Feasibility assessment of using the MiToS staging system for conducting economic evaluation in amyotrophic lateral sclerosis.

OBJECTIVES: This study assessed the feasibility of using the Milano-Torino staging (MiToS) system for conducting economic evaluation to measure health outcomes in amyotrophic lateral sclerosis (ALS). METHODS: A Markov model was developed using the MiToS system and evaluated with a hypothetical treatment versus standard of care. Health utilities and transition probabilities were derived from the literature. Four-time horizons (1, 5, 10, and 20 years) were examined. Treatment effects of 20-35% relative risk reduction (RRR) of progressing to the next MiToS stage were assessed. Three patient distribution scenarios were tested: (1) all patients began in stage 0; (2) patient distribution based on real-world TONiC study; (3) distribution based on the PRO-ACT database. Health outcomes (quality-adjusted life-years [QALYs], life-years [LYs]) were reported with a 3% discount rate. RESULTS: A time horizon of 10 years fully captured treatment benefits: incremental QALYs were 0.28-0.60, 0.21-0.45, and 0.26-0.55 for scenarios 1-3, respectively; incremental LYs were 0.56-1.17, 0.46-0.97, and 0.53-1.11, respectively. CONCLUSION: MiToS-based staging can be used for conducting economic analyses in ALS. Estimated incremental QALY and LY gains were meaningful within the context of ALS, for hypothetical treatments with RRR of 20-35%.

Exercise interventions on health-related quality of life for people with cancer during active treatment.

