Leesa F. Wockner

Augmented renal clearance in the ICU: results of a multicenter observational study of renal function in critically ill patients with normal plasma creatinine concentrations*.

Objective:To describe the prevalence and natural history of augmented renal clearance in a cohort of recently admitted critically ill patients with normal plasma creatinine concentrations. Design:Multicenter, prospective, observational study. Setting:Four, tertiary-level, university-affiliated, ICUs in Australia, Singapore, Hong Kong, and Portugal. Patients:Study participants had to have an expected ICU length of stay more than 24 hours, no evidence of absolute renal impairment (admission plasma creatinine < 120 µmol/L), and no history of prior renal replacement therapy or chronic kidney disease. Convenience sampling was used at each participating site. Interventions:Eight-hour urinary creatinine clearances were collected daily, as the primary method of measuring renal function. Augmented renal clearance was defined by a creatinine clearance more than or equal to 130 mL/min/1.73 m2. Additional demographic, physiological, therapeutic, and outcome data were recorded prospectively. Measurements and Main Results:Nine hundred thirty-two patients were admitted to the participating ICUs over the study period, and 281 of which were recruited into the study, contributing 1,660 individual creatinine clearance measures. The mean age (95% CI) was 54.4 years (52.5–56.4 yr), Acute Physiology and Chronic Health Evaluation II score was 16 (15.2–16.7), and ICU mortality was 8.5%. Overall, 65.1% manifested augmented renal clearance on at least one occasion during the first seven study days; the majority (74%) of whom did so on more than or equal to 50% of their creatinine clearance measures. Using a mixed-effects model, the presence of augmented renal clearance on study day 1 strongly predicted (p = 0.019) sustained elevation of creatinine clearance in these patients over the first week in ICU. Conclusions:Augmented renal clearance appears to be a common finding in this patient group, with sustained elevation of creatinine clearance throughout the first week in ICU. Future studies should focus on the implications for accurate dosing of renally eliminated pharmaceuticals in patients with augmented renal clearance, in addition to the potential impact on individual clinical outcomes.

Critical appraisal of the diagnosis of the sessile serrated adenoma.

The sessile serrated adenoma (SSA) is a relatively recently described polyp that can present diagnostic difficulties for the practicing pathologist. The frequency of SSA diagnoses varies dramatically in the reported literature. In addition, the histologic interface between the microvesicular hyperplastic polyp (MVHP) and the SSA continues to be a diagnostic problem. The trend in recent years has been toward a lower threshold for SSA diagnosis. Herein, we have performed a cross-sectional study of 6340 colorectal polyps received at a high-volume community-based pathology practice over a 3-month period. After central review, with strict application of the diagnostic criteria outlined in the 2010 edition of the World Health Organization Classification of Tumours of the Digestive Tract, we found that SSAs represented 12.1% of all polyps. In addition, we developed novel diagnostic subcategories in an attempt to determine the most appropriate cutoff for the interface between the MVHP and the SSA. We found that serrated polyps (MVHPs or SSAs) with any SSA-like crypts had clinical features more in common with the SSA than the MVHP and that this diagnostic cutoff showed good reproducibility between pathologists. This supports the position of a recent consensus publication proposing that polyps with as few as 1 SSA-type crypt should be diagnosed as an SSA. Applying these criteria to our cohort yields an overall SSA rate of 14.7%. In summary, we believe that SSAs continue to be underdiagnosed in pathologic practice and that this may result in inadequate surveillance and thus contribute to interval colorectal carcinomas.

Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease

Koyama et al. show that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes, mediated by donor CD103+CD11b− DCs that migrate from the colon under the influence of CCR7. This antigen presentation imprints gut-homing integrin signatures on donor T cells, leading to their migration to the GI tract where they mediate fulminant disease.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

The D-Health Trial: A randomized trial of vitamin D for prevention of mortality and cancer

BACKGROUND Vitamin D, specifically serum 25(OH)D has been associated with mortality, cancer and multiple other health endpoints in observational studies, but there is a paucity of clinical trial evidence sufficient to determine the safety and effectiveness of population-wide supplementation. We have therefore launched the D-Health Trial, a randomized trial of vitamin D supplementation for prevention of mortality and cancer. Here we report the methods and describe the trial cohort. METHODS The D-Health Trial is a randomized placebo-controlled trial, with planned intervention for 5years and a further 5years of passive follow-up through linkage with health and death registers. Participants aged 65-84years were recruited from the general population of Australia. The intervention is monthly oral doses of 60,000IU of cholecalciferol or matching placebo. The primary outcome is all-cause mortality. Secondary outcomes are total cancer incidence and colorectal cancer incidence. RESULTS We recruited 21,315 participants to the trial between February 2014 and May 2015. The participants in the two arms of the trial were well-balanced at baseline. Comparison with Australian population statistics shows that the trial participants were less likely to report being in fair or poor health, to be current smokers or to have diabetes than the Australian population. However, the proportion overweight or with health conditions such as arthritis and angina was similar. CONCLUSIONS Observational data cannot be considered sufficient to support interventions delivered at a population level. Large-scale randomized trials such as the D-Health Trial are needed to inform public health policy and practice.

Genome-wide DNA methylation analysis of formalin-fixed paraffin embedded colorectal cancer tissue

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin‐embedded (FFPE) tissue to interrogate methylation levels on a genome‐wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and “restoration” of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the “restoration” method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

Chromosomal Instability in BRAF Mutant, Microsatellite Stable Colorectal Cancers

The BRAF oncogene is mutated in 15% of sporadic colorectal cancers. Approximately half of these BRAF mutant cancers demonstrate frequent frameshift mutations termed microsatellite instability (MSI), but are diploid and chromosomally stable. BRAF wild type cancers are typically microsatellite stable (MSS) and instead acquire chromosomal instability (CIN). In these cancers, CIN is associated with a poor outcome. BRAF mutant cancers that are MSS, typically present at an advanced stage and have a particularly poor prognosis. We have previously demonstrated clinical and molecular similarities between MSS cancers with or without a BRAF mutation, and therefore hypothesised that CIN may also be frequent in BRAF mutant/MSS cancers. BRAF mutant/MSS (n = 60), and BRAF wild type/MSS CRCs (n = 90) were investigated for CIN using loss of heterozygosity analysis over twelve loci encompassing chromosomal regions 5q, 8p, 17p and 18q. CIN was frequent in BRAF mutant/MSS cancers (41/57, 72%), which was comparable to the rate found in BRAF wild type/MSS cancers (74/90, 82%). The greatest loss in BRAF mutant/MSS cancers occurred at 8p (26/44, 59%), and the least at 5q (19/49, 39%). CIN in BRAF mutant/MSS cancers correlated with advanced stage (AJCC III/IV: 15/17, 88%; p = 0.02); showed high rates of co-occurrence with the CpG Island Methylator Phenotype (17/23, 74%); and CIN at 18q and 8p associated with worse survival (p = 0.02, p<0.05). This study demonstrates that CIN commonly occurs in advanced BRAF mutant/MSS colorectal cancers where it may contribute to poorer survival, and further highlights molecular similarities occurring between these and BRAF wild type cancers.

Adult Survivorship of the Dengue Mosquito Aedes aegypti Varies Seasonally in Central Vietnam

The survival characteristics of the mosquito Aedes aegypti affect transmission rates of dengue because transmission requires infected mosquitoes to survive long enough for the virus to infect the salivary glands. Mosquito survival is assumed to be high in tropical, dengue endemic, countries like Vietnam. However, the survival rates of wild populations of mosquitoes are seldom measured due the difficulty of predicting mosquito age. Hon Mieu Island in central Vietnam is the site of a pilot release of Ae. aegypti infected with a strain of Wolbachia pipientis bacteria (wMelPop) that induces virus interference and mosquito life-shortening. We used the most accurate mosquito age grading approach, transcriptional profiling, to establish the survival patterns of the mosquito population from the population age structure. Furthermore, estimations were validated on mosquitoes released into a large semi-field environment consisting of an enclosed house, garden and yard to incorporate natural environmental variability. Mosquito survival was highest during the dry/cool (January-April) and dry/hot (May-August) seasons, when 92 and 64% of Hon Mieu mosquitoes had survived to an age that they were able to transmit dengue (12 d), respectively. This was reduced to 29% during the wet/cool season from September to December. The presence of Ae. aegypti older than 12 d during each season is likely to facilitate the observed continuity of dengue transmission in the region. We provide season specific Ae. aegypti survival models for improved dengue epidemiology and evaluation of mosquito control strategies that aim to reduce mosquito survival to break the dengue transmission cycle.

RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p<0.0001). ZNRF3 was mutated in 16/54(30%) BRAF mutant/MSI and 5/33(15%) BRAF mutant/microsatellite stable compared to 0/27 BRAF wild type cancers (p=0.004). An RNF43 frameshift mutation (X659fs) occurred in 80% BRAF mutant/MSI cancers. This high rate was verified in a second series of 25/35(71%) BRAF mutant/MSI cancers. RNF43 and ZNRF3 had lower transcript expression in BRAF mutant compared to BRAF wildtype cancers and less cytoplasmic protein expression in BRAF mutant/MSI compared to other subtypes. Treatment with a porcupine inhibitor reduced RNF43/ZNRF3 mutant colony growth by 50% and synergised with a MEK inhibitor to dramatically reduce growth. This study suggests inactivation of RNF43 and ZNRF3 is important in serrated tumorigenesis and has identified a potential therapeutic strategy for this cancer subtype.

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar‐simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild‐type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.