Serum protein profiling reveals baseline and pharmacodynamic biomarker signatures associated with clinical outcome in mCRC patients treated with chemotherapy ± cediranib.

BACKGROUND: This study evaluated soluble serum proteins as biomarkers to subset patients with metastatic colorectal cancer (mCRC) treated with chemotherapy±cediranib, a vascular endothelial growth factor (VEGF) signalling inhibitor (VEGFi). Exploring biomarkers at pre- and on-treatment may identify patient subgroups showing clinical benefit on cediranib combination. METHODS: Two hundred and seven serum proteins were analysed in 588 mCRC patients at pre- and on-treatment with chemotherapy (FOLFOX/CAPOX)±cediranib 20 mg. Patients were enrolled in the phase III trial HORIZON II. We correlated baseline biomarker signatures and pharmacodynamic (PD) biomarkers with PFS and OS. RESULTS: We identified a baseline signature (BS) of 47 biomarkers that included VEGFA, VEGFD, VEGFR2, VEGFR3 and TIE-2, which defined two distinct subgroups of patients. Patients treated with chemotherapy plus cediranib who had 'high' BS had shorter PFS (HR=1.82, P=0.003) than patients with 'low' BS. This BS did not correlate with PFS of the patients treated with chemotherapy plus placebo. In addition, we identified a profile of 16 PD proteins on treatment associated with PFS (HR=0.58, P<0.001) and OS (HR=0.52, P<0.001) in patients treated with chemotherapy plus cediranib. This PD profile did not correlate with PFS and OS in patients treated with chemotherapy plus placebo. CONCLUSIONS: Serum proteins may represent relevant biomarkers to predict the outcome of patients treated with VEGFi-based therapies. We report a BS and PD biomarkers that may identify mCRC patients showing increased benefit of combining cediranib with chemotherapy. These exploratory findings need to be validated in future prospective studies.

Effects of dairy slurry on silage fermentation characteristics and nutritive value of alfalfa.

Dairy producers frequently ask questions about the risks associated with applying dairy slurry to growing alfalfa (Medicago sativa L.). Our objectives were to determine the effects of applying dairy slurry on the subsequent nutritive value and fermentation characteristics of alfalfa balage. Dairy slurry was applied to 0.17-ha plots of alfalfa; applications were made to the second (HARV1) and third (HARV2) cuttings during June and July of 2012, respectively, at mean rates of 42,400 ± 5271 and 41,700 ± 2397 L/ha, respectively. Application strategies included (1) no slurry, (2) slurry applied directly to stubble immediately after the preceding harvest, (3) slurry applied after 1 wk of post-ensiled regrowth, or (4) slurry applied after 2 wk of regrowth. All harvested forage was packaged in large, rectangular bales that were ensiled as wrapped balage. Yields of DM harvested from HARV1 (2,477 kg/ha) and HARV2 (781 kg/ha) were not affected by slurry application treatment. By May 2013, all silages appeared to be well preserved, with no indication of undesirable odors characteristic of clostridial fermentations. Clostridium tyrobutyricum, which is known to negatively affect cheese production, was not detected in any forage on either a pre- or post-ensiled basis. On a pre-ensiled basis, counts for Clostridium cluster 1 were greater for slurry-applied plots than for those receiving no slurry, and this response was consistent for HARV1 (4.44 vs. 3.29 log10 genomic copies/g) and HARV2 (4.99 vs. 3.88 log10 genomic copies/g). Similar responses were observed on a post-ensiled basis; however, post-ensiled counts also were greater for HARV1 (5.51 vs. 5.17 log10 genomic copies/g) and HARV2 (5.84 vs. 5.28 log10 genomic copies/g) when slurry was applied to regrowth compared with stubble. For HARV2, counts also were greater following a 2-wk application delay compared with a 1-wk delay (6.23 vs. 5.45 log10 genomic copies/g). These results suggest that the risk of clostridial fermentations in alfalfa silages is greater following applications of slurry. Based on pre- and post-ensiled clostridial counts, applications of dairy slurry on stubble are preferred (and less risky) compared with delayed applications on growing alfalfa.

Prognostic/predictive value of 207 serum factors in colorectal cancer treated with cediranib and/or chemotherapy.

BACKGROUND: The prognostic and predictive value of multiple serum biomarkers was evaluated using samples from a randomised phase III study (HORIZON II) investigating chemotherapy with or without cediranib in metastatic colorectal cancer (mCRC). METHODS: Baseline levels of 207 protein markers were measured in serum samples from 582 HORIZON II (FOLFOX/XELOX plus cediranib 20 mg (n=330) or placebo (n=252)) patients. Median baseline values of each biomarker were used to categorise patients as high or low. Markers were then assessed for their association with efficacy, defined by progression-free survival (PFS) and overall survival (OS). A generalised boosted regression model identified markers of particular interest. RESULTS: Correlation of protein levels with PFS and OS suggested that multiple factors had a prognostic value, independent of treatment arm, including IL-6, IL-8, C-reactive protein (CRP), ICAM-1 and carcinoembryonic antigen (CEA). Among the angiogenesis regulators, low levels of vascular endothelial growth factor (VEGF), VEGF-D, VEGFR-1, VEGFR-3, NRP1 and Tie-2 correlated with better outcome. CONCLUSION: This large data set generated using serum samples from mCRC patients treated with chemotherapy and VEGF inhibitors, defines baseline characteristics for 207 serum proteins. Multiple prognostic factors were identified that could be disease related or predict which patients derive most benefit from 5-fluorouracil (5-FU)-based chemotherapy, meriting further exploration in prospective studies.

