Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine ...clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.
Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor ...delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
The pharmaceutical industry is in the process of re-inventing its pipeline in an attempt to overcome its increasing phase II and III attrition rates. Here, we describe how systems pharmacology can be ...used as a risk assessment tool to alleviate this problem before bringing in larger investments. We propose that this translational research tool could provide a valuable, complementary addition to other emerging innovative approaches for target identification and validation in discovery and, ultimately, for aiding clinical trial optimization.
Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide ...copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations.
In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing).
Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%.
Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.
The repair of DNA damage by homologous recombination (HR) is a key pathway for the maintenance of genetic stability in mammalian cells, especially during and following DNA replication. The central HR ...protein is RAD51, which ensures high fidelity DNA repair by facilitating strand exchange between damaged and undamaged homologous DNA segments. Several RAD51-like proteins, including XRCC2, appear to help with this process, but their roles are not well understood. Here we show that XRCC2 is highly conserved and that most substantial truncations of the protein destroy its ability to function. XRCC2 and its partner protein RAD51L3 are found to interact with RAD51 in the 2-hybrid system, and XRCC2 is shown to be important but not essential for the accumulation of RAD51 at the sites of DNA damage. We visualize the localization of XRCC2 protein at the same sites of DNA damage for the first time using specialized irradiation conditions. Our data indicate that an important function of XRCC2 is to enhance the activity of RAD51, so that the loss of XRCC2 results in a severe delay in the early response of RAD51 to DNA damage.
Secretory and membrane‐bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation ...of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double‐deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double‐deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high‐level production of recombinant proteins.
The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® ...Array platform is designed to produce actionable mutation calls in archival material.
We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls.
A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5-10%) than product claims indicated.
This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results.
Pulmonary arterial hypertension is a proliferative vascular disease, characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Prostacyclin ...(PGI2) analogues have anti-proliferative effects on distal human pulmonary artery smooth muscle cells (PASMCs), which are dependent on intracellular cAMP stimulation. We therefore sought to investigate the involvement of the main cAMP-specific enzymes, phosphodiesterase type 4 (PDE4), responsible for cAMP hydrolysis.
Distal human PASMCs were derived from pulmonary arteries by explant culture (n = 14, passage 3-12). Responses to platelet-derived growth factor-BB (5-10 ng/ml), serum, PGI2 analogues (cicaprost, iloprost) and PDE4 inhibitors (roflumilast, rolipram, cilomilast) were determined by measuring cAMP phosphodiesterase activity, intracellular cAMP levels, DNA synthesis, apoptosis (as measured by DNA fragmentation and nuclear condensation) and matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) production.
Expression of all four PDE4A-D genes was detected in PASMC isolates. PDE4 contributed to the main proportion (35.9 +/- 2.3%, n = 5) of cAMP-specific hydrolytic activity demonstrated in PASMCs, compared to PDE3 (21.5 +/- 2.5%), PDE2 (15.8 +/- 3.4%) or PDE1 activity (14.5 +/- 4.2%). Intracellular cAMP levels were increased by PGI2 analogues and further elevated in cells co-treated with roflumilast, rolipram and cilomilast. DNA synthesis was attenuated by 1 microM roflumilast (49 +/- 6% inhibition), rolipram (37 +/- 6%) and cilomilast (30 +/- 4%) and, in the presence of 5 nM cicaprost, these compounds exhibited EC50 values of 4.4 (2.6-6.1) nM (Mean and 95% confidence interval), 59 (36-83) nM and 97 (66-130) nM respectively. Roflumilast attenuated cell proliferation and gelatinase (MMP-2 and MMP-9) production and promoted the anti-proliferative effects of PGI2 analogues. The cAMP activators iloprost and forskolin also induced apoptosis, whereas roflumilast had no significant effect.
PDE4 enzymes are expressed in distal human PASMCs and the effects of cAMP-stimulating agents on DNA synthesis, proliferation and MMP production is dependent, at least in part, on PDE4 activity. PDE4 inhibition may provide greater control of cAMP-mediated anti-proliferative effects in human PASMCs and therefore could prove useful as an additional therapy for pulmonary arterial hypertension.
The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Several proteins showing sequence similarity ...to Rad51 have previously been identified in both yeast and human cells. In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange. Here, we report the purification of one of the representatives of the RAD51 family in human cells. We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence. We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2. By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct. Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.
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