Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis.

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics. We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples. Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV mutation load.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Estimates of potential childhood lead exposure from contaminated soil using the US EPA IEUBK Model in Sydney, Australia.

Surface soils in portions of the Sydney (New South Wales, Australia) urban area are contaminated with lead (Pb) primarily from past use of Pb in gasoline, the deterioration of exterior lead-based paints, and industrial activities. Surface soil samples (n=341) were collected from a depth of 0-2.5cm at a density of approximately one sample per square kilometre within the Sydney estuary catchment and analysed for lead. The bioaccessibility of soil Pb was analysed in 18 samples. The blood lead level (BLL) of a hypothetical 24 month old child was predicted at soil sampling sites in residential and open land use using the United States Environmental Protection Agency (US EPA) Integrated Exposure Uptake and Biokinetic (IEUBK) model. Other environmental exposures used the Australian National Environmental Protection Measure (NEPM) default values. The IEUBK model predicted a geometric mean BLL of 2.0±2.1µg/dL using measured soil lead bioavailability measurements (bioavailability =34%) and 2.4±2.8µg/dL using the Australian NEPM default assumption (bioavailability =50%). Assuming children were present and residing at the sampling locations, the IEUBK model incorporating soil Pb bioavailability predicted that 5.6% of the children at the sampling locations could potentially have BLLs exceeding 5µg/dL and 2.1% potentially could have BLLs exceeding 10µg/dL. These estimations are consistent with BLLs previously measured in children in Sydney.

LPL gene expression is associated with poor prognosis in CLL and closely related to NOTCH1 mutations.

INTRODUCTION: Chronic lymphocytic leukemia is a heterogeneous yet incurable disease. Whole-genome and whole-exome sequencing studies have revealed recurrently occurring somatic mutations in some genes. Several other prognostic markers have previously been tested for their prognostic value in CLL. LPL is among these markers. AIM: To evaluate LPL gene expression together with the well-established prognostic markers of CLL and investigate correlations with more recently identified prognostic markers, NOTCH1 and TP53 mutations. METHODS: On 149 patients, LPL gene expression was analyzed by real-time RT-PCR. Exon 34 of NOTCH1 was PCR-amplified and directly sequenced. RESULTS: LPL gene expression could be measured as a categorical variable (LPL+/LPL-) and was associated with time to treatment (P < 0.001) and overall survival (P = 0.007). In patients otherwise classified as having a good prognosis according to established and new prognostic markers, 3 of 4 patients, who received treatment within 24 months after diagnosis, were LPL+ (P = 0.03). There was a strong correlation between NOTCH1 mutation and LPL+ (P = 0.005). The unfavorable prognosis of LPL+ was maintained in CLL with wild-type NOTCH1. CONCLUSIONS: NOTCH1 mutations are tightly associated with LPL gene expression. LPL expression is independently associated with poor outcome in CLL and can be measured as a categorical variable.

Unravelling a 'miner's myth' that environmental contamination in mining towns is naturally occurring.

Australia has a long history of metal mining and smelting. Extraction and processing have resulted in elevated levels of toxic metals surrounding mining operations, which have adverse health effects, particularly to children. Resource companies, government agencies and employees often construct 'myths' to down play potential exposure risks and responsibility arising from operating emissions. Typical statements include: contaminants are naturally occurring, the wind blows emissions away from residential areas, contaminants are not bioavailable, or the problem is a legacy issue and not related to current operations. Evidence from mining and smelting towns shows that such 'myths' are exactly that. In mining towns, the default and primary defence against contamination is that elevated metals in adjacent urban environments are from the erosion and weathering of the ore bodies over millennia-hence 'naturally occurring'. Not only is this a difficult argument to unravel from an evidence-based perspective, but also it causes confusion and delays remediation work, hindering efforts to reduce harmful exposures to children. An example of this situation is from Broken Hill, New South Wales, home to one of the world's largest lead-zinc-silver ore body, which has been mined continuously for over 130 years. Environmental metal concentration and lead isotopic data from soil samples collected from across Broken Hill are used to establish the nature and timing of lead contamination. We use multiple lines of evidence to unravel a 'miner's myth' by evaluating current soil metal concentrations and lead isotopic compositions, geological data, historical environmental assessments and old photographic evidence to assess the impacts from early smelting along with mining to the surface soils in the city.

MDM2 phenotypic and genotypic profiling, respective to TP53 genetic status, in diffuse large B-cell lymphoma patients treated with rituximab-CHOP immunochemotherapy: a report from the International DLBCL Rituximab-CHOP Consortium Program.

MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53 genetically-defined large cohort of de novo DLBCL patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) chemotherapy, we assessed MDM2 and p53 expression by immunohistochemistry (n = 478), MDM2 gene amplification by fluorescence in situ hybridization (n = 364), and a single nucleotide polymorphism in the MDM2 promoter, SNP309, by SNP genotyping assay (n = 108). Our results show that MDM2 overexpression, unlike p53 overexpression, is not a significant prognostic factor in overall DLBCL. Both MDM2 and p53 overexpression do not predict for an adverse clinical outcome in patients with wild-type p53 but predicts for significantly poorer survival in patients with mutated p53. Variable p53 activities may ultimately determine the survival differences, as suggested by the gene expression profiling analysis. MDM2 amplification was observed in 3 of 364 (0.8%) patients with high MDM2 expression. The presence of SNP309 did not correlate with MDM2 expression and survival. This study indicates that evaluation of MDM2 and p53 expression correlating with TP53 genetic status is essential to assess their prognostic significance and is important for designing therapeutic strategies that target the MDM2-p53 interaction.

Environmental arsenic, cadmium and lead dust emissions from metal mine operations: Implications for environmental management, monitoring and human health.

Although blood lead values in children are predominantly falling globally, there are locations where lead exposure remains a persistent problem. One such location is Broken Hill, Australia, where the percentage of blood lead values >10 µg/dL in children aged 1-4 years has risen from 12.6% (2010), to 13% (2011) to 21% (2012). The purpose of this study was to determine the extent of metal contamination in places accessible to children. This study examines contemporary exposure risks from arsenic, cadmium, lead, silver and zinc in surface soil and dust, and in pre- and post-play hand wipes at six playgrounds across Broken Hill over a 5-day period in September 2013. Soil lead (mean 2,450 mg/kg) and zinc (mean 3,710 mg/kg) were the most elevated metals in playgrounds. Surface dust lead concentrations were consistently elevated (mean 27,500 µg/m(2)) with the highest lead in surface dust (59,900 µg/m(2)) and post-play hand wipes (60,900 µg/m(2)) recorded close to existing mining operations. Surface and post-play hand wipe dust values exceeded national guidelines for lead and international benchmarks for arsenic, cadmium and lead. Lead isotopic compositions ((206)Pb/(207)Pb, (208)Pb/(207)Pb) of surface dust wipes from the playgrounds revealed the source of lead contamination to be indistinct from the local Broken Hill ore body. The data suggest frequent, cumulative and ongoing mine-derived dust metal contamination poses a serious risk of harm to children.

Clinical Progression of Metabolic-Associated Fatty Liver Disease Is Rare in a Danish Tertiary Liver Center.

Data concerning non-invasive discrimination of simple steatosis from steatohepatitis in metabolic-associated fatty liver disease (MAFLD) and risk of disease progression in patients with MAFLD are conflicting. We aimed to investigate these factors in an MAFLD cohort at a Danish tertiary liver centre. We retrospectively assessed 129 patients with biopsy-proven MAFLD. Patients were divided according to the presence of simple steatosis or steatohepatitis in liver biopsies. Histological and clinical progression were assessed during follow-up. Patients with steatohepatitis had higher BMIs, liver stiffness, HbA1c and soluble (sCD163) and were more prone to have metabolic syndrome at baseline compared with simple steatosis patients. Of the 129 patients, 31 had a follow-up biopsy after a median of 287 days; simple steatosis progressed to steatohepatitis in 7 cases, while 2 regressed. Twenty patients had the same fibrosis stage according to the follow-up biopsy, seven progressed and four regressed. Only 14 patients progressed clinically (median follow-up time was 3.8 years). Clinical progression was associated with female sex, high creatinine, high sCD163 and disease severity in the diagnostic liver biopsy. Steatohepatitis was associated with metabolic and inflammatory parameters including fibroscan. Disease progression was seen in only 11% of cases and was mainly related to more severe histological disease at baseline.

Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.

AIM: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM). RESULTS: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p < 0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p < 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. CONCLUSION: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

VegeSafe: A community science program measuring soil-metal contamination, evaluating risk and providing advice for safe gardening.