Recent skin self-examination and doctor visits in relation to melanoma risk and tumour depth.

BACKGROUND: Little is known about the potential benefit of skin self-examination for melanoma prevention and early detection. OBJECTIVES: To determine whether skin self-examination is associated with reduced melanoma risk, self-detection of tumours, and reduced risk of deeper melanomas. METHODS: We used data from a population-based case-control study (423 cases, 678 controls) to assess recent skin self-examination in relation to self-detection, melanoma risk and tumour depth ( ≤1 mm; > 1 mm). Logistic regression was used to estimate odds ratios (ORs) and confidence intervals (CIs) for associations of interest. RESULTS: Skin self-examination conducted 1-11 times during a recent year was associated with a possible decrease in melanoma risk (OR 0·74; 95% CI 0·54-1·02). Melanoma risk was decreased for those who conducted skin self-examination and saw a doctor (OR 0·52; 95% CI 0·30-0·90). Among cases, those who examined their skin were twice as likely to self-detect the melanoma (OR 2·23; 95% CI 1·47-3·38), but self-detection was not associated with shallower tumours. Tumour depth was reduced for those who conducted skin self-examination 1-11 times during a recent year (OR 0·39; 95% CI 0·18-0·81), but was not influenced by seeing a doctor, or by conducting skin self-examination and seeing a doctor. CONCLUSIONS: Risk of a deeper tumour and possibly risk of melanoma were reduced by skin self-examination 1-11 times annually. Melanoma risk was markedly reduced by skin self-examination coupled with a doctor visit. We cannot, however, exclude the possibility that our findings reflect bias or confounding. Additional studies are needed to elucidate the potential benefits of skin self-examination for melanoma prevention and early detection.

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation.

AIMS: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. METHODS AND RESULTS: Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l(-1) into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were > or =84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation. CONCLUSION: Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. SIGNIFICANCE AND IMPACT OF THE STUDY: Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.

Hydrologic, land cover, and seasonal patterns of waterborne pathogens in Great Lakes tributaries.

Great Lakes tributaries are known to deliver waterborne pathogens from a host of sources. To examine the hydrologic, land cover, and seasonal patterns of waterborne pathogens (i.e. protozoa (2), pathogenic bacteria (4) human viruses, (8) and bovine viruses (8)) eight rivers were monitored in the Great Lakes Basin over 29 months from February 2011 to June 2013. Sampling locations represented a wide variety of land cover classes from urban to agriculture to forest. A custom automated pathogen sampler was deployed at eight sampling locations which provided unattended, flow-weighted, large-volume (120-1630 L) sampling. Human and bovine viruses and pathogenic bacteria were detected by real-time qPCR in 16%, 14%, and 1.4% of 290 samples collected while protozoa were never detected. The most frequently detected pathogens were: bovine polyomavirus (11%), and human adenovirus C, D, F (9%). Human and bovine viruses were present in 16.9% and 14.8% of runoff-event samples (n = 189) resulting from precipitation and snowmelt, and 13.9% and 12.9% of low-flow samples (n = 101), respectively, indicating multiple delivery mechanisms could be influential. Data indicated human and bovine virus prevalence was different depending on land cover within the watershed. Occurrence, concentration, and flux of human viruses were greatest in samples from the three sampling locations with greater than 25% urban influence than those with less than 25% urban influence. Similarly, occurrence, concentration, and flux of bovine viruses were greatest in samples from the two sampling locations with greater than 50 cattle/km2 than those with less than 50 cattle/km2. In seasonal analysis, human and bovine viruses occurred more frequently in spring and winter seasons than during the fall and summer. Concentration, occurrence, and flux in the context of hydrologic condition, seasonality, and land use must be considered for each watershed individually to develop effective watershed management strategies for pathogen reduction.

Multi-drug resistance in chronic lymphocytic leukemia.

We evaluated 45 chronic lymphocyte leukemia (CLL) patients for the presence of multi-drug resistance (MDR) by the ex vivo techniques: 1) a functional assay utilizing doxorubicin (dox) retention with modulation; 2) a cytotoxicity assay (MTT) with modulation; 3) and four monoclonal antibodies. Ex vivo tests were correlated with disease stage and prior treatment, and were repeated as patients became resistant to alkylating agents, fludarabine and VAD chemotherapy (infusion of vincristine, dox, and oral dexamethasone). The majority of patients (64.4%) were in early stage and were untreated (62.2%). P-glycoprotein (p-gp 170) was detected most frequently by the monoclonal antibody MRK-16 (48%) and by functional modulation of dox retention by PSC-833 (40.6%) and by functional modulation of the MTT assay with vincristine (0.29) and dox (0.39) with PSC-833 at 1.0 microg/mL. Functional modulation of dox retention with PSC-833 was significantly associated with stage, but not with either the MTT assay or any of the monoclonal antibodies. None of the tests correlated with prior chