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The effect of increasing intravenous doses of theophylline and enprofylline, a new xanthine derivative, on bronchial responsiveness to methacholine was studied in eight asthmatic patients. Methacholine provocations were carried out on three days before and after increasing doses of theophylline, enprofylline, and placebo, a double blind study design being used. Methacholine responsiveness was determined as the provocative concentration of methacholine causing a fall of 20% in FEV1 (PC20). The patients were characterised pharmacokinetically before the main study to provide an individual dosage scheme for each patient that would provide rapid steady state plasma concentration plateaus of 5, 10, and 15 mg/l for theophylline and 1.25, 2.5, and 3.75 mg/l for enprofylline. Dose increments in the main study were given at 90 minute intervals. FEV1 showed a small progressive decrease after placebo; it remained high in relation to placebo after both drugs and this effect was dose related. Methacholine PC20 values decreased after placebo; mean values were higher after theophylline and enprofylline than after placebo (maximum difference 2.0 and 1.7 doubling doses of methacholine); the effect of both drugs were dose related. Thus enprofylline and theophylline when given intravenously cause a small dose related increase in FEV1 and methacholine PC20 when compared with placebo. [ABSTRACT FROM PUBLISHER]

The present study reports the changes in configuration of maximal flow-volume curves after eight weeks' treatment with inhaled corticosteroids in 14 asthmatic patients. The configuration was compared with that seen after bronchodilatation following inhalation of a single dose of ipratropium bromide. After inhaled corticosteroids the shape of the flow-volume curves was less bowed toward the volume axis, whereas the shape of the flow-volume curves after inhalation of ipratropium bromide showed no significant change. A significant correlation was observed between the decrease in blood eosinophil cell count and the straightening of the flow-volume curves, quantitatively expressed as shape factor and slope ratio. It is concluded that these changes in flow-volume curve configuration reflect a decrease in inhomogeneously distributed inflammatory airway narrowing. [ABSTRACT FROM PUBLISHER]

Pulmonary function tests, including spirometry, transfer factor of the lungs for carbon monoxide (TlCO), and the two components of TlCO, the diffusing capacity of the alveolocapillary membrane (Dm) and pulmonary capillary blood volume (Vc), were carried out in a group of patients with testicular carcinoma during and after treatment with the Einhorn regimen. The lung function parameters of patients who developed bleomycin-induced pneumonitis were compared with those recorded in a group of patients who did not develop this syndrome. We suggest that bleomycin-induced damage to the pulmonary capillary vasculature can be monitored by measuring Vc and that ensuing fibrosis can be measured by recording Dm. The decrease in Dm is probably compensated for by an increase in Vc, leading to a smaller change in TlCO. [ABSTRACT FROM AUTHOR]

Correction: The original version of this article [1] unfortunately contained a typographical error. The 'Alzheimer's Disease Neuroimaging Initiative' was erroneously included as 'Alzheimer's Disease Neuroimaging Initative' in the author list of the article. [ABSTRACT FROM AUTHOR]

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to investigate whether systemic lupus erythematosus (SLE) patients with interstitial lung disease represent a particular subset of patients characterized by the presence of clinical, serologic, and nailfold capillary patterns overlapping scleroderma.Patients and Methods: In 57 consecutive patients with SLE, a standardized detailed history was obtained and a physical examination performed, directed at signs and symptoms of connective tissue diseases, in particular scleroderma. Additionally, pulmonary function testing, chest radiography, radionuclide transit studies of the esophagus, nailfold capillary microscopy, and detailed serologic studies directed at the antigenic specificities of antinuclear antibodies were performed. Patients were divided into three groups based on the results of pulmonary function testing, i.e., normal lung function, restriction, or isolated impairment of diffusion. Clinical, serologic, and nailfold capillary microscopic findings were compared among these three groups.Results: Twenty patients had normal lung function, 19 had restrictive lung function loss, and 9 had an isolated impairment of the diffusing capacity (T1,CO). Patients with obstructive lung disease (n = 9) were excluded from analysis. Sclerodermatous changes of the hands were associated with a restrictive lung function pattern. Interstitial changes on chest radiograph were associated with isolated impairment of T1,CO. Nailfold capillary abnormalities correlated with decreased T1,CO and Dm, the component of T1,CO representing the diffusing capacity of the alveolocapillary membrane. Antibodies to U1-RNA were associated with restrictive lung function and decreased T1,CO.Conclusion: We conclude that interstitial lung disease is present in a subset of SLE patients characterized by an increased prevalence of scleroderma traits and anti-(U1)RNA antibodies. Microvascular changes may contribute to the development of interstitial lung disease in SLE as well as in scleroderma. [ABSTRACT FROM AUTHOR]

