The role of TET-mediated DNA hydroxymethylation in prostate cancer.

Ten-eleven translocation (TET) proteins are recently characterized dioxygenases that regulate demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further derivatives. The recent finding that 5hmC is also a stable and independent epigenetic modification indicates that these proteins play an important role in diverse physiological and pathological processes such as neural and tumor development. Both the genomic distribution of (hydroxy)methylation and the expression and activity of TET proteins are dysregulated in a wide range of cancers including prostate cancer. Up to now it is still unknown how changes in TET and 5(h)mC profiles are related to the pathogenesis of prostate cancer. In this review, we explore recent advances in the current understanding of how TET expression and function are regulated in development and cancer. Furthermore, we look at the impact on 5hmC in prostate cancer and the potential underlying mechanisms. Finally, we tried to summarize the latest techniques for detecting and quantifying global and locus-specific 5hmC levels of genomic DNA.

Association Of Plasma And Urinary Mutant DNA With Clinical Outcomes In Muscle Invasive Bladder Cancer.

Muscle Invasive Bladder Cancer (MIBC) has a poor prognosis. Whilst patients can achieve a 6% improvement in overall survival with Neo-Adjuvant Chemotherapy (NAC), many do not respond. Body fluid mutant DNA (mutDNA) may allow non-invasive identification of treatment failure. We collected 248 liquid biopsy samples including plasma, cell pellet (UCP) and supernatant (USN) from spun urine, from 17 patients undergoing NAC. We assessed single nucleotide variants and copy number alterations in mutDNA using Tagged-Amplicon- and shallow Whole Genome- Sequencing. MutDNA was detected in 35.3%, 47.1% and 52.9% of pre-NAC plasma, UCP and USN samples respectively, and urine samples contained higher levels of mutDNA (p = <0.001). Longitudinal mutDNA demonstrated tumour evolution under the selective pressure of NAC e.g. in one case, urine analysis tracked two distinct clones with contrasting treatment sensitivity. Of note, persistence of mutDNA detection during NAC predicted disease recurrence (p = 0.003), emphasising its potential as an early biomarker for chemotherapy response.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study.

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome. METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone. FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions. INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

N-acetyl-L-aspartyl-L-glutamate peptidase-like 2 is overexpressed in cancer and promotes a pro-migratory and pro-metastatic phenotype.

N-acetyl-L-aspartyl-L-glutamate peptidase-like 2 (NAALADL2) is a member of the glutamate carboxypeptidase II family, best characterized by prostate-specific membrane antigen (PSMA/NAALAD1). Using immunohistochemistry (IHC), we have shown overexpression of NAALADL2 in colon and prostate tumours when compared with benign tissue. In prostate cancer, NAALADL2 expression was associated with stage and Grade, as well as circulating mRNA levels of the NAALADL2 gene. Overexpression of NAALADL2 was shown to predict poor survival following radical prostatectomy. In contrast to PSMA/NAALAD1, NAALADL2 was localized at the basal cell surface where it promotes adhesion to extracellular matrix proteins. Using stable knockdown and overexpression cell lines, we have demonstrated NAALADL2-dependent changes in cell migration, invasion and colony-forming potential. Expression arrays of the knockdown and overexpression cell lines have identified nine genes that co-expressed with NAALADL2, which included membrane proteins and genes known to be androgen regulated, including the prostate cancer biomarkers AGR2 and SPON2. Androgen regulation was confirmed in a number of these genes, although NAALADL2 itself was not found to be androgen regulated. NAALADL2 was also found to regulate levels of Ser133 phosphorylated C-AMP-binding protein (CREB), a master regulator of a number of cellular processes involved in cancer development and progression. In combination, these data suggest that changes in expression of NAALADL2 can impact upon a number of pro-oncogenic pathways and processes, making it a useful biomarker for both diagnosis and prognosis.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Peroxiredoxin-3 is overexpressed in prostate cancer and promotes cancer cell survival by protecting cells from oxidative stress.

OBJECTIVE: We have previously identified peroxiredoxin-3 (PRDX-3) as a cell-surface protein that is androgen regulated in the LNCaP prostate cancer (PCa) cell line. PRDX-3 is a member of the peroxiredoxin family that are responsible for neutralising reactive oxygen species. EXPERIMENTAL DESIGN: PRDX-3 expression was examined in tissue from 32 patients using immunohistochemistry. Subcellular distribution was determined using confocal microscopy. PRDX-3 expression was determined in antiandrogen-resistant cell lines by western blotting and quantitative RT-PCR. The pathways of PRDX-3 overexpression and knockdown on apoptosis and response to oxidative stress were investigated using protein arrays. RESULTS: PRDX-3 is upregulated in a number of endocrine-regulated tumours; in particular in PCa and prostatic intraepithelial neoplasia. Although the majority of PRDX-3 is localised to the mitochondria, we have confirmed that PRDX-3 at the cell membrane is androgen regulated. In antiandrogen-resistant LNCaP cell lines, PRDX-3 is upregulated at the protein but not RNA level. Resistant cells also possess an upregulation of the tricarboxylic acid (TCA) pathway and resistance to H2O2-induced apoptosis through a failure to activate pro-apoptotic pathways. Knockdown of PRDX-3 restored H2O2 sensitivity. CONCLUSION: Our results suggest that PRDX-3 has an essential role in regulating oxidation-induced apoptosis in antiandrogen-resistant cells. PRDX-3 may have potential as a therapeutic target in castrate-independent PCa.

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer.

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Moderate neutropenia with adjuvant CMF confers improved survival in early breast cancer.

Despite the extensive literature clearly demonstrating the survival benefit for adjuvant chemotherapy in women with operable breast cancer, there are few data confirming this in routine practice. Some studies have suggested that not all women gain to the same extent, with older women showing a smaller benefit and lower doses achieving poorer outcomes. We therefore reviewed the case notes of 750 women treated over a 15-year period at The Edinburgh Cancer Centre with the same intravenous CMF (cyclophosphamide, methotrexate and 5-fluorouracil) regimen, to identify patient- and treatment-related factors influencing outcome in routine practice. The actuarial 10-year survival for these women was 59.3%, with the anticipated poorer outcome for those with more involved ipsilateral axillary nodes, higher grade and ER-negative tumours. There was no evidence that a lower delivered dose intensity or older age at presentation resulted in a poorer survival. Of particular interest was the observation that 45% of patients who had grade 2/3 neutropenia had a 10% absolute survival advantage over those with no neutropenia (P<0.001). This strongly suggests that some degree of neutropenia has more influence on outcome than age or delivered dose intensity.

Is there a relationship between HLA type and prognostic factors in breast cancer?

No convincing association exists between HLA type and breast cancer development but certain HLA types have been suggested to be associated with poor risk disease. Here, the HLA type (class I and II) for 141 breast cancer patients was compared to a control population of 100 individuals and to the prognostic indicators for the patients. No association was found between HLA type and breast cancer development. Consideration of individual HLA/prognostic factor relationships previously reported confirmed that HLA-B7 was over-represented in premenopausal oestrogen-receptor (ER)-positive, grade 3 tumours (p = 0.04) and that HLA-A1 correlated positively with Nottingham Prognostic Index (p < 0.05) and with ER-negative disease (p < 0.05). These findings suggest that some previously identified associations between HLA type I and particular prognostic factors may be real, if weak but appear to conflict with the only other sizeable study investigating HLA type II in breast cancer (which negatively correlated HLA-DR 11 with early onset disease).

Effects of humidification on nasal symptoms and compliance in sleep apnea patients using continuous positive airway pressure.

