Philippe Nizard

The surface functionalization of silver nanoparticles (NPs) by Raman reporters has stimulated a wide interest in recent years for the design of Surface-Enhanced Raman Spectroscopy (SERS) labels. However, silver NPs are prone to oxidation and aggregation, which strongly limits their applications. The design of stable SERS tags based on Ag NPs still represents a major challenge for Raman bioimaging. We address this issue herein by taking advantage of aryl diazonium salt chemistry to obtain stable Ag NPs functionalized by multifunctional polyaryl layers bearing different Raman reporters (–NO₂, –CN, –CCH). The resulting SERS-encoded Ag NPs were characterized by UV-vis absorption, transmission electron microscopy (TEM) and SERS. The formation of multilayers at the surface of Ag NPs gives access to new spectrally distinguishable SERS codes thus broadening the library of available Raman tags. Proof-of-concept Raman imaging experiments were performed on cancer cells (HeLa) after NP uptake, highlighting the large potentials of diazonium salt chemistry to design Ag NPs-based SERS labels for Raman bioimaging.

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

Summary: The BRAF^V600E mutation is reported in half of patients with Langerhans cell histiocytosis (LCH). This study investigated the detection of the BRAF^V600E allele in circulating cell‐free (ccf) DNA in a paediatric LCH cohort. Children with BRAF^V600E‐mutated LCH were investigated to detect ccf BRAF^V600E at diagnosis (n = 48) and during follow‐up (n = 17) using a picolitre‐droplet digital PCR assay. At diagnosis, ccf BRAF^V600E was positive in 15/15 (100%) patients with risk‐organ positive multisystem (RO+ MS) LCH, 5/12 (42%) of patients with RO− MS LCH and 3/21 (14%) patients with single‐system (SS) LCH (P < 0·001, Fisher's exact test). The positive BRAF^V600E load was higher for RO+ patients (mean, 2·90%; range, 0·04–11·4%) than for RO− patients (mean, 0·16%; range, 0·01–0·39) (P = 0·003, Mann–Whitney U test). After first‐line vinblastine‐steroid induction therapy, 7/7 (100%) of the non‐responders remained positive for ccf BRAF^V600E compared to 2/4 (50%) of the partial‐responders and 0/4 of the complete responders (P = 0·002, Fisher's exact test). Six children treated with vemurafenib showed a clinical response that was associated with a decrease in the ccf BRAF^V600E load at day 15. Thus, ccf BRAF^V600E is a promising biomarker for monitoring the response to therapy for children with RO+ MS LCH or RO− LCH resistant to first‐line chemotherapy.

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.

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 KRAS-mutated 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. Clin Cancer Res; 21(5); 1087–97. ©2014 AACR.

Cell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells.

Key Points: 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.

Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)‐associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER‐associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.