Background: The gene C9orf72 harbors a non-coding hexanucleotide repeat expansion known to cause amyotrophic lateral sclerosis and frontotemporal dementia. While previous studies have estimated the length of this repeat expansion in multiple tissues, technological limitations have impeded researchers from exploring additional features, such as methylation levels., Methods: We aimed to characterize C9orf72 repeat expansions using a targeted, amplification-free long-read sequencing method. Our primary goal was to determine the presence and subsequent quantification of observed methylation in the C9orf72 repeat expansion. In addition, we measured the repeat length and purity of the expansion. To do this, we sequenced DNA extracted from blood for 27 individuals with an expanded C9orf72 repeat., Results: For these individuals, we obtained a total of 7,765 on-target reads, including 1,612 fully covering the expanded allele. Our in-depth analysis revealed that the expansion itself is methylated, with great variability in total methylation levels observed, as represented by the proportion of methylated CpGs (13 to 66%). Interestingly, we demonstrated that the expanded allele is more highly methylated than the wild-type allele (P-Value = 2.76E-05) and that increased methylation levels are observed in longer repeat expansions (P-Value = 1.18E-04). Furthermore, methylation levels correlate with age at collection (P-Value = 3.25E-04) as well as age at disease onset (P-Value = 0.020). Additionally, we detected repeat lengths up to 4,088 repeats (~ 25 kb) and found that the expansion contains few interruptions in the blood., Conclusions: Taken together, our study demonstrates robust ability to quantify methylation of the expanded C9orf72 repeat, capturing differences between individuals harboring this expansion and revealing clinical associations., Competing Interests: Declarations. Ethics approval and consent to participate: All subjects agreed to be in the study, and biological specimens were obtained after informed consent with approval from the Mayo Clinic Institutional Review Board (IRB). Consent for publication: Not applicable. Competing interests: MDJ and RR hold a patent on methods to screen for the C9orf72 hexanucleotide repeat expansion., (© 2024. The Author(s).)

Motivation: Alternative splicing generates multiple RNA isoforms from a single gene, enriching genetic diversity and impacting gene function. Effective visualization of these isoforms and their expression patterns is crucial but challenging due to limitations in existing tools. Traditional genome browsers lack programmability, while other tools offer limited customization, produce static plots, or cannot simultaneously display structures and expression levels. RNApysoforms was developed to address these gaps by providing a Python-based package that enables concurrent visualization of RNA isoform structures and expression data. Leveraging plotly and polars libraries, it offers an interactive, customizable, and faster-rendering framework suitable for web applications, enhancing the analysis and dissemination of RNA isoform research., Availability and Implementation: RNApysoforms is a Python package available at (https://github.com/UK-SBCoA-EbbertLab/RNApysoforms) via an open-source MIT license. It can be easily installed using the piip package installer for Python. Thorough documentation and usage vignettes are available at: https://rna-pysoforms.readthedocs.io/en/latest/.

Objective: To systematically review and meta-analyze bulk RNA sequencing studies comparing Alzheimer's disease (AD) patients with controls in human brain tissue, assessing study quality and identifying key genes and pathways., Methods: We searched PubMed, Web of Science, and Scopus on September 23, 2023, for studies using bulk RNAseq on primary human brain tissue from AD patients and controls. Excluded were non-primary tissue, re-analyses without new data, limited RNA types and gene panels. Quality was assessed with a 10-category tool. Meta-analysis used high-quality datasets., Results: From 3,266 records, 24 studies met criteria. Meta-analysis found 571 differentially expressed genes (DEGs) in temporal lobe and 189 in frontal lobe; overlapping pathways included "Tube morphogenesis" and "Neuroactive ligand-receptor interaction.", Limitations: Study heterogeneity and limited data tables constrained the review., Conclusions: Rigorous methods are vital in AD transcriptomic studies. Findings enhance understanding of transcriptomic changes, aiding biomarker and therapeutic development., Registration: PROSPERO (CRD42023466522)., Competing Interests: Competing interests Statement: The authors report no competing interests.