STUDY OBJECTIVES: To evaluate the effects of humidification on nasal symptoms and compliance in sleep apnea patients using continuous positive airway pressure (CPAP). DESIGN: A randomized, crossover design was employed. SETTING: The study was conducted at two suburban community-based hospital sleep laboratories. PATIENTS: Data were collected on 38 obstructive sleep apnea patients (mean age, 44.1 years) in whom CPAP was a novel treatment. INTERVENTIONS: The interventions were heated humidity, cold passover humidity, and a washout period without humidity. MEASUREMENTS AND RESULTS: Patients were titrated with heated humidity or cold passover humidity in the laboratory and subsequently initiated on humidity. Objective compliance, self-report of factors affecting CPAP use, satisfaction with CPAP, feeling upon awakening, and daytime sleepiness were assessed at the completion of each 3-week treatment period and a 2-week washout period. Outcome measures were assessed with one-way analysis of variance followed by Scheffe post hoc comparisons. Significant main effects were observed for compliance (F2,37 = 5.2; p = 0.008), satisfaction with CPAP (F2,37 = 4.5; p = 0.01), and feeling refreshed on awakening (F2,37 = 4.4; p = 0.02). A significant decrease in daytime sleepiness was observed between baseline and each of the conditions (F3,37 = 55.5; p<0.0001), but Epworth sleepiness scale scores did not differ between conditions (all p values >0.56). CPAP use with heated humidity (5.52+/-2.1 h/night) was greater than CPAP use without humidity (4.93+/-2.2 h/night; p = 0.008). Compliance differences were not observed between CPAP use with cold passover humidity and CPAP use without humidity. Patients were more satisfied with CPAP when it was used with heated or cold passover humidity (p< or =0.05). However, only heated humidity resulted in feeling more refreshed on awakening (p<0.05). No significant differences were observed among the three groups on the global adverse side effect score (F2,37 = 2.5; p = 0.09). Specific side effects such as dry mouth or throat and dry nose were reported less frequently when CPAP was used with heated humidity compared to CPAP use without humidity (p<0.001). CONCLUSIONS: Compliance with CPAP is enhanced when heated humidification is employed. This is likely due to a reduction in side effects associated with upper airway symptoms and a more refreshed feeling upon awakening. Compliance gains may be realized sooner if patients are started with heated humidity at CPAP initiation.

A method to determine case manager caseloads in long-term care.

Case management has come to the forefront of home- and community-based long-term care (LTC) as an attempt to reduce the costs of LTC and the fragmentation of the home- and community-based LTC system. The cost of case management itself, however, must also receive attention. A key variable contributing to the cost of a case management program is the size of the case managers' caseloads; the higher the caseloads, the lower the costs, all other things being equal. However, caseloads that are too high can reduce the quality of care. Thus, a program's recommended caseload is an important factor. This article presents a method for determining a recommended caseload for a home- and community-based LTC program, using a case study. This method can be adapted for use by programs that have a different caseload history from that of the program in the case study.

Assessment of eversion and plantar flexion strength after repair of Achilles tendon rupture using peroneus brevis tendon transfer.

Eight patients with Achilles tendon ruptures, one of which was a late discovery, underwent peroneus brevis tendon transfers to repair their injuries. Subsequently, both the repaired and normal extremities of all eight patients were tested for eversion and plantar flexion strength. The Cybex 340 isokinetic system (Lumex Corp, Bayshore, New York) was used at both 30 degrees per second and 120 degrees per second. Patients' affected sides were then assessed subjectively for function compared with their opposite normal sides. Results for eversion strength showed a 17.4% deficit at 30 degrees per second and a 14.9% deficit at 120 degrees per second on the repaired extremity. Only the group with the 14.9% deficit showed a statistically significant difference using the paired t test (P < 0.05). Results for plantar flexion strength showed a 1.5% difference at 30 degrees per second and a 5.8% difference at 120 degrees per second. None of these differences were statistically significant. Subjective assessment showed no functional compromise in eversion strength, plantar flexion strength, activities of daily living, or ankle stability. Conclusions are that mild objective eversion weakness and no objective plantar flexion weakness can be expected after this procedure; however, subjective assessment reveals no functional compromise.

The cold pressor test: vascular and myocardial response patterns and their stability.

The purposes of the present study were to compare the cardiovascular response patterns evoked by three versions of the cold pressor test (either forehead stimulation or hand or foot immersion) and to determine the reproducibility of the responses over a 2-week interval. Blood pressure, heart rate, stroke volume, cardiac output, total peripheral resistance, and systolic time intervals were obtained during rest and during the cold pressor test in 42 young men. Across conditions, the pressor response was supported by peripheral resistance increases with concomitant stroke volume decreases. Although the response patterns were generally similar across sites, exceptions were apparent for heart rate. Forehead stimulation was characterized by no significant change in heart rate, whereas limb (hand or foot) immersion was associated with significant heart rate acceleration. The responses elicited by the three cold pressor test conditions were reliable and showed little evidence of attenuation over the test-retest interval.