BACKGROUND: People with cancer undergoing active treatment experience numerous disease- and treatment-related adverse outcomes and poorer health-related quality of life (HRQoL). Exercise interventions are hypothesized to alleviate these adverse outcomes. HRQoL and its domains are important measures of cancer survivorship, both during and after the end of active treatment for cancer. OBJECTIVES: To evaluate the effectiveness of exercise on overall HRQoL outcomes and specific HRQoL domains among adults with cancer during active treatment. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed MEDLINE, EMBASE, CINAHL, PsycINFO, PEDRO, LILACS, SIGLE, SportDiscus, OTSeeker, Sociological Abstracts from inception to November 2011 with no language or date restrictions. We also searched citations through Web of Science and Scopus, PubMed's related article feature, and several websites. We reviewed reference lists of included trials and other reviews in the field. SELECTION CRITERIA: We included all randomized controlled trials (RCTs) and quasi-randomized controlled clinical trials (CCTs) comparing exercise interventions with usual care or other type of non-exercise comparison intervention to maintain or enhance, or both, overall HRQoL or at least one distinct domain of HRQoL. Included trials tested exercise interventions that were initiated when adults with cancer were undergoing active cancer treatment or were scheduled to initiate treatment. DATA COLLECTION AND ANALYSIS: Five paired review authors independently extracted information on characteristics of included trials, data on effects of the intervention, and assessed risk of bias based on predefined criteria. Where possible, we performed meta-analyses for HRQoL and HRQoL domains for the reported difference between baseline values and follow-up values using standardized mean differences (SMDs) and a random-effects model by length of follow-up. We also reported the SMD at follow-up between the exercise and control groups. Because investigators used many different HRQoL and HRQoL domain instruments and often more than one for the same domain, we selected the more commonly used instrument to include in the SMD meta-analyses. We also report the mean difference for each type of instrument separately. MAIN RESULTS: We included 56 trials with 4826 participants randomized to an exercise (n = 2286) or comparison (n = 1985) group. Cancer diagnoses in trial participants included breast, prostate, gynecologic, hematologic, and other. Thirty-six trials were conducted among participants who were currently undergoing active treatment for their cancer, 10 trials were conducted among participants both during and post active cancer treatment, and the remaining 10 trials were conducted among participants scheduled for active cancer treatment. Mode of exercise intervention differed across trials and included walking by itself or in combination with cycling, resistance training, or strength training; resistance training; strength training; cycling; yoga; or Qigong. HRQoL and its domains were assessed using a wide range of measures.The results suggest that exercise interventions compared with control interventions have a positive impact on overall HRQoL and certain HRQoL domains. Exercise interventions resulted in improvements in: HRQoL from baseline to 12 weeks' follow-up (SMD 0.33; 95% CI 0.12 to 0.55) or when comparing difference in follow-up scores at 12 weeks (SMD 0.47; 95% CI 0.16 to 0.79); physical functioning from baseline to 12 weeks' follow-up (SMD 0.69; 95% CI 0.16 to 1.22) or 6 months (SMD 0.28; 95% CI 0.00 to 0.55); or when comparing differences in follow-up scores at 12 weeks (SMD 0.28; 95% CI 0.11 to 0.45) or 6 months (SMD 0.29; 95% CI 0.07 to 0.50); role function from baseline to 12 weeks' follow-up (SMD 0.48; 95% CI 0.07 to 0.90) or when comparing differences in follow-up scores at 12 weeks (SMD 0.17; 95% CI 0.00 to 0.34) or 6 months (SMD 0.32; 95% CI 0.03 to 0.61); and, in social functioning at 12 weeks' follow-up (SMD 0.54; 95% CI 0.03 to 1.05) or when comparing differences in follow-up scores at both 12 weeks (SMD 0.16; 95% CI 0.04 to 0.27) and 6 months (SMD 0.24; 95% CI 0.03 to 0.44). Further, exercise interventions resulted in a decrease in fatigue from baseline to 12 weeks' follow-up (SMD -0.38; 95% CI -0.57 to -0.18) or when comparing difference in follow-up scores at follow-up of 12 weeks (SMD -0.73; 95% CI -1.14 to -0.31). Since there is consistency of findings on both types of measures (change scores and difference in follow-up scores) there is greater confidence in the robustness of these findings.When examining exercise effects by subgroups, exercise interventions had significantly greater reduction in anxiety for survivors with breast cancer than those with other types of cancer. Further, there was greater reduction in depression, fatigue, and sleep disturbances, and improvement in HRQoL, emotional wellbeing (EWB), physical functioning, and role function for cancer survivors diagnosed with cancers other than breast cancer but not for breast cancer. There were also greater improvements in HRQoL and physical functioning, and reduction in anxiety, fatigue, and sleep disturbances when prescribed a moderate or vigorous versus a mild exercise program.Results of the review need to be interpreted cautiously owing to the risk of bias. All the trials reviewed were at high risk for performance bias. In addition, the majority of trials were at high risk for detection, attrition, and selection bias. AUTHORS' CONCLUSIONS: This systematic review indicates that exercise may have beneficial effects at varying follow-up periods on HRQoL and certain HRQoL domains including physical functioning, role function, social functioning, and fatigue. Positive effects of exercise interventions are more pronounced with moderate- or vigorous-intensity versus mild-intensity exercise programs. The positive results must be interpreted cautiously because of the heterogeneity of exercise programs tested and measures used to assess HRQoL and HRQoL domains, and the risk of bias in many trials. Further research is required to investigate how to sustain positive effects of exercise over time and to determine essential attributes of exercise (mode, intensity, frequency, duration, timing) by cancer type and cancer treatment for optimal effects on HRQoL and its domains.

Exercise interventions on health-related quality of life for cancer survivors.

