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Background: Circulating tumour DNA (ctDNA) analysis promises to improve the clinical care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 patients with advanced-stage cutaneous melanoma through multiple cycles of immunotherapy. Method: A melanoma-specific ctDNA next-generation sequencing (NGS) panel, droplet digital polymerase chain reaction (ddPCR) and mass spectrometry analysis were used to identify ctDNA mutations in longitudinal blood plasma samples from Aotearoa New Zealand (NZ) patients receiving immunotherapy for melanoma. These technologies were used in conjunction to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. Results: During the course of immunotherapy treatment, a high level of dynamic mutational complexity was identified in blood plasma, including multiple BRAF mutations in the same patient, clinically relevant BRAF mutations emerging through therapy and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis–reanalysis concordance, as well as concordance between different ctDNA measurement technologies. In addition, we observed > 90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared with standard EDTA blood collection protocols with rapid processing. We also found that the undetectability of ctDNA at a proportion of treatment cycles was associated with durable clinical benefit (DCB). Conclusion: We found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically relevant mutations, adding support for expanded clinical trials of this technology in a variety of oncology settings. [ABSTRACT FROM AUTHOR]

miRNAs are a well-studied class of non-coding RNAs, predominantly functioning to down-regulate gene expression from messenger RNA (mRNA) in a targeted manner by binding to complementary sequence on the target mRNA. Many miRNAs have been linked to the development of hallmarks of cancer. miRNAs represent valuable therapeutic targets to exploit in the search for novel cancer treatments, due to their ubiquitous expression and their ability to tightly regulate the gene expression of a whole host of genes and pathways in a single hit. The miRNA system may be harnessed for therapeutic use either through replacement of tumour suppressive miRNAs lost in cancer, or through inhibition of oncogenic miRNAs overexpressed in cancer. There is a large body of work investigating optimal systemic and localised delivery strategies, and while miRNA therapeutics show promise, it is clear that further developments to delivery strategies may be required to allow safe translation of miRNAs to the clinic. The information gleaned from miRNA signatures as biomarkers is already proving invaluable in the fight to better understand and treat individual tumours, and there is great promise to the applications of these small, but mighty molecules in the future of cancer therapeutics. [ABSTRACT FROM AUTHOR]

During axon pathfinding, growth cones commonly show changes in sensitivity to guidance cues that follow a cell-intrinsic timetable. The cellular timer mechanisms that regulate such changes are, however, poorly understood. Here we have investigated microRNAs (miRNAs) in the timing control of sensitivity to the semaphorin Sema3A in Xenopus laevis retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant neuropilin-1 (NRP1) receptor expression and caused cell-autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was newly identified as a target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 is important in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that miRNAs may act broadly as linear timers in vertebrate neuronal development. [ABSTRACT FROM AUTHOR]

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms. [ABSTRACT FROM AUTHOR]

A correction to the article "A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans" that was published in the September 12, 2010 issue is presented.