Even though alternative RNA splicing was discovered nearly 50 years ago (1977), we still understand very little about most isoforms arising from a single gene, including in which tissues they are expressed and if their functions differ. Human gene annotations suggest remarkable transcriptional complexity, with approximately 252,798 distinct RNA isoform annotations from 62,710 gene bodies (Ensembl v109; 2023), emphasizing the need to understand their biological effects. For example, 256 gene bodies have ≥50 annotated isoforms and 30 have ≥100, where one protein-coding gene ( MAPK10 ) even has 192 distinct RNA isoform annotations. Whether such isoform diversity results from biological redundancy or spurious alternative splicing (i.e., noise), or whether individual isoforms have specialized functions (even if subtle) remains a mystery for most genes. Recent studies by Aguzzoli-Heberle et al., Leung et al., and Glinos et al. demonstrated long-read RNAseq enables improved RNA isoform quantification for essentially any tissue, cell type, or biological condition ( e.g., disease, development, aging, etc.), making it possible to better assess individual isoform expression and function. While each study provided important discoveries related to RNA isoform diversity, deeper exploration is needed. We sought to quantify and characterize real isoform usage across tissues (compared to annotations). We used long-read RNAseq data from 58 GTEx samples across nine tissues (three brain, two heart, muscle, lung, liver, and cultured fibroblasts) generated by Glinos et al. and found considerable isoform diversity within and across tissues. Cerebellar hemisphere was the most transcriptionally complex tissue (22,522 distinct isoforms; 3,726 unique); liver was least diverse (12,435 distinct isoforms; 1,039 unique). We highlight gene clusters exhibiting high tissue-specific isoform diversity per tissue ( e.g. , TPM1 expresses 19 in heart's atrial appendage). We also validated 447 of the 700 new isoforms discovered by Aguzzoli-Heberle et al. and found that 88 were expressed in all nine tissues, while 58 were specific to a single tissue. This study represents a broad survey of the RNA isoform landscape, demonstrating isoform diversity across nine tissues and emphasizes the need to better understand how individual isoforms from a single gene body contribute to human health and disease., Competing Interests: Competing interests The authors report no competing interests.

With the introduction of population-based bowel cancer screening, rectal cancer is diagnosed at earlier stages, yet standard treatment still requires the same extensive surgery that is used for more advanced stages. Organ preserving treatment is rapidly developing and is subject of investigation in numerous clinical trials. The STAR-TREC trial is an international, multi-centre randomised trial investigating organ preservation using (chemo)radiotherapy. Patients with small mrT1-3bN0V0M0 tumours are randomized between three arms: standard TME, organ preservation with SCRT or with CRT. In this trial, the clinical target volume has been tailored to the early staged disease of the included patients. This mesorectal irradiation volume includes the mesorectum and pre-sacral lymph nodes at the level of the tumour, two centimetres below and cranially up to the S2-3 interspace level. In contrast to conventional irradiation volumes, the lateral lymph nodes and the nodes along the superior rectal artery are excluded. As a result, the dose to the bowel, bladder, anal sphincter and the neurovascular plexus in the lower pelvis is substantially decreased, especially when combined with modern irradiation techniques, such as dynamic arc therapy. These lower doses are expected to lead to decreasing acute and late toxicity and beneficial functional outcomes. The implementation of this novel target volume will be accompanied by an extensive quality assurance program in the STAR-TREC trial. We describe the rationale behind the novel, mesorectal only radiotherapy treatment used in the STAR-TREC trial specifically tailored for early stage disease, with the goal of organ preservation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Purpose: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials., Methods and Materials: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom., Results: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival., Conclusions: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is an age-associated neurodegenerative disease characterized by amyloidosis, tauopathy, and activation of microglia, the brain resident innate immune cells. We show that a RiboTag translational profiling approach can bypass biases due to cellular enrichment/cell sorting. Using this approach in models of amyloidosis, tauopathy, and aging, we revealed a common set of alterations and identified a central APOE-driven network that converged on CCL3 and CCL4 across all conditions. Notably, aged females demonstrated a significant exacerbation of many of these shared transcripts in this APOE network, revealing a potential mechanism for increased AD susceptibility in females. This study has broad implications for microglial transcriptomic approaches and provides new insights into microglial pathways associated with different pathological aspects of aging and AD., (© 2018 Kang et al.)