BACKGROUND: Cancer survivors experience numerous disease and treatment-related adverse outcomes and poorer health-related quality of life (HRQoL). Exercise interventions are hypothesized to alleviate these adverse outcomes. HRQoL and its domains are important measures for cancer survivorship. OBJECTIVES: To evaluate the effectiveness of exercise on overall HRQoL and HRQoL domains among adult post-treatment cancer survivors. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, MEDLINE, EMBASE, CINAHL, PsycINFO, PEDRO, LILACS, SIGLE, SportDiscus, OTSeeker, and Sociological Abstracts from inception to October 2011 with no language or date restrictions. We also searched citations through Web of Science and Scopus, PubMed's related article feature, and several websites. We reviewed reference lists of included trials and other reviews in the field. SELECTION CRITERIA: We included all randomized controlled trials (RCTs) and controlled clinical trials (CCTs) comparing exercise interventions with usual care or other nonexercise intervention to assess overall HRQoL or at least one HRQoL domain in adults. Included trials tested exercise interventions that were initiated after completion of active cancer treatment. We excluded trials including people who were terminally ill, or receiving hospice care, or both, and where the majority of trial participants were undergoing active treatment for either the primary or recurrent cancer. DATA COLLECTION AND ANALYSIS: Five paired review authors independently extracted information on characteristics of included trials, data on effects of the intervention, and assessed risk of bias based on predefined criteria. Where possible, meta-analyses results were performed for HRQoL and HRQoL domains for the reported difference between baseline values and follow-up values using standardized mean differences (SMD) and a random-effects model by length of follow-up. We also reported the SMDs between mean follow-up values of exercise and control group. Because investigators used many different HRQoL and HRQoL domain instruments and often more than one for the same domain, we selected the more commonly used instrument to include in the SMD meta-analyses. We also report the mean difference for each type of instrument separately. MAIN RESULTS: We included 40 trials with 3694 participants randomized to an exercise (n = 1927) or comparison (n = 1764) group. Cancer diagnoses in study participants included breast, colorectal, head and neck, lymphoma, and other. Thirty trials were conducted among participants who had completed active treatment for their primary or recurrent cancer and 10 trials included participants both during and post cancer treatment. Mode of the exercise intervention included strength training, resistance training, walking, cycling, yoga, Qigong, or Tai Chi. HRQoL and its domains were measured using a wide range of measures.The results suggested that exercise compared with control has a positive impact on HRQoL and certain HRQoL domains. Exercise resulted in improvement in: global HRQoL at 12 weeks' (SMD 0.48; 95% confidence interval (CI) 0.16 to 0.81) and 6 months' (0.46; 95% CI 0.09 to 0.84) follow-up, breast cancer concerns between 12 weeks' and 6 months' follow-up (SMD 0.99; 95% CI 0.41 to 1.57), body image/self-esteem when assessed using the Rosenberg Self-Esteem scale at 12 weeks (MD 4.50; 95% CI 3.40 to 5.60) and between 12 weeks' and 6 months' (mean difference (MD) 2.70; 95% CI 0.73 to 4.67) follow-up, emotional well-being at 12 weeks' follow-up (SMD 0.33; 95% CI 0.05 to 0.61), sexuality at 6 months' follow-up (SMD 0.40; 95% CI 0.11 to 0.68), sleep disturbance when comparing follow-up values by comparison group at 12 weeks' follow-up (SMD -0.46; 95% CI -0.72 to -0.20), and social functioning at 12 weeks' (SMD 0.45; 95% CI 0.02 to 0.87) and 6 months' (SMD 0.49; 95% CI 0.11 to 0.87) follow-up. Further, exercise interventions resulted in decreased anxiety at 12 weeks' follow-up (SMD -0.26; 95% CI -0.07 to -0.44), fatigue at 12 weeks' (SMD -0.82; 95% CI -1.50 to -0.14) and between 12 weeks' and 6 months' (SMD -0.42; 95% CI -0.02 to -0.83) follow-up, and pain at 12 weeks' follow-up (SMD -0.29; 95% CI -0.55 to -0.04) when comparing follow-up values by comparison group.Positive trends and impact of exercise intervention existed for depression and body image (when analyzing combined instruments); however, because few studies measured these outcomes the robustness of findings is uncertain.No conclusions can be drawn regarding the effects of exercise interventions on HRQoL domains of cognitive function, physical functioning, general health perspective, role function, and spirituality.Results of the review need to be interpreted cautiously owing to the risk of bias. All the trials reviewed were at high risk for performance bias. In addition, the majority of trials were at high risk for detection, attrition, and selection bias. AUTHORS' CONCLUSIONS: This systematic review indicates that exercise may have beneficial effects on HRQoL and certain HRQoL domains including cancer-specific concerns (e.g. breast cancer), body image/self-esteem, emotional well-being, sexuality, sleep disturbance, social functioning, anxiety, fatigue, and pain at varying follow-up periods. The positive results must be interpreted cautiously due to the heterogeneity of exercise programs tested and measures used to assess HRQoL and HRQoL domains, and the risk of bias in many trials. Further research is required to investigate how to sustain positive effects of exercise over time and to determine essential attributes of exercise (mode, intensity, frequency, duration, timing) by cancer type and cancer treatment for optimal effects on HRQoL and its domains.

A Systematic Review and Meta-Analysis of Studies of Defibrotide Prophylaxis for Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome.

