Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM

<p>Abstract</p> <p>Background</p> <p>The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM.</p> <p>Methods</p> <p>Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the <it>CDKN2A </it>(<it>p16</it>) and <it>RARB </it>promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample.</p> <p>Results</p> <p><it>CDKN2A </it>promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the <it>RARB </it>promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess <it>RARB </it>methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP.</p> <p>Conclusion</p> <p>MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.</p

Neodymium-140 DOTA-LM3:Evaluation of an <i>In Vivo</i> Generator for PET with a Non-Internalizing Vector

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior

¹⁸F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal cancer therapy

Within the field of nanoparticle-assisted photothermal cancer therapy, focus has mostly been on developing novel heat-generating nanoparticles with the right optical and dimensional properties. Comparison and evaluation of their performance in tumor-bearing animals are commonly assessed by changes in tumor volume; however, this is usually a late-occurring event. This study implements 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging to perform early evaluation of the treatment outcome of photothermal therapy. Silica-gold nanoshells (NS) are administered intravenously to nude mice bearing human neuroendocrine tumor xenografts and the tumors are irradiated by a near-infrared laser. The animals are positron emission tomography scanned with 2-deoxy-2-[F-18]fluoro-D-glucose one day before and one day after treatment. Using this setup, a significant decrease in tumor uptake of 2-deoxy-2-[F-18]fluoro-D-glucose is found already one day after therapy in the group receiving NS and laser treatment compared to control animals. At this time point no change in tumor volume can be detected. Moreover, the change in tumor uptake, is used to stratify the animals into responders and non-responders, where the responding group matched improved survival. Overall, these findings support the use of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging for preclinical and clinical evaluation and optimization of photothermal therapy

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials

Genome-wide association analysis of more than 120,000 individuals identifies 15 new susceptibility loci for breast cancer.

Genome-wide association studies (GWAS) and large-scale replication studies have identified common variants in 79 loci associated with breast cancer, explaining ∼14% of the familial risk of the disease. To identify new susceptibility loci, we performed a meta-analysis of 11 GWAS, comprising 15,748 breast cancer cases and 18,084 controls together with 46,785 cases and 42,892 controls from 41 studies genotyped on a 211,155-marker custom array (iCOGS). Analyses were restricted to women of European ancestry. We generated genotypes for more than 11 million SNPs by imputation using the 1000 Genomes Project reference panel, and we identified 15 new loci associated with breast cancer at P < 5 × 10(-8). Combining association analysis with ChIP-seq chromatin binding data in mammary cell lines and ChIA-PET chromatin interaction data from ENCODE, we identified likely target genes in two regions: SETBP1 at 18q12.3 and RNF115 and PDZK1 at 1q21.1. One association appears to be driven by an amino acid substitution encoded in EXO1.BCAC is funded by Cancer Research UK (C1287/A10118, C1287/A12014) and by the European Community's Seventh Framework Programme under grant agreement 223175 (HEALTH-F2-2009-223175) (COGS). Meetings of the BCAC have been funded by the European Union COST programme (BM0606). Genotyping on the iCOGS array was funded by the European Union (HEALTH-F2-2009-223175), Cancer Research UK (C1287/A10710, C8197/A16565), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer program and the Ministry of Economic Development, Innovation and Export Trade of Quebec, grant PSR-SIIRI-701. Combination of the GWAS data was supported in part by the US National Institutes of Health (NIH) Cancer Post-Cancer GWAS initiative, grant 1 U19 CA148065-01 (DRIVE, part of the GAME-ON initiative). For a full description of funding and acknowledgments, see the Supplementary Note.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.324