ABSTRACT
BACKGROUND: Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. PATIENTS AND METHODS: We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. RESULTS: Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. CONCLUSION: We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.
Subject(s)
DNA/drug effects , Formaldehyde/chemistry , Methanol/chemistry , Neoplasms/diagnosis , Tissue Fixation/methods , Base Sequence , DNA/analysis , High-Throughput Nucleotide Sequencing , Humans , Middle Aged , Paraffin Embedding , Sequence Analysis, DNAABSTRACT
In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene). The changes in intensity and lambda(max) in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, Km=6.3 x 10(-8) M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 microM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.
Subject(s)
Antiviral Agents/administration & dosage , Cytomegalovirus/drug effects , Intracellular Fluid/drug effects , Oligonucleotides, Antisense/administration & dosage , Serum Albumin, Bovine/administration & dosage , Animals , Antiviral Agents/pharmacokinetics , Cattle , Cell Line , Cytomegalovirus/metabolism , Dose-Response Relationship, Drug , Humans , Intracellular Fluid/metabolism , Oligonucleotides, Antisense/pharmacokinetics , Serum Albumin, Bovine/pharmacokineticsABSTRACT
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