Purpose: Multiple phase 2 trials of neoadjuvant treatment intensification in locally advanced rectal cancer have reported promising efficacy signals, but these have not translated into improved cancer outcomes in phase 3 trials. Improvements in phase 2 trial design are needed to reduce these false-positive signals. This systematic review evaluated the design of phase 2 trials of neoadjuvant long-course radiation or chemoradiation therapy treatment intensification in locally advanced rectal cancer., Methods and Materials: The PubMed, EMBASE, MEDLINE, and Cochrane Library databases were searched for published phase 2 trials of neoadjuvant treatment intensification from 2004 to 2016. Trial clinical design and outcomes were assessed, with statistical design and compliance rated using a previously published system. Multivariable meta-regression analysis of pathologic complete response (pCR) was conducted., Results: We identified 92 eligible trials. Patients with American Joint Committee on Cancer stage II and III equivalent disease were eligible in 87 trials (94.6%). In 43 trials (46.7%), local staging on magnetic resonance imaging was mandated. Only 12 trials (13.0%) were randomized, with 8 having a standard-treatment control arm. Just 51 trials (55.4%) described their statistical design, with 21 trials (22.8%) failing to report their sample size derivation. Most trials (n=84, 91.3%) defined a primary endpoint, but 15 different primary endpoints were used. All trials reported pCR rates. Only 38 trials (41.3%) adequately reported trial statistical design and compliance. Meta-analysis revealed a pooled pCR rate of 17.5% (95% confidence interval, 15.7%-19.4%) across treatment arms of neoadjuvant long-course radiation or chemoradiation therapy treatment intensification and substantial heterogeneity among the reported effect sizes (I 2 = 55.3%, P<.001). Multivariable meta-regression analysis suggested increased pCR rates with higher radiation therapy doses (adjusted P=.025)., Conclusions: Improvement in the design of future phase 2 rectal cancer trials is urgently required. A significant increase in randomized trials is essential to overcome selection bias and determine novel schedules suitable for phase 3 testing. This systematic review provides key recommendations to guide future treatment intensification trial design in rectal cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: HCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6., Objective: To evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing., Study Design: This study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database., Results: Agreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven "not detected" by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure., Conclusions: The PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Ebbert et al. reported gene-gene interactions between rs11136000-rs670139 (CLU-MS4A4E) and rs3865444-rs670139 (CD33-MS4A4E). We evaluate these interactions in the largest data set for an epistasis study., Methods: We tested interactions using 3837 cases and 4145 controls from Alzheimer's Disease Genetics Consortium using meta-analyses and permutation analyses. We repeated meta-analyses stratified by apolipoprotein E (APOE) ε4 status, estimated combined odds ratio (OR) and population attributable fraction (cPAF), and explored causal variants., Results: Results support the CLU-MS4A4E interaction and a dominant effect. An association between CLU-MS4A4E and APOE ε4 negative status exists. The estimated synergy factor, OR, and cPAF for rs11136000-rs670139 are 2.23, 2.45, and 8.0, respectively. We identified potential causal variants., Discussion: We replicated the CLU-MS4A4E interaction in a large case-control series and observed APOE ε4 and possible dominant effect. The CLU-MS4A4E OR is higher than any Alzheimer's disease locus except APOE ε4, APP, and TREM2. We estimated an 8% decrease in Alzheimer's disease incidence without CLU-MS4A4E risk alleles and identified potential causal variants., (Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Cholesterol has been implicated in the pathogenesis of late-onset Alzheimer's disease (LOAD) and the cholesteryl ester transfer protein (CETP) is critical to cholesterol regulation within the cell, making CETP an Alzheimer's disease candidate gene. Several studies have suggested that CETP I405V (rs5882) is associated with cognitive function and LOAD risk, but findings vary and most studies have been conducted using relatively small numbers of samples. To test whether this variant is involved in cognitive function and LOAD progression, we genotyped 4486 subjects with up to 12 years of longitudinal cognitive assessment. Analyses revealed an average 0.6-point decrease per year in the rate of cognitive decline for each additional valine (p < 0.011). We failed to detect the association between CETP I405V and LOAD status (p < 0.28). We conclude that CETP I405V is associated with preserved cognition over time but is not associated with LOAD status., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Reported odds ratios and population attributable fractions (PAF) for late-onset Alzheimer's disease (LOAD) risk loci (BIN1, ABCA7, CR1, MS4A4E, CD2AP, PICALM, MS4A6A, CD33, and CLU) come from clinically ascertained samples. Little is known about the combined PAF for these LOAD risk alleles and the utility of these combined markers for case-control prediction. Here we evaluate these loci in a large population-based sample to estimate PAF and explore the effects of additive and nonadditive interactions on LOAD status prediction performance., Methods: 2419 samples from the Cache County Memory Study were genotyped for APOE and nine LOAD risk loci from AlzGene.org. We used logistic regression and receiver operator characteristic analysis to assess the LOAD status prediction performance of these loci using additive and nonadditive models and compared odds ratios and PAFs between AlzGene.org and Cache County., Results: Odds ratios were comparable between Cache County and AlzGene.org when identical single nucleotide polymorphisms were genotyped. PAFs from AlzGene.org ranged from 2.25% to 37%; those from Cache County ranged from .05% to 20%. Including non-APOE alleles significantly improved LOAD status prediction performance (area under the curve = .80) over APOE alone (area under the curve = .78) when not constrained to an additive relationship (p < .03). We identified potential allelic interactions (p values uncorrected): CD33-MS4A4E (synergy factor = 5.31; p < .003) and CLU-MS4A4E (synergy factor = 3.81; p < .016)., Conclusions: Although nonadditive interactions between loci significantly improve diagnostic ability, the improvement does not reach the desired sensitivity or specificity for clinical use. Nevertheless, these results suggest that understanding gene-gene interactions may be important in resolving Alzheimer's disease etiology., (Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The use of cerebrospinal fluid levels of Aβ42 and pTau181 as endophenotypes for genetic studies of Alzheimer's disease (AD) has led to successful identification of both rare and common AD risk variants. In addition, this approach has provided meaningful hypotheses for the biological mechanisms by which known AD risk variants modulate the disease process. In this article we discuss these successes and outline challenges to effective and continued applications of this approach. We contrast the statistical power of this approach with traditional case-control designs and discuss solutions to address challenges in quality control and data analysis for these phenotypes. Finally, we discuss the potential for the use of this approach with larger samples as well as the incorporation of next generation sequencing and for future work with other endophenotypes for AD.

Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.

Recent studies have identified the rs75932628 (R47H) variant in TREM2 as an Alzheimer's disease risk factor with estimated odds ratio ranging from 2.9 to 5.1. The Cache County Memory Study is a large, population-based sample designed for the study of memory and aging. We genotyped R47H in 2974 samples (427 cases and 2540 control subjects) from the Cache County study using a custom TaqMan assay. We observed 7 heterozygous cases and 12 heterozygous control subjects with an odds ratio of 3.5 (95% confidence interval, 1.3-8.8; p = 0.0076). The minor allele frequency and population attributable fraction for R47H were 0.0029 and 0.004, respectively. This study replicates the association between R47H and Alzheimer's disease risk in a large, population-based sample, and estimates the population frequency and attributable risk of this rare variant., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Current HIV-1 sequencing-based methods for detecting drug resistance-associated mutations are open and susceptible to contamination. Informatic identification of clinical sequences that are nearly identical to one another may indicate specimen-to-specimen contamination or another laboratory-associated issue., Objectives: To design an informatic tool to rapidly identify potential contamination in the clinical laboratory using sequence analysis and to establish reference ranges for sequence variation in the HIV-1 protease and reverse transcriptase regions among a U.S. patient population., Study Design: We developed an open-source tool named HIV Contamination Detection (HIVCD). HIVCD was utilized to make pairwise comparisons of nearly 8000 partial HIV-1 pol gene sequences from patients across the United States and to calculate percent identities (PIDs) for each pair. ROC analysis and standard deviations of PID data were used to determine reference ranges for between-patient and within-patient comparisons and to guide selection of a threshold for identifying abnormally high PID between two unrelated sequences., Results: The PID reference range for between-patient comparisons ranged from 83.8 to 95.7% while within-patient comparisons ranged from 96 to 100%. Interestingly, 48% of between-patient sequence pairs with a PID>96.5 were geographically related. The selected threshold for abnormally high PIDs was 96 (AUC=0.993, sensitivity=0.980, specificity=0.999). During routine use, HIVCD identified a specimen mix-up and the source of contamination of a negative control., Conclusions: In our experience, HIVCD is easily incorporated into laboratory workflow, useful for identifying potential laboratory errors, and contributes to quality testing. This type of analysis should be incorporated into routine laboratory practice., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p < 0.001; and interaction test, p = 0.006). No significant interactions between treatment and the PAM50 subtypes or the various clinical-pathological variables, including Ki-67 and histologic grade, were identified. Finally, similar OS results were obtained in the CALGB data set, although the interaction test did not reach statistical significance (p = 0.109). The PAM50 proliferation score identifies a subset of patients with a low proliferation status that may derive a larger benefit from weekly paclitaxel.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. ©2012 AACR., (©2012 AACR.)

