A three-gene DNA methylation biomarker accurately classifies early stage prostate cancer.

BACKGROUND: We identify and validate accurate diagnostic biomarkers for prostate cancer through a systematic evaluation of DNA methylation alterations. MATERIALS AND METHODS: We assembled three early prostate cancer cohorts (total patients = 699) from which we collected and processed over 1300 prostatectomy tissue samples for DNA extraction. Using real-time methylation-specific PCR, we measured normalized methylation levels at 15 frequently methylated loci. After partitioning sample sets into independent training and validation cohorts, classifiers were developed using logistic regression, analyzed, and validated. RESULTS: In the training dataset, DNA methylation levels at 7 of 15 genomic loci (glutathione S-transferase Pi 1 [GSTP1], CCDC181, hyaluronan, and proteoglycan link protein 3 [HAPLN3], GSTM2, growth arrest-specific 6 [GAS6], RASSF1, and APC) showed large differences between cancer and benign samples. The best binary classifier was the GAS6/GSTP1/HAPLN3 logistic regression model, with an area under these curves of 0.97, which showed a sensitivity of 94%, and a specificity of 93% after external validation. CONCLUSION: We created and validated a multigene model for the classification of benign and malignant prostate tissue. With false positive and negative rates below 7%, this three-gene biomarker represents a promising basis for more accurate prostate cancer diagnosis.

Pseudarthrosis in adult and pediatric spinal deformity surgery: a systematic review of the literature and meta-analysis of incidence, characteristics, and risk factors.

We conducted a systematic review with meta-analysis and qualitative synthesis. This study aims to characterize pseudarthrosis after long-segment fusion in spinal deformity by identifying incidence rates by etiology, risk factors for its development, and common features. Pseudarthrosis can be a painful and debilitating complication of spinal fusion that may require reoperation. It is poorly characterized in the setting of spinal deformity. The MEDLINE, EMBASE, and Cochrane databases were searched for clinical research including spinal deformity patients treated with long-segment fusions reporting pseudarthrosis as a complication. Meta-analysis was performed on etiologic subsets of the studies to calculate incidence rates for pseudarthrosis. Qualitative synthesis was performed to identify characteristics of and risk factors for pseudarthrosis. The review found 162 articles reporting outcomes for 16,938 patients which met inclusion criteria. In general, the included studies were of medium to low quality according to recommended reporting standards and study design. Meta-analysis calculated an incidence of 1.4% (95% CI 0.9-1.8%) for pseudarthrosis in adolescent idiopathic scoliosis, 2.2% (95% CI 1.3-3.2%) in neuromuscular scoliosis, and 6.3% (95% CI 4.3-8.2%) in adult spinal deformity. Risk factors for pseudarthrosis include age over 55, construct length greater than 12 segments, smoking, thoracolumbar kyphosis greater than 20°, and fusion to the sacrum. Choice of graft material, pre-operative coronal alignment, post-operative analgesics, and sex have no significant impact on fusion rates. Older patients with greater deformity requiring more extensive instrumentation are at higher risk for pseudarthrosis. Overall incidence of pseudarthrosis requiring reoperation is low in adult populations and very low in adolescent populations.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.