BACKGROUND AND OBJECTIVES: Defibrotide is approved to treat severe veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) after haematopoietic cell transplantation in patients aged > 1 month in the European Union and for VOD/SOS with renal/pulmonary dysfunction post-haematopoietic cell transplantation in the United States. This meta-analysis estimated the incidence and risk of VOD/SOS after intravenous defibrotide prophylaxis using the published literature. METHODS: PubMed, Embase and Web of Science were searched through 30 November 2021 for defibrotide studies in VOD/SOS "prevention" or "prophylaxis," excluding phase I studies, case reports, studies with fewer than ten patients and reviews. RESULTS: The search identified 733 records; 24 met inclusion criteria, of which 20 (N = 3005) evaluated intravenous defibrotide for VOD/SOS prophylaxis. Overall VOD/SOS incidence with intravenous defibrotide was 5%, with incidences of 5% in adults and 8% in paediatric patients. In eight studies with data on intravenous defibrotide prophylaxis vs controls (e.g. heparin, no prophylaxis), VOD/SOS incidence in controls was 16%. The risk ratio for developing VOD/SOS with defibrotide prophylaxis vs controls was 0.30 (95% confidence interval 0.12-0.71; p = 0.006). CONCLUSIONS: This analysis suggests a low incidence of VOD/SOS following intravenous defibrotide prophylaxis, regardless of age group, and a lower relative risk for VOD/SOS with defibrotide prophylaxis vs controls in patient populations at high risk of VOD/SOS.

A Meta-Analysis Evaluating the Incidence of Bleeding Events With Intravenous Defibrotide Treatment Outside the Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome Setting.

Defibrotide is approved to treat hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) with renal/pulmonary dysfunction following hematopoietic cell transplantation (HCT) in adult and pediatric patients in the United States, and to treat severe hepatic VOD/SOS post HCT in adult and pediatric patients aged >1 month in the European Union. The defibrotide prescribing information warns that defibrotide may increase bleeding risk in VOD/SOS patients. To broaden our understanding of the incidence of bleeding with defibrotide, we performed a meta-analysis of the published literature of defibrotide use outside of the post-HCT VOD/SOS setting. Of 1857 records identified, 125 reported on defibrotide; 23 contained data on bleeding events. The estimated overall incidence of bleeding events was 1% (95% confidence interval [CI]: 0%-2%) and 8% (95% CI: 3%-14%) in studies using intravenous defibrotide and studies with controls, respectively. The risk ratio for bleeding events with intravenous defibrotide versus controls was 0.36 (95% CI: 0.24-0.52; P < .00001) among studies with data on intravenous defibrotide and controls. This meta-analysis of defibrotide use outside of the post-HCT VOD/SOS setting suggests that the incidence of bleeding with defibrotide is lower than controls.

Systematic review and meta-analysis: efficacy and safety of early biologic treatment in adult and paediatric patients with Crohn's disease.

BACKGROUND: There is an increasing body of evidence showing that earlier use of biologics improves clinical outcomes in Crohn's disease (CD). AIM: To perform a systematic review and meta-analysis to assess the impact of early biologic use in the treatment of CD. METHODS: PubMed and Embase databases were searched for English language papers and conference abstracts published through April 30, 2019. Studies were selected for inclusion if patients initiated biologics within 2 years of a CD diagnosis or if earlier biologics use (top-down) was compared with a conventional step-up strategy. Random-effects meta-analyses were conducted to compare clinical remission (CR), relapse and endoscopic healing rates between early biologic treatment (<2 years of disease duration or top-down treatment strategy) and late/conventional treatment (biologic use after >2 years of disease duration or conventional step-up treatment strategy). RESULTS: A total of 3069 records were identified, of which 47 references met the selection criteria for systematic review. A total of 18 471 patients were studied, with a median follow-up of 64 weeks (range 10-416). Meta-analysis found that early use of biologics was associated with higher rates of clinical remission (OR 2.10 [95% CI: 1.69-2.60], n = 2763, P < .00001), lower relapse rates (OR 0.31 [95% CI: 0.14-0.68], n = 596, P = .003) and higher mucosal healing rates (OR 2.37 [95% CI: 1.78-3.16], n = 994, P < .00001) compared with late/conventional management. CONCLUSIONS: Early biologic treatment is associated with improved clinical outcomes in both adult and paediatric CD patients, not only in prospective clinical trials but also in real-world settings.

Publisher Correction: Systematic review of defibrotide studies in the treatment of veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS).