Background: Many methodologies have been used in research to identify the "intrinsic" subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions., Methods: We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and "intrinsic" subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments., Results: ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis., Conclusions: The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.

To determine the outcomes of patients managed with different routes of enteral feeding during chemoradiotherapy for oropharynx cancer. The hospital and dietetic records of consecutive patients with oropharynx squamous cell carcinoma treated between January 2007 and June 2009 with concurrent chemoradiotherapy were reviewed retrospectively. One hundred and four patients were analysed. Seventy-one received a prophylactic gastrostomy, 21 were managed with a strategy of NG tube as required and 12 received a therapeutic gastrostomy. Patients with a prophylactic gastrostomy commenced enteral feeding a median of 24 days after commencing radiotherapy, compared with a median of 41 days (p<0.001) for the NG as required group. Comparing prophylactic gastrostomy, NG as required and therapeutic gastrostomy, median number of unplanned inpatient days were 6, 14 and 7, respectively (p<0.01 for prophylactic gastrostomy vs. NG as required). Mean percentage weight loss at the end of treatment (6.1% vs. 7.1% vs. 5.2%, respectively) and at 6 months post-radiotherapy (11.7%, 14.3% and 8.9%) were similar in all groups (p=0.23). There was no significant difference in type of diet post-radiotherapy between prophylactic gastrostomy and NG as required groups (p=0.22). Median duration of enteral feeding was 181, 64 and 644 days, respectively (p<0.01 for prophylactic gastrostomy vs. NG as required). Use of a prophylactic gastrostomy (p<0.01) and higher T stage (p<0.01) were associated with increased duration of enteral feeding on a multivariate analysis. These data reinforce concerns regarding the detrimental impact of prophylactic gastrostomy placement upon long-term enteral feed dependence., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Recent studies suggest that intrinsic breast cancer subtypes may differ in their responsiveness to specific chemotherapy regimens. We examined this hypothesis on NCIC.CTG MA.5, a clinical trial randomizing premenopausal women with node-positive breast cancer to adjuvant CMF (cyclophosphamide-methotrexate-fluorouracil) versus CEF (cyclophosphamide-epirubicin-fluorouracil) chemotherapy., Experimental Design: Intrinsic subtype was determined for 476 tumors using the quantitative reverse transcriptase PCR PAM50 gene expression test. Luminal A, luminal B, HER2-enriched (HER2-E), and basal-like subtypes were correlated with relapse-free survival (RFS) and overall survival (OS), estimated using Kaplan-Meier plots and log-rank testing. Multivariable Cox regression analyses determined significance of interaction between treatment and intrinsic subtypes., Results: Intrinsic subtypes were associated with RFS (P = 0.0005) and OS (P < 0.0001) on the combined cohort. The HER2-E showed the greatest benefit from CEF versus CMF, with absolute 5-year RFS and OS differences exceeding 20%, whereas there was a less than 2% difference for non-HER2-E tumors (interaction test P = 0.03 for RFS and 0.03 for OS). Within clinically defined Her2(+) tumors, 79% (72 of 91) were classified as the HER2-E subtype by gene expression and this subset was strongly associated with better response to CEF versus CMF (62% vs. 22%, P = 0.0006). There was no significant difference in benefit between CEF and CMF in basal-like tumors [n = 94; HR, 1.1; 95% confidence interval (CI), 0.6-2.1 for RFS and HR, 1.3; 95% CI, 0.7-2.5 for OS]., Conclusion: HER2-E strongly predicted anthracycline sensitivity. The chemotherapy-sensitive basal-like tumors showed no added benefit for CEF over CMF, suggesting that nonanthracycline regimens may be adequate in this subtype although further investigation is required., (©2012 AACR.)

