G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5–1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%.Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8-12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL-positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase-3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up-regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia.

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Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20.Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.

Introduction: Gastroenteropancreatic neuroendocrine carcinomas (GEPNEC) are characterised by a heterogeneous molecular profile and a poor prognosis. Circulating tumour DNA (ctDNA) analysis may be useful for NEC management. This study aimed at describing ctDNA mutations, to assess their predictive value for response to chemotherapies, and their change according to disease progression. Methods: The CIRCAN-NEC study included patients with GEPNEC or NEC from an unknown primary, scheduled to begin first or second-line chemotherapy. Blood samples were collected prior to chemotherapy initiation, at first evaluation, and during disease progression. ctDNA were sequenced by next-generation sequencing (NGS). Molecular response was defined as a decrease of at least 30% of the mutant allele fraction (MAF). Results: All 24 patients included received platinum-etoposide first-line chemotherapy; 19 received a FOLFIRI-based post first-line regimen. Twenty-two patients had at least one driver mutation: TP53 (n=21), RB1 (n=2), KRAS (n=4), BRAF (n=3). Ten (42%) had a “adenocarcinoma-like” profile. Five of six patients with matching ctDNA/tissue NGS harboured at least one concordant mutation (44% concordance at the gene level). The concordance rate between ctDNA-mutation/immunohistochemistry-profile was 64% (7/11) for TP53/p53+ and 14% (1/7) for RB1/pRb-. In this pilot study including few patients by subgroups, patients with KRAS (HR=3.60, 95%CI [1.06-12.04]) and BRAF (HR=4.25, 95%CI [1.11-16.40]) mutations had shorter progression-free survival (PFS) under platinum-etoposide, while the two patients with RB1 mutations had shorter PFS under FOLFIRI-based chemotherapy. Twenty-eight periods of treatment were assessed: 10 patients had a molecular response (7/10 had a morphological response), which was associated with longer PFS (HR=0.37, 95%CI [0.15; 0.91]). Conclusion: This pilot study shows a high sensitivity of ctDNA assessment, which is encouraging for the future management of GEPNEC (tumour molecular diagnosis and evaluation of disease progression).

Magnetophoresis-based microfluidic devices offer simple and reliable manipulation of micro-scale objects and provide a large panel of applications, from selective trapping to high-throughput sorting. However, the fabrication and integration of micro-scale magnets in microsystems involve complex and expensive processes. Here we report on an inexpensive and easy-to-handle fabrication process of micrometer-scale permanent magnets, based on the self-organization of NdFeB particles in a polymer matrix (polydimethylsiloxane, PDMS). A study of the inner structure by X-ray tomography revealed a chain-like organization of the particles leading to an array of hard magnetic microstructures with a mean diameter of 4 µm. The magnetic performance of the self-assembled micro-magnets was first estimated by COMSOL simulations. The micro-magnets were then integrated into a microfluidic device where they act as micro-traps. The magnetic forces exerted by the micro-magnets on superparamagnetic beads were measured by colloidal probe atomic force microscopy (AFM) and in operando in the microfluidic system. Forces as high as several nanonewtons were reached. Adding an external millimeter-sized magnet allowed target magnetization and the interaction range to be increased. Then, the integrated micro-magnets were used to study the magnetophoretic trapping efficiency of magnetic beads, providing efficiencies of 100% at 0.5 mL/h and 75% at 1 mL/h. Finally, the micro-magnets were implemented for cell sorting by performing white blood cell depletion.

Circulating cell-free DNA (cfDNA) has the potential to be a specific biomarker for the therapeutic management of lung cancer patients. Here, a new sequencing error-reduction method based on molecular amplification pools (MAPs) was utilized to analyze cfDNA in lung cancer patients. We determined the accuracy of MAPs plasma sequencing with respect to droplet digital polymerase chain reaction assays (ddPCR), and tested whether actionable mutation discovery is improved by next-generation sequencing (NGS) in a clinical setting. This study reports data from 356 lung cancer patients receiving plasma testing as part of routine clinical management. Sequencing of cfDNA via MAPs had a sensitivity of 98.5% and specificity 98.9%. The ddPCR assay was used as the reference, since it is an established, accurate assay that can be performed contemporaneously on the same plasma sample. MAPs sequencing detected somatic variants in 261 of 356 samples (73%). Non-actionable clonal hematopoiesis-associated variants were identified via sequencing in 21% of samples. The accuracy of this cfDNA sequencing approach was similar to that of ddPCR assays in a clinical setting, down to an allele frequency of 0.1%. Due to broader coverage and high sensitivity for insertions and deletions, sequencing via MAPs afforded important detection of additional actionable mutations.