The original version of this article included some minor typesetting errors in Table 1-a corrected version of which is provided in this correction article and has been corrected in the original article-and one error in text, which has been corrected in the original article. Furthermore, the article was erroneously published with the copyright holders as 'Springer Nature Limited', however, since the article is Open Access, the copyright holders should in fact be 'The Author(s)'. This has also been corrected in the original article.

Systematic review of defibrotide studies in the treatment of veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS).

Veno-occlusive disease (VOD), also called sinusoidal obstruction syndrome (SOS), is a potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT) conditioning or high-dose nontransplant chemotherapy. VOD/SOS with multi-organ dysfunction (MOD) is associated with a mortality rate of > 80%. Defibrotide (25 mg/kg/day) is approved to treat hepatic VOD/SOS with renal or pulmonary dysfunction post HSCT in the United States and to treat severe hepatic VOD/SOS in patients > 1 month of age in the European Union. A random effects model was used for pooling data from 17 systematically chosen defibrotide studies. For patients in these reports (n = 2598), and those in the subset of 10 reports of patients treated with ~ 25 mg/kg/day (n = 1691), estimated Day + 100 survival rates were 54% and 56%, respectively. Among those patients treated with ~ 25 mg/kg/day, estimated Day + 100 survival was 44% among patients with MOD and 71% in patients without MOD; survival was 41% and 70%, respectively, for the population of patients receiving any dose of defibrotide. Safety results were not pooled owing to differences in reporting methodology but were generally consistent with the known tolerability profile of defibrotide. This analysis provides the largest assessment of survival in patients treated with defibrotide for VOD/SOS with or without MOD.

A dimeric peptide that binds selectively to prostate-specific membrane antigen and inhibits its enzymatic activity.

Prostate-specific membrane antigen (PSMA) is highly expressed by both normal and malignant prostate epithelial cells and by the neovasculature of many tumor types; however, it is not expressed by normal endothelial cells or other normal tissues. PSMA, therefore, represents an attractive candidate for selectively targeted therapies for prostate and/or other solid tumors. As an alternative approach to antibody-based anti-PSMA therapies, small peptides that bind selectively to PSMA-producing cells can be used to deliver cytotoxic drugs, protein toxins, and viruses selectively to malignant sites while minimizing systemic toxicity to normal tissues. Small peptides are relatively inexpensive to produce, not immunogenic, and easily coupled to cytotoxic agents. In the present study, a random phage library consisting of linear 12 amino acid peptides was used to identify peptides that bound selectively to PSMA. From a series of monomeric peptides, one with the sequence WQPDTAHHWATL was used to show binding of soluble peptide to PSMA. A dimeric version of this peptide showed markedly enhanced binding to soluble PSMA and an IC50 of 2.2 micromol/L for inhibition of PSMA enzymatic activity. Fluorescently labeled dimeric peptide bound selectively to PSMA-producing prostate cancer cells in vitro with no significant binding to non-PSMA-producing cells. Molecular modeling of the dimeric peptide revealed that histidine residues in close vicinity can efficiently coordinate a divalent ion and hold the peptide in a favorable configuration for binding and subsequent inhibition. These dimeric peptides, therefore, represent putative PSMA-selective targeting agents that are currently being evaluated for selective binding in vivo.

Improved methods for the generation of human gene knockout and knockin cell lines.

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of beta-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.

Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS: We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION: Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-beta2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-beta2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.

Detection of promoter hypermethylation of multiple genes in the tumor and bronchoalveolar lavage of patients with lung cancer.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients. EXPERIMENTAL DESIGN: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-beta 2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-beta 2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-beta 2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-beta 2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation. CONCLUSION: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Epigenetic silencing of S100A2 in bladder and head and neck cancers.

S100A2, a member of the S100 protein family, is known to be downregulated in a number of human cancers, leading to its designation as a potential tumor suppressor gene. Here, we investigated the expression and methylation status of S100A2 in head&neck and bladder cancer. Reduced mRNA and protein expression was observed in 8 head&neck and bladder cancer cell lines. To explore the mechanism responsible for the downregulation of S100A2, we treated six cell lines with 5-aza-2'-deoxycytidine. We found S100A2 is silenced in association with aberrant promoter-region methylation and its expression is restored with 5-aza-2'-deoxycytidine treatment. Of 31 primary head&neck cancer cases and 31 bladder cancer cases, promoter methylation was detected in 90% and 80% of cases, respectively. Interestingly, only 1/9 of normal head&neck tissues and 2/6 of normal bladder tissues showed promoter methylation. S100A2 promoter methylation can be detected in urine and is more frequent in bladder cancer patients than in healthy subjects (96% vs 48% respectively). Moreover, increased methylation of S100A2 is linked to the progression of the tumor in bladder cancer (p<0.01). Together, this data shows that methylation-associated inactivation of S100A2 is frequent and may be an important event in the tumorigenesis of head&neck and bladder cancer.