The extent of metal contamination in Sydney residential garden soils was evaluated using data collected during a three-year Macquarie University community science program called VegeSafe. Despite knowledge of industrial and urban contamination amongst scientists, the general public remains under-informed about the potential risks of exposure from legacy contaminants in their home garden environment. The community was offered free soil metal screening, allowing access to soil samples for research purposes. Participants followed specific soil sampling instructions and posted samples to the University for analysis with a field portable X-ray Fluorescence (pXRF) spectrometer. Over the three-year study period, >5200 soil samples, primarily from vegetable gardens, were collected from >1200 Australian homes. As anticipated, the primary soil metal of concern was lead; mean concentrations were 413 mg/kg (front yard), 707 mg/kg (drip line), 226 mg/kg (back yard) and 301 mg/kg (vegetable garden). The Australian soil lead guideline of 300 mg/kg for residential gardens was exceeded at 40% of Sydney homes, while concentrations >1000 mg/kg were identified at 15% of homes. The incidence of highest soil lead contamination was greatest in the inner city area with concentrations declining towards background values of 20-30 mg/kg at 30-40 km distance from the city. Community engagement with VegeSafe participants has resulted in useful outcomes: dissemination of knowledge related to contamination legacies and health risks; owners building raised beds containing uncontaminated soil and in numerous cases, owners replacing all of their contaminated soil.

Tracing changes in atmospheric sources of lead contamination using lead isotopic compositions in Australian red wine.

Air quality data detailing changes to atmospheric composition from Australia's leaded petrol consumption is spatially and temporally limited. In order to address this data gap, wine was investigated as a potential proxy for atmospheric lead conditions. Wine spanning sixty years was collected from two wine regions proximal to the South Australian capital city, Adelaide, and analysed for lead concentration and lead and strontium isotopic composition for source apportionment. Maximum wine lead concentrations (328 µg/L) occur prior to the lead-in-air monitoring in South Australia in the later 1970s. Wine lead concentrations mirror available lead-in-air measurements and show a declining trend reflecting parallel reductions in leaded petrol emissions. Lead from petrol dominated the lead in wine ((206)Pb/(207)Pb: 1.086; (208)Pb/(207)Pb: 2.360) until the introduction of unleaded petrol, which resulted in a shift in the wine lead isotopic composition closer to vineyard soil ((206)Pb/(207)Pb: 1.137; (208)Pb/(207)Pb: 2.421). Current mining activities or vinification processes appear to have no impact with recent wine samples containing less than 4 µg/L of lead. This study demonstrates wine can be used to chronicle changes in environmental lead emissions and is an effective proxy for atmospherically sourced depositions of lead in the absence of air quality data.

Handheld X-ray Fluorescence Spectrometers: Radiation Exposure Risks of Matrix-Specific Measurement Scenarios.

This study investigates X-ray intensity and dispersion around handheld X-ray fluorescence (XRF) instruments during the measurement of a range of sample matrices to establish radiation exposure risk during operation. Four handheld XRF instruments representing three manufacturers were used on four smooth, flat-lying materials of contrasting matrix composition. Dose rates were measured at 10, 20, 30, and 40 cm intervals every 30° around the instrument at 0 and 45° from the horizontal, as well as vertically from the instrument screen. The analysis of polyethylene recorded dose rates 156 times higher (on average) than steel measurements and 34 times higher than both quartz sand and quartz sandstone. A worst-case exposure scenario was assumed where a user analyses a polyethylene material at arms reach for 1 h each working day for one year. This scenario resulted in an effective body dose of 73.5 µSv, equivalent to three to four chest X-rays (20 µSv) a year, 20 times lower than the average annual background radiation exposure in Australia and well below the annual exposure limit of 1 mSv for non-radiation workers. This study finds the advantages of using handheld XRF spectrometers far outweighs the risk of low radiation exposure linked to X-ray scattering from samples.

Environmental contamination in an Australian mining community and potential influences on early childhood health and behavioural outcomes.

Arsenic, cadmium and lead in aerosols, dusts and surface soils from Australia's oldest continuous lead mining town of Broken Hill were compared to standardised national childhood developmental (year 1) and education performance measures (years 3,5,7,9). Contaminants close to mining operations were elevated with maximum lead levels in soil: 8900 mg/kg; dust wipe: 86,061 µg/m(2); dust deposition: 2950 µg/m(2)/day; aerosols: 0.707 µg/m(3). The proportion of children from Broken Hill central, the area with the highest environmental contamination, presented with vulnerabilities in two or more developmental areas at 2.6 times the national average. Compared with other school catchments of Broken Hill, children in years 3 and 5 from the most contaminated school catchment returned consistently the lowest educational scores. By contrast, children living and attending schools associated with lower environmental contamination levels recorded higher school scores and lower developmental vulnerabilities. Similar results were identified in Australia's two other major lead mining and smelting cities of Port Pirie and Mount Isa.

Evaluating the efficacy of playground washing to reduce environmental metal exposures.

