Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis.Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRASmutated allele fraction.Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors.Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

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.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Miniaturized devices for the extraction of DNA have been used for assessing genetic material in biological, forensic and environmental samples. However, the ability to isolate trace amounts of highly fragmented DNA from biological fluids remains a challenge. The current work reports a microfluidic approach that combines on line a dynamic magnetic extraction procedure with droplet-based digital PCR (ddPCR). This strategy maximizes the surface area for DNA binding within the chip, in order to capture short DNA fragments, with the possibility of recovering the purified samples into picoliter volumes for high sensitivity mutation detection. The application of this technology to capture circulating cell-free DNA (ccfDNA) from serum samples of cancer patients is demonstrated herein, with efficiencies comparable to standard column-based DNA extraction methods. This technology uses lesser amounts of required material and reagents, and has a higher potential for automation and multiplex DNA analysis. Furthermore, this approach can also be extended for the detection of other circulating biomarkers, such as nucleic acid sequences with aberrant methylation patterns or miRNA.

In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.

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Synthetic approaches based on the patterned deposition of volatile molecules from the vapor phase have been used extensively in the creation of surface-chemical gradients; however, the ability to generate diffusion-controlled 1D and 2D gradients from multiple sources remains a challenge. The current work reports a one-step approach to the synthesis of continuous and periodic chemical gradients with simple and intricate geometries using multiple sources within custom reaction chambers. Specifically, this approach provides precise, simultaneous control over the physico-chemical conditions (e.g., concentration, evaporation rate, and direction of diffusion flux of the chemical moieties) and the geometrical parameters (e.g., size, shape, and position) during surface functionalization, thus enabling materials with predictable surface-chemical gradients applicable to the manipulation and/or organization of liquid droplets and that can generate assemblies of functional solids (e.g., silver nanoparticles) that are transferrable via stamping. These surfaces can be useful to various fields, for example, molecular diagnostics and microfabrication. Furthermore, this work extends the application of these surfaces to the precise placement and manipulation of gas bubbles that can have potential use in, for example, controlling bubble nucleation in processes designed to manage heat transfer.Received: ((will be filled in by the editorial staff))Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff)) and showed that they are transferrable to various surfaces, and applicable to, for example, the manipulation of liquids droplets and gas bubbles.

The lamination of mechanically stiff structures to elastic materials is prevalent in biological systems and popular in many emerging synthetic systems, such as soft robotics, microfluidics, stretchable electronics, and pop-up assemblies. The disparate mechanical and chemical properties of these materials have made it challenging to develop universal synthetic procedures capable of reliably adhering to these classes of materials together. Herein, a simple and scalable procedure is described that is capable of covalently laminating a variety of commodity ("off-the-shelf") thermoplastic sheets to silicone rubber films. When combined with laser printing, the nonbonding sites can be "printed" onto the thermoplastic sheets, enabling the direct fabrication of microfluidic systems for actuation and liquid handling applications. The versatility of this approach in generating thin, multifunctional laminates is demonstrated through the fabrication of milliscale soft actuators and grippers with hinged articulation and microfluidic channels with built-in optical filtering and pressure-dependent geometries. This method of fabrication offers several advantages, including technical simplicity, process scalability, design versatility, and material diversity. The concepts and strategies presented herein are broadly applicable to the soft robotics, microfluidics, and advanced and additive manufacturing communities where hybrid rubber/plastic structures are prevalent.