis, detection of recurrence after surgery and monitoring for tumor response or therapeutic resistance in metastatic setting. Such recent developments offer new perspectives for personalized medicine in colorectal cancer but still needs some standardized detection methods and further studies to validate its use in clinical routine.
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http://dx.doi.org/10.1016/j.clinre.2018.03.002DOI Listing
October 2018

Mutation and Methylation Analysis of Circulating Tumor DNA Can Be Used for Follow-up of Metastatic Colorectal Cancer Patients.

Clin Colorectal Cancer 2018 06 22;17(2):e369-e379. Epub 2018 Feb 22.

Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium; Department of Oncology, Antwerp University Hospital, Antwerp, Belgium. Electronic address:

Background: Targeted therapies, although contributing to survival improvement in metastatic colorectal cancer (mCRC), are expensive and may cause adverse effects. Therefore, confirming that patients are responding to these therapies is extremely important. Currently, follow-up is performed using radiographic evaluation, which has its limitations. Liquid biopsies, reflecting real-time tumor characteristics, hold great potential in monitoring tumor disease.

Patients And Methods: Blood samples were collected at different time points during treatment of 24 mCRC patients. Mutation and NPY methylation picoliter droplet-based digital PCR (ddPCR) assays were performed on circulating DNA to investigate whether these assays can be used for disease monitoring.

Results: The results of the mutation and methylation assays were correlated with each other and corresponded with the results of radiographic evaluation. There was a steep decrease in circulating tumor DNA levels immediately after treatment initiation. Furthermore, circulating tumor DNA levels were increased in progressive samples and were undetectable in patients undergoing curative surgery.

Conclusion: This prospective study showed that tumor-specific mutation and NPY methylation ddPCR assays performed on circulating DNA can be used for the follow-up of mCRC patients during treatment and could complement current follow-up methods. The analysis of NPY methylation is promising, as it has the additional advantage that no prior knowledge of tumor mutations is needed.
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http://dx.doi.org/10.1016/j.clcc.2018.02.006DOI Listing
June 2018

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.

Anal Chem 2018 03 9;90(6):3766-3774. Epub 2018 Mar 9.

INSERM UMR-S1147, CNRS SNC5014 , Paris Descartes University , 45 rue des Saints-Pères , Paris , France.

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.
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http://dx.doi.org/10.1021/acs.analchem.7b04034DOI Listing
March 2018

Phenotypes and survival in Erdheim-Chester disease: Results from a 165-patient cohort.

Am J Hematol 2018 May 10;93(5):E114-E117. Epub 2018 Feb 10.

Internal Medicine Department 2, Pitié-Salpêtrière Hospital, French National Centre for Rare Systemic Diseases, AP-HP, Paris, 75013, France.

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http://dx.doi.org/10.1002/ajh.25055DOI Listing
May 2018

Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective.

Expert Rev Mol Diagn 2018 01 13;18(1):7-17. Epub 2017 Nov 13.

a INSERM UMR-S1147, CNRS SNC5014; Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer , Paris , France.

Introduction: Early detection of cancers through the analysis of ctDNA could have a significant impact on morbidity and mortality of cancer patients. However, using ctDNA for early cancer diagnosis is challenging partly due to the low amount of tumor DNA released in the circulation and its dilution within DNA originating from non-tumor cells. Development of new technologies such as droplet-based digital PCR (ddPCR) or optimized next generation sequencing (NGS) has greatly improved the sensitivity, specificity and precision for the detection of rare sequences. Areas covered: This paper will focus on the potential application of ddPCR and optimized NGS to detect ctDNA for detection of cancer recurrence and minimal residual disease as well as early diagnosis of cancer patients. Expert commentary: Compared to tumor tissue biopsies, blood-based ctDNA analyses are minimally invasive and accessible for regular follow-up of cancer patients. They are also described as a better picture of patients' pathology allowing to highlight both tumor heterogeneity and multiple tumor sites. After a brief introduction on the application of the follow-up of ctDNA using genetic or epigenetic biomarkers for prognosis and surveillance of cancer patients, potential perspectives of using ctDNA for early diagnosis of cancers will be presented.
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http://dx.doi.org/10.1080/14737159.2018.1400384DOI Listing
January 2018

Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.

Nat Protoc 2017 Sep 24;12(9):1912-1932. Epub 2017 Aug 24.

LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.

Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 μl of reaction mix.
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http://dx.doi.org/10.1038/nprot.2017.092DOI Listing
September 2017