Using total quality management in long-term care case management.

Long-term care (LTC) case management has emerged as a way of addressing problems in the long term care system. Case managers serve as links between chronically ill and disabled individuals and the services these individuals need but of which they often are unaware. In this capacity, case managers address the fragmentation in the long-term care system. In guiding clients to cost-effective ways of meeting their long-term care needs, case managers also address the need for cost containment in long-term care. To ensure that case management is effectively addressing these and other problems, attention to quality is essential. However, quality assurance programs too often focus on minutiae rather than on true indicators of quality. In addition, people who are not involved in the related areas frequently make decisions about quality. These decisions impose additional, and sometimes unnecessary, restrictions on the people who are required to implement them. Total quality management (TQM) offers a new approach. In this article, a proposal is made to incorporate TQM principles into LTC case management. The LTC case management demonstration project currently underway in Virginia serves as the case study for this proposal. The proposal demonstrates a process in which quality can be built into a case management system in a way that involves the people who will implement the resultant changes. The proposal shows how case management staff members can learn to identify areas where quality is impaired and to use techniques to aid them in identifying the causes of problems. A procedure for testing solutions and for incorporating the findings into the project is described.

Differential effects of neuroleptic drugs on the delayed matching-to-sample performance of pigeons.

The effects of clozapine, thiothixene, sulpiride, chlorpromazine and loxapine were examined in pigeons responding under a delayed matching-to-sample (DMTS) procedure using 0-, 2- and 8-sec delay intervals. Chlorpromazine (3-100 mg/kg), thiothixene (0.03-1.7 mg/kg), clozapine (0.1-5.6 mg/kg) and loxapine (0.1-10 mg/kg) produced dose-related decreases in the percent of correct responses (accuracy). With the exception of chlorpromazine, the relative magnitude of the accuracy-decreasing effects were unrelated to the length of the delay interval and the nondrug levels of accuracy. In contrast to these accuracy-decreasing effects, sulpiride (3-300 mg/kg) failed to decrease accuracy across the range of doses evaluated. Chlorpromazine, loxapine and clozapine increased response rates at low doses and then decreased response rates as the dose was increased. Thiothixene and sulpiride only decreased response rates in a dose-dependent fashion. The order of potency for the rate-suppressing effects of these drugs was thiothixene greater than clozapine = loxapine greater than chlorpromazine greater than sulpiride. The results of the present investigation suggest that, despite similar dopamine antagonist properties, neuroleptics produce qualitatively different effects in pigeons responding under DMTS procedures.

Antagonism of the analgesic effects of mu and kappa opioid agonists in the squirrel monkey.

The effects of several mu and kappa opioid agonists were examined alone and in combination with the opioid antagonist quadazocine in squirrel monkeys responding under a schedule of shock titration. Under this procedure, shock was scheduled to increase once every 15 sec from 0.01 to 2.0 mA in 30 steps. Five responses on a lever during the 15-sec shock period terminated the shock for 15 sec, after which the shock resumed at the next lower intensity. The intensity below which the monkeys maintained the shock 50% of the time (median shock level) and the rate of responding in the presence of shock were determined under control conditions and after administration of the mu agonists, l-methadone and fentanyl and the kappa agonists, bremazocine, ethylketocyclazocine, ketocyclazocine and U50,488. When examined alone, intermediate doses of mu and kappa agonists increased median shock level. At the highest doses of these compounds responding was eliminated and shock rose to its peak intensity. When the mu and kappa agonists were examined in combination with quadazocine, dose-effect curves for median shock level and for rate of responding were shifted to the right in a dose-dependent fashion. A comparison of the pA2 values for quadazocine on median shock level and on rate of responding revealed similar values for the two measures; however, pA2 values differed depending on the agonist examined. That is, the pA2 values for quadazocine in combination with l-methadone and fentanyl on median shock level were 7.43 and 7.61, respectively; whereas the pA2 value for quadazocine in combination with bremazocine and U50,488 were 6.53 and 6.43, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)