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. colicells, expressing either the reporter enzyme β-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of ∼300 droplets s⁻¹. The false positive error rate of the sorter at this throughput was <1 in 10⁴ droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. colicells, 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 (∼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.

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan^® assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan^®probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRASgene in the presence of a 200 000-fold excess of unmutated KRASgenes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan^®probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAScodon 12 in parallel in a single experiment.

BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Multiplex miRNA detection is a promising way to non-invasive diagnostics. In this review, we discuss the recent advances to the multiplexing of miRNA quantification.

Purpose: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with a broad spectrum of clinical manifestations and outcomes in children. The somatic BRAF^V600E mutation occurs frequently, but clinical significance remains to be determined. Patients and Methods: BRAF^V600E mutation was investigated in a French LCH cohort. We analyzed associations between mutation status and clinical presentation, extent of disease, reactivation rate, response to therapy, and long-term permanent sequelae. Results: Among 315 patients with successfully determined BRAF status, 173 (54.6%) carried a BRAF^V600E mutation. Patients with BRAF^V600E manifested more severe disease than did those with wild-type BRAF. Patients with BRAF^V600E comprised 87.8% of patients (43 of 49) with multisystem LCH with risk organ involvement (liver, spleen, hematology), 68.6% of patients (35 of 51) with multisystem LCH without risk organ involvement, 43.9% of patients (86 of 196) with single-system LCH, and 42.1% of patients (8 of 19) with lung-involved LCH (P < .001). BRAF^V600E mutation was also associated with organ involvement that could lead to permanent, irreversible damage, such as neurologic (75%) and pituitary (72.9%) injuries. Compared with patients with wild-type BRAF, patients with BRAF^V600E more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% v 3.3%; P = .001), showed a higher reactivation rate (5-year reactivation rate, 42.8% v 28.1%; P = .006), and had more permanent, long-term consequences from disease or treatment (27.9% v 12.6%; P = .001). Conclusion: In children with LCH, BRAF^V600E mutation was associated with high-risk features, permanent injury, and poor short-term response to chemotherapy. Further population-based studies should be undertaken to confirm our observations and to assess the impact of BRAF inhibitors for this subgroup of patients who may benefit from targeted therapy.

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.