Washing and wet mopping is often advocated as a remedial treatment to limit exposure to lead dust. Here, surface and pre- and post-play wipes were measured to ascertain dust metal exposures (arsenic, cadmium, copper, lead and zinc) following play routines at four playgrounds in the smelter city of Port Pirie, South Australia, which are washed regularly. Although post-play hand wipe metals were 55.9% (95% CI: -0.78, -0.34) lower on wash days, loadings increased ∼5.1% (95% CI: 1.2, 11.7) per hour after washing. Despite washing, post-play hand lead exceeded a conservative value of 800 µg/m(2) within 24 h or sooner, with loadings increasing in proximity to the smelter. Post-play lead loadings were always >1000 µg/m(2) at the playground closest to smelter. Playground washing results in short-lived exposure reduction and effective treatment requires elimination of smelter emissions.

High expression of PI3K core complex genes is associated with poor prognosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world. Autophagy is a highly conserved process in eukaryotic cells. In CLL autophagy is involved in mediating the effect of chemotherapy but the role of autophagy in CLL pathogenesis remains unknown. In the present study, we used real-time RT-PCR to analyze expression of the PIK3C3, PIK3R4, and BECN1 genes. These genes encode the components of the PI3K core complex, which is central to initiation of autophagy. A consecutive series of 149 well-characterized CLL cases from Region of Southern Denmark were included in the study. All three genes were observed to be independent markers of prognosis in CLL with high expression being associated with more aggressive disease. With this clear association with outcome in CLL, these genes thereby represent promising candidates for future functional studies on the role of autophagy in CLL, and they may further represent targets of treatment.

Lead isotopic compositions of ash sourced from Australian bushfires.

This study identifies natural and industrial lead remobilized in ash deposits from three bushfires in relatively pristine areas of Australia in 2011 using lead isotopic compositions ((208)Pb/(207)Pb; (206)Pb/(207)Pb). Lead concentrations in the ash ranged from 1 to 36 mg/kg, bracketing the range of lead (4-23 mg/kg) in surface soils (0-2 cm), subsurface (40-50 cm) soils and rocks. The lead isotopic compositions of ash and surface soil samples were compared to subsurface soils and local bedrock samples. The data show that many of the ash and surface soil lead isotopic compositions were a mixture of natural lead and legacy industrial lead depositions (such as leaded petrol combustion). However, some of the ash samples at each of the sites had lead isotopic compositions that did not fit a simple two end-member mixing model, indicating other, unidentified sources.

Environmental lead exposure risks associated with children's outdoor playgrounds.

This study examines exposure risks associated with lead smelter emissions at children's public playgrounds in Port Pirie, South Australia. Lead and other metal values were measured in air, soil, surface dust and on pre- and post-play hand wipes. Playgrounds closest to the smelter were significantly more lead contaminated compared to those further away (t(27.545) = 3.76; p = .001). Port Pirie post-play hand wipes contained significantly higher lead loadings (maximum hand lead value of 49,432 µg/m(2)) than pre-play hand wipes (t(27) = 3.57, p = .001). A 1% increase in air lead (µg/m(3)) was related to a 0.713% increase in lead dust on play surfaces (95% CI, 0.253-1.174), and a 0.612% increase in post-play wipe lead (95% CI, 0.257-0.970). Contaminated dust from smelter emissions is determined as the source and cause of childhood lead poisoning at a rate of approximately one child every third day.

COPE-ICD: patient experience of participation in an ICD specific rehabilitation programme.

PURPOSE: Evaluating rehabilitation programmes from the patient's perspective is much needed, as the patients are the most important stakeholders in the health care system. A comprehensive rehabilitation programme, COPE-ICD programme, consists of exercise training and nursing consultations during a one year period post ICD implantation. The purpose of this paper is to describe the experience and meaning of participating in a comprehensive ICD-specific rehabilitation programme. METHODS: Qualitative interviews were conducted with 10 patients representing the participating population, and later transcribed. The analysis was inspired by Ricoeur's theory of interpretation, which consists of three levels: naive reading, structured analysis and critical interpretation and discussion. RESULTS: The overall concept was that participating in the COPE-ICD programme meant feeling inspired and secure through individualized care. Four themes emerged: Knowledge: patients gained much needed understanding; Physical attention: patients interpreted body signals and adjusted their exercise behaviour; Trust: patients regained trust, felt secure and dared to live again; Strategies of living: patients' coping was supported through reflection and professional dialogue, and they dealt with the risk of shock or death. CONCLUSION: Participating in an ICD-specific rehabilitation programme can make patients feel inspired and secure through individualized care. They discover that they have to rethink some of their strategies of living and they gain support in the reflection and coping needed for that. Patients gain insight and they develop a special physical attention whereby they continue healthy activities through interpreting body signals and adjusting exercise behaviour. They learn to trust their body again.