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.
This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C
), second (C
) and/or third (C
) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation (
) or hypermethylation (
). 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.
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 C
had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5-12.6;
< 0.0001). By analyzing the evolution of the ctDNA concentration between C
and C
or C
(C
), 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" (
= 58) versus "bad ctDNA responder" (
) had a better objective response rate (
< 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09-0.40;
< 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11-0.57;
< 0.001).
This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC.
.
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.
Display omitted
•A reversibly sealed microfluidic platform for 3D tumor microtissue formation and analysis is developed.•After the assay, the resealable chip allows selective spheroid retrieval for ...in-depth analysis.•On and off-chip characterization of 3D microtissue is carried out.•Cytotoxicity effect of drug was analyzed by using three different methods (optical, fluorescence and scanning electron microscopy).•We analyzed the SEM images to study the morphology parameters of spheroids subjected to drug treatments.
Nowadays, microfluidic 3D cell culture is widely used to mimic complex microtissue and dynamic environment, performing more realistic in vitro assays for drug testing. Herein, we developed a novel microfluidic platform for tumor microtissue culture, drug response analysis and versatile microscopic characterization. By reversibly bonding the chip, we go beyond the on/off chip tradeoff, which allows us to perform both fluorescence and SEM characterization of tumor microtissues on a simple platform. The microfluidic chip consists of spherical microwells connected via microchannels, bonded through a magnetic system. Colorectal cancer HT-29 cells were cultured as spherical microtissues on chip and their growth kinetics monitored. The cytotoxic activity of Camptothecin was evaluated by in situ live/dead fluorescence staining and quantification of morphology parameters. Finally, we demonstrated the possibility to collect the 3D tumor microtissues and characterize their surface damaged by the drug using scanning electron microscopy. This reversibly sealed microfluidic platform thus enables to grow sets of 3D tumor microtissues in a controlled dynamic microenvinroment, and subsequently to retrieve the 3D tumor microtissues after chemotherapeutic treatment for in-depth analysis.
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.
Erdheim-Chester disease (ECD) is a rare histiocytic disorder that is challenging to diagnose and treat. We performed molecular analysis of BRAF in the largest cohort of ECD patients studied to date ...followed by N/KRAS, PIK3CA, and AKT1 mutational analysis in BRAF wild-type patients. Forty-six of 80 (57.5%) of patients were BRAFV600E-mutant. NRAS mutations were detected in 3 of 17 ECD BRAFV600E wild-type patients. PIK3CA mutations (p.E542K, p.E545K, p.A1046T, and p.H1047R) were detected in 7 of 55 patients, 4 of whom also had BRAF mutations. Mutant NRAS was present in peripheral blood CD14+ cells, but not lymphoid cells, from an NRASQ61R mutant patient. Our results underscore the central role of RAS-RAF-MEK-ERK activation in ECD and identify an important role of activation of RAS-PI3K-AKT signaling in ECD. These results provide a rationale for targeting mutant RAS or PI3K/AKT/mTOR signaling in the subset of ECD patients with NRAS or PIK3CA mutations.
•PIK3CA and NRAS mutations are recurrent in BRAFV600E wild-type ECD patients.•57.5% (46/80) of ECD patients have a BRAFV600E mutation, and an additional 10.9% and 3.7% have PIK3CA and NRAS mutations, respectively.
Most cell studies are performed at a population level, relying on the assumption of a normal distribution of the function and fate of a cell among a population. However, technologies allowing ...single-cell analysis (SCA) have recently arisen and have led to increasing evidence of cell population heterogeneity and its importance. Tremendous amounts of new data could now be uncovered to redefine our understanding of cell omics. Microfluidics has emerged as a major technological player in this new era and is gradually increasing in use among biology laboratories, mainly due to the single-cell high-throughput handling solutions it offers. In this review, we assess its use and relevance for omics analysis at the single-cell level, with a specific focus on compartment-based microfluidic approaches.
We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell ...sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s(-1). To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme beta-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of approximately 300 droplets s(-1). The false positive error rate of the sorter at this throughput was <1 in 10(4) droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( approximately 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.
PDF