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression through binding to messenger RNAs (mRNA) thereby promoting mRNA degradation or altered translation. A single-nucleotide polymorphism (SNP) located within a miRNA-binding site could thus alter mRNA translation and influence cancer risk and treatment response. The common SNPs located within the 3'-untranslated regions of 20 DNA repair genes were analysed for putative miRNA-binding sites using bioinformatics algorithms, calculating the difference in Gibbs free binding energy (ΔΔG) for each wild-type versus variant allele. Seven SNPs were selected to be genotyped in germ line DNAs both from a bladder cancer case-control series (752 cases and 704 controls) and 202 muscle-invasive bladder cancer radiotherapy cases. The PARP-1 SNP rs8679 was also genotyped in a breast cancer case-control series (257 cases and 512 controls). Without adjustment for multiple testing, multivariate analysis demonstrated an association with increased bladder cancer risk with PARP1 rs8679 (P(trend) = 0.05) while variant homozygotes of PARP1 rs8679 were also noted to have an increased breast cancer risk (P = 0.03). In the radiotherapy cases, carriers of the RAD51 rs7180135 minor allele had improved cancer-specific survival (hazard ratio 0.52, 95% confidence interval 0.31-0.87, P = 0.01). This is the first report of associations between DNA repair gene miRNA-binding site SNPs with bladder and breast cancer risk and radiotherapy outcomes. If validated, these findings may give further insight into the biology of bladder carcinogenesis, allow testing of the RAD51 SNP as a potential predictive biomarker and also reveal potential targets for new cancer treatments.

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277&#95;279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

Radical radiotherapy and surgery achieve similar cure rates in muscle-invasive bladder cancer, but the choice of which treatment would be most beneficial cannot currently be predicted for individual patients. The primary aim of this study was to assess whether expression of any of a panel of DNA damage signaling proteins in tumor samples taken before irradiation could be used as a predictive marker of radiotherapy response, or rather was prognostic. Protein expression of MRE11, RAD50, NBS1, ATM, and H2AX was studied by immunohistochemistry in pretreatment tumor specimens from two cohorts of bladder cancer patients (validation cohort prospectively acquired) treated with radical radiotherapy and one cohort of cystectomy patients. In the radiotherapy test cohort (n = 86), low tumor MRE11 expression was associated with worse cancer-specific survival compared with high expression [43.1% versus 68.7% 3-year cause-specific survival (CSS), P = 0.012] by Kaplan-Meier analysis. This was confirmed in the radiotherapy validation cohort (n = 93; 43.0% versus 71.2%, P = 0.020). However, in the cystectomy cohort (n = 88), MRE11 expression was not associated with cancer-specific survival, commensurate with MRE11 being a predictive marker. High MRE11 expression in the combined radiotherapy cohort had a significantly better cancer-specific survival compared with the high-expression cystectomy cohort (69.9% versus 53.8% 3-year CSS, P = 0.021). In this validated immunohistochemistry study, MRE11 protein expression was shown and confirmed as a predictive factor associated with survival following bladder cancer radiotherapy, justifying its inclusion in subsequent trial designs. MRE11 expression may ultimately allow patient selection for radiotherapy or cystectomy, thus improving overall cure rates., (©2010 AACR.)

The activation of regulated exocytosis occurs by a rise in cytosolic Ca(2+) concentration. Synaptotagmins act as the Ca(2+) sensors, whereas the machinery that allows fusion of secretory vesicles with the plasma membrane consists of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including syntaxin 1, SNAP-25, and VAMP. Within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18-1. The exact role of Munc18-1 and the steps within exocytosis in which it acts have been intensively investigated. Current evidence suggests that Munc18-1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.

Rab proteins are GTPases that transit between GTP- and GDP-bound states. In the GTP-bound form they can recruit specific effector to membrane domains. It is possible that the exchange of Rab effectors between membranes and cytosol would be determined by the exchange of the particular Rab partner. We have compared the cycling of three Rab3/27 effectors, Granuphilin, Noc2, and Rabphilin, in PC12 cells using fluorescence recovery after photobleaching of EGFP-tagged proteins. All three effectors become localised to secretory granules. Granuphilin and Noc2 showed little or no exchange between secretory granules and cytosol whereas Rabphilin showed rapid and complete exchange. Both Noc2 and Rabphilin were found to be recruited to granules by Rab27 but the data suggest that Rabphilin did not form stable complexes with Rab27 on secretory granules and so Rab effector cycling between membranes and cytosol can be independent of that of the Rab protein.

Genetic and epigenetic defects in Wnt/beta-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/beta-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly derepressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/beta-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/beta-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/beta-catenin signaling in colorectal cancer.