Digital HLA allelotyping.

Stem cell transplantation is a potentially curative therapy for a number of neoplastic disorders. Successful transplantation of bone marrow, and other sources of stem cells, depends upon the extent to which human leukocyte antigen (HLA) alleles in donor and recipient are matched. HLA incompatibility can result in cytotoxic T-cell mediated graft failure or graft versus host disease. It has been shown that accurate matching between donor and recipient minimizes both of these outcomes and results in improved patient survival. Therefore, methods for high resolution HLA analysis have substantial clinical impact. Heterozygosity at the HLA loci interferes with current methods of DNA sequence-based HLA typing. We present a novel method for HLA analysis, which we term Digital HLA Allelotyping, or dig-HLA. This method employs a strategy in which the expressed HLA alleles are randomly sorted and individually identified.

Deficiency of Bloom syndrome helicase activity is radiomimetic.

Bloom syndrome is caused by homozygous mutations in BLM, which encodes a RecQ DNA helicase. Patient-derived cells deficient in BLM helicase activity exhibit genetic instability--apparent cytogenetically as sister chromatid exchanges--and activated DNA damage signaling. In this report, we show that BLM-knockout colorectal cancer cells exhibited endogenous, ATM-dependent double-strand DNA break responses similar to those recently observed in Bloom syndrome patient-derived cells. Xenograft tumors established from BLM-deficient cancer cells were not radiosensitive, but exhibited growth impairment that was comparable to that of wild type tumors treated with a single, high dose of ionizing radiation. These results suggest that pharmacological inhibitors of BLM would have a radiomimetic effect and that transient inhibition of BLM activity might be a viable strategy for anticancer therapy.

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites.

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.

Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertility.

During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3'untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3'UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3'UTR of Tnp2 gene was replaced by 3' 3'UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3'UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility.

Quantitation of promoter methylation of multiple genes in urine DNA and bladder cancer detection.

BACKGROUND: The noninvasive identification of bladder tumors may improve disease control and prevent disease progression. Aberrant promoter methylation (i.e., hypermethylation) is a major mechanism for silencing tumor suppressor genes and other cancer-associated genes in many human cancers, including bladder cancer. METHODS: A quantitative fluorogenic real-time polymerase chain reaction (PCR) assay was used to examine primary tumor DNA and urine sediment DNA from 15 patients with bladder cancer and 25 control subjects for promoter hypermethylation of nine genes (APC, ARF, CDH1, GSTP1, MGMT, CDKN2A, RARbeta2, RASSF1A, and TIMP3) to identify potential biomarkers for bladder cancer. We then used these markers to examine urine sediment DNA samples from an additional 160 patients with bladder cancers of various stages and grades and from an additional 69 age-matched control subjects. Data were analyzed on the basis of a prediction model and were internally validated using a jacknife procedure. All statistical tests were two-sided. RESULTS: For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Four genes displayed 100% specificity. Of the 175 bladder cancer patients, 121 (69%, 95% confidence interval [CI] = 62% to 76%) displayed promoter methylation in at least one of these genes (CDKN2A, ARF, MGMT, and GSTP1), whereas all control subjects were negative for such methylation (100% specificity, 95% CI = 96% to 100%). A logistic prediction model using the methylation levels of all remaining five genes was developed and internally validated for subjects who were negative on the four-gene panel. This combined, two-stage predictor produced an internally validated ROC curve with an overall sensitivity of 82% (95% CI = 75 % to 87%) and specificity of 96% (95% CI = 90% to 99%). CONCLUSION: Testing a small panel of genes with the quantitative methylation-specific PCR assay in urine sediment DNA is a powerful noninvasive approach for the detection of bladder cancer. Larger independent confirmatory cohorts with longitudinal follow-up will be required in future studies to define the impact of this technology on early detection, prognosis, and disease monitoring before clinical application.