Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

Region-specific changes in prodynorphin mRNA and ir-dynorphin A levels after kindled seizures.

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Kindled seizure-evoked somatostatin release in the hippocampus: inhibition by MK-801

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Early changes in prodynorphin mRNA and ir-dynorphin A levels after kindled seizures in the rat.

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Mechanisms of action of CHF3381 in the forebrain

1. Aim of this study was to gain insight into the mechanism of action of CHF3381, a novel putative antiepileptic and neuroprotective drug. 2. CHF3381 blocked NMDA currents in primary cultures of cortical neurons: maximal effect was nearly −80% of the NMDA-evoked current, with EC(50) of approximately 5 μM. This effect was selective, reversible, use-dependent and elicited at the concentrations reached in the rodent brain after peripheral administration of therapeutic doses. 3. CHF3381 also inhibited voltage-gated Na(+) currents in an apparently voltage-dependent manner. However, this effect could be obtained only at relatively high concentrations (100 μM). 4. Consistent with the mild effects on voltage-gated Na(+) channels, CHF3381 (100 μM) failed to affect electrical stimulation-evoked glutamate overflow in hippocampal slices. In contrast, the anti-convulsant agent and Na(+) channel blocker lamotrigine (100 μM) inhibited stimulation-evoked glutamate overflow by approximately 50%. 5. CHF3381 reduced kindled seizure-induced c-fos mRNA levels within the same brain regions, and to a similar level, as the selective NMDA receptor antagonist MK801, providing circumstantial evidence to the idea that CHF3381 blocks NMDA receptors in vivo. 6. The present mechanistic studies suggest that the primary mechanism of action of CHF3381 in the forebrain is blockade of NMDA receptors. On this basis, this compound may have a potential use in other diseases caused by or associated with a pathologically high level of NMDA receptor activation

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect