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.

Oxidative stress induces mitochondrial DNA damage and cytotoxicity through independent mechanisms in human cancer cells.

Intrinsic oxidative stress through increased production of reactive oxygen species (ROS) is associated with carcinogenic transformation, cell toxicity, and DNA damage. Mitochondrial DNA (mtDNA) is a natural surrogate to oxidative DNA damage. MtDNA damage results in the loss of its supercoiled structure and is readily detectable using a novel, supercoiling-sensitive real-time PCR method. Our studies have demonstrated that mtDNA damage, as measured by DNA strand breaks and copy number depletion, is very sensitive to exogenous H2O2 but independent of endogenous ROS production in both prostate cancer and normal cells. In contrast, aggressive prostate cancer cells exhibit a more than 10-fold sensitivity to H2O2-induced cell toxicity than normal cells, and a cascade of secondary ROS production is a critical determinant to the differential response. We propose a new paradigm to account for different mechanisms governing cellular oxidative stress, cell toxicity, and DNA damage with important ramifications in devising new techniques and strategies in prostate cancer prevention and treatment.

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.

A panel of TMPRSS2:ERG fusion transcript markers for urine-based prostate cancer detection with high specificity and sensitivity.

BACKGROUND: The TMPRSS2:ERG fusion is both prevalent and unique to prostate cancer (PCa) and has great potential for noninvasive diagnosis of PCa in bodily fluids. OBJECTIVES: To evaluate the specificity and sensitivity of the TMPRSS2:ERG fusion in urine from diverse clinical contexts and to explore potential clinical applications. DESIGN, SETTING, AND PARTICIPANTS: A total of 101 subjects were enrolled in 2008 from urologic oncology clinics to form three study groups: 44 PCa free, 46 confirmed PCa, and 11 negative prostate biopsies. The PCa-free group included females, healthy young men, and post-radical prostatectomy (RP) patients. The confirmed PCa group was composed of patients under active surveillance, scheduled for treatment, or with metastatic disease. MEASUREMENTS: Urine was collected after attentive digital rectal exam (DRE) and coded to blind group allocation for laboratory test. RNA from urine sediments was analyzed using a panel of four TMPRSS2:ERG fusion markers with quantitative polymerase chain reaction (qPCR). RESULTS AND LIMITATIONS: Our fusion markers demonstrated very high technical specificity and sensitivity for detecting a single fusion-positive cancer cell (VCaP) in the presence of at least 3000 cells in urine sediments. In clinical analysis, there were no fusion-positive samples in the PCa-free group (0 of 44 samples), while there were 16 of 46 (34.8%) fusion-positive samples in the confirmed PCa group. The fusion incidence varied significantly among the three PCa subgroups. The clinical sensitivity increased to 45.4% in cancer patients prior to treatments. The fusion markers were detected in 2 of 11 (18.2%) biopsy-negative patients, suggesting potentially false negative biopsies. This study is not prospective and is limited in sample sizes. CONCLUSIONS: Our novel panel of TMPRSS2:ERG fusion markers provided a very specific and sensitive tool for urine-based detection of PCa. Theses markers can potentially be used to diagnose patients with PCa who have negative biopsies.

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.

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair.

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.

Mitochondrial mutagenesis and oxidative stress in human prostate cancer.

Prostate cancer is the most common cancer diagnosed in men in the United States, but the primary cause and the molecular events leading to prostate carcinogenesis are poorly understood. Using the approach of laser capture microdissection, we revealed extensive somatic mitochondrial DNA (mtDNA) mutations in prostatic neoplastic lesions. Inspection of the lesion associated mutations not only provided new insights into the genetics of prostate cancer, but also revealed new patterns of mtDNA mutation in prostate carcinogenesis. Further analysis on a high frequency of multiple mutational events observed in the same neoplastic lesion revealed an unusually rapid process in mitochondrial mutagenesis, suggesting a new process of mitochondrial hyper-mutagenesis in cancer cells, likely mediated by cellular oxidative stress. Thus, active mitochondrial mutagenesis in prostate cancer suggests a prominent role of increased cellular oxidative stress in neoplastic transformation and the increased susceptibility of neoplastic cells to oxidative damage.

Simultaneous generation of multiple mitochondrial DNA mutations in human prostate tumors suggests mitochondrial hyper-mutagenesis.

Multiple somatic mitochondrial DNA mutations are frequently reported in human tumors, but the process leading to homoplasmic transformation and accumulation of multiple mutations in the same tumor cell lineage remains a mystery. We address possible mechanisms responsible for the generation of multiple mitochondrial (mt)DNA mutations observed in a high frequency of prostate tumors using sensitive mutant-specific PCR coupled with laser capture microdissection. Analysis of prostate tumors with multiple mtDNA mutations in the control region indicates that the mutations are locally confined, that the multiple mutations exist on the same molecules and that more than one mtDNA mutant species co-exists in the same neoplastic lesion. These results suggest an unusually rapid process in mtDNA mutagenesis during tumor progression. On the basis of prostate tumor cell kinetics, we propose a unique process of mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, to account for a burst of multiple mtDNA mutations in human prostate tumors.

Genetic differentiation at nuclear and mitochondrial loci among large white-headed gulls: sex-biased interspecific gene flow?

We measured genetic differentiation among species of large white-headed gulls using mitochondrial (cytochrome b haplotypes) and nuclear (microsatellites) markers. Additional information was added using a previously published study of allozymes on the same species. Levels of differentiation among species at nuclear markers are much lower than would be expected for avian species and are not concordant with the level of differentiation in mitochondrial markers. This discrepancy is best explained by a combination of recent species origin and interspecific gene flow after speciation. The data also suggest that female-mediated gene flow is reduced compared to male-mediated gene flow, either due to behavioral bias or due to stronger counterselection of female hybrids in accordance with Haldane's rule for ZW species. Whatever the reasons for the low differentiation of the species' nuclear gene pools, the extensive similarity of their nuclear genome demonstrates that selection on a limited number of characters is an important factor in establishing and maintaining clear-cut phenotypic differences between these species and suggests that the number of loci involved in this process is quite low. This situation may not be exceptional in birds, indeed a number of studies have found similarly low level of differentiation in nuclear markers among congeneric bird species, although usually based on a single set of markers. Because hybridization is a widespread phenomenon in birds, many of these cases might be due to interspecific gene flow.

Extensive somatic mitochondrial mutations in primary prostate cancer using laser capture microdissection.

Prostate cancer is the second leading cause of cancer deaths among men in the United States,but the precise molecular events leading to prostate carcinogenesis are not well understood. We isolated histologically defined cell populations from prostate cancer and its preinvasive lesions using laser capture microdissection, and performed genetic analysis on the mitochondrial genome, a sensitive cytoplasmic DNA. An extremely high incidence of somatic mutation (90% of prostatectomy cancer specimens) was found in the control region (the displacement loop) of mitochondrial DNA. The massive induction of lesion-associated mutations suggests active mitochondrial mutagenesis in both prostate cancer and its preinvasive lesions. Inspection of these mutations provides new insights into prostate cancer genetics and reveals unique patterns of somatic mutations in prostatic neoplastic lesions.