Early detection of clinically significant prostate cancer at diagnosis: a prospective study using a novel panel of TMPRSS2:ETS fusion gene markers.

We explore noninvasive clinical applications of multiple disease-specific fusion markers recently discovered in prostate cancer to predict the risk of cancer occurrence and aggressiveness at diagnosis. A total of 92 men who were prostate-specific antigen (PSA) screened and scheduled for diagnostic biopsy were enrolled for this study. Prospectively collected urine was blind coded for laboratory tests. RNA from urine sediments was analyzed using a panel of 6 TMPRSS2:ETS fusion markers with a sensitive quantitative PCR platform. The pathology reported 39 biopsy-positive cases from 92 patients (42.4%). In urine test, 10 unique combinations of fusion types were detected in 32 of 92 (34.8%) prebiopsy samples. A novel combination of fusion markers, termed Fx (III, IV, ETS), was identified with a sensitivity of 51.3% and an odds ratio of 10.1 in detecting cancer on biopsy. Incorporating a categorical variable of Fx (III, IV, ETS) with urine PCA3 and serum PSA, a regression model was developed to predict biopsy outcomes with an overall accuracy of 77%. Moreover, the overexpression of Fx (III, IV, or ETS) was shown to be an independent predictor to the high-grade cancer, with a predictive accuracy of 80% when coupled with PSA density. The individualized risk scores further stratified a high-risk group that is composed of 92% high-grade cancers and a low-risk group that harbors mainly clinically insignificant cancers. In conclusion, we have identified a novel combination of fusion types very specific to the clinically significant prostate cancer and developed effective regression models to predict biopsy outcomes and aggressive cancers at diagnosis.

Simultaneous quantification of mitochondrial DNA damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.

Systemic oxidative stress is associated with a wide range of pathological conditions. Oxidative DNA damage is frequently measured in circulating lymphocytes. Mitochondrial DNA (mtDNA) is known to be more sensitive to oxidative damage than nuclear DNA but is rarely used for direct measurement of DNA damage in clinical studies. Based on the supercoiling-sensitive real-time PCR method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtDNA structural damage and copy number change in isolated lymphocytes in a single test. We show that lymphocytes have significantly less mtDNA content and relatively lower baseline levels of damage than cancer cell lines. In an ex vivo challenge experiment, we demonstrate, for the first time, that exogenous H2O2 induces a significant increase in mtDNA damage in lymphocytes from healthy individuals, but no repair activity is observed after 1 h recovery. We further demonstrate that whole blood may serve as a convenient alternative to the isolated lymphocytes in mtDNA analysis. Thus, the blood analysis with the multiple mtDNA end-points proposed in the current study may provide a simple and sensitive test to interrogate the nature and extent of systemic oxidative stress for a broad spectrum of clinical investigations.

RNA preservation in decalcified cochlear samples.

HYPOTHESIS: Decalcification of cochlear samples in Morse's solution after methacarn fixation provides greater RNA quantification and morphologic preservation of cochlear structures as compared with EDTA and formic acid decalcifying solutions after methacarn fixation. BACKGROUND: A variety of fixatives and decalcifying agents can fragment or chemically alter RNA in samples inhibiting their isolation and quantification. Morphologic alterations can also be observed in light microscopy analyses. The cochlea is embedded in the bone; hence, fixation and decalcification steps are mandatory to obtain histologic sections and preserve the cochlea for morphologic evaluation. METHODS: Cochlear samples obtained in a RNase-free environment were processed in 4 combinations of decalcifying agents in combination with methacarn fixation. Samples in Protocols 1, 2, and 3 were fixed in methacarn for 4 hours at 4°C, followed by decalcification at 4°C with Morse's solution, 10% ethylenediaminetetraacetic acid, and 5% formic acid solution, respectively. Samples processed with protocol 4 were decalcified in Morse's solution at 4°C followed by fixation for 4 hours at 4°C. Real-time PCR analysis was performed on total RNA extracted. Histology sections were evaluated for morphology preservation of cochlear structures. RESULTS: RNA was isolated in all samples. Relative expression levels were greatest with Protocol 1 and lowest with Protocol 3. Morphology preservation was adequate with Protocols 1, 2, and 3. CONCLUSION: Of the 4 protocols evaluated, methacarn fixation followed by decalcification in Morse's solution provided the greatest genetic expression levels as well as the best tissue morphology preservation in the cochlea.

Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study.

BACKGROUND: There is good evidence to show that varicocele repair can improve conventional sperm parameters, as well as, sperm DNA integrity, in infertile men with a clinical varicocele. OBJECTIVE: To examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity and sperm mitochondrial DNA (mtDNA) copy number. DESIGN, SETTING, AND PARTICIPANTS: A prospective study done between March 2007 and January 2008. We evaluated a consecutive series of infertile men (n = 14) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and poor motility (<25 % rapid progressive and <50 % progressive). SURGICAL PROCEDURE: Microsurgical sub-inguinal varicocelectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Conventional sperm parameters, sperm mtDNA copy number (by real time PCR) and sperm chromatin structure assay (SCSA) parameters (%DFI,% HDS) before and 4 months after microsurgical varicocelectomy. RESULTS AND LIMITATIONS: Sperm concentration and SCSA parameters (%DFI and %HDS) improved significantly after surgery (P < 0.05). Sperm mitochondrial DNA copy number decreased significantly after surgery (27 ± 30 to 9 ± 6 copies per sperm, respectively, P = 0.032). There was a significant negative correlation between mitochondrial DNA copy number and sperm motility (r = - 0.71, P = 0.002). CONCLUSION: These findings support the concept that correction of a varicocele can improve spermatogenesis and sperm function, as mitochondrial DNA copy number has been suggested to reflect the efficiency of spermatogenesis and has been inversely related to sperm motility.

Mitochondrial DNA damage is sensitive to exogenous H(2)O(2) but independent of cellular ROS production in prostate cancer cells.

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H(2)O(2) induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O(2)(-)), is shown to be a critical determinant of H(2)O(2)-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H(2)O(2) exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H(2)O(2) is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro.

Measuring mtDNA damage using a supercoiling-sensitive qPCR approach.

Compromised mitochondrial DNA structural integrity can have functional consequences for mitochondrial gene expression and replication leading to metabolic and degenerative diseases, aging, and cancer. Gel electrophoresis coupled with Southern blot and probe hybridization and long PCR are established methods for detecting mtDNA damage. But each has its respective shortcomings: gel electrophoresis is at best semi-quantitative and long PCR does not offer information on the structure. To overcome these limitations, we developed a new method with real-time PCR to accurately quantify the mtDNA structural damage/repair and copy number change. We previously showed that the different mtDNA structures (supercoiled, relaxed circular, and linear) have profound influences on the outcome of the real-time PCR amplification. The supercoiled structure is inhibitory to the PCR amplification, while relaxed structures are readily amplified. We will illustrate the use of this new method by quantifying the kinetics of mtDNA damage and repair in LNCaP prostate cancer cells induced by exogenous H2O2 treatments. The use of this new method on clinical samples for spontaneous mtDNA damage level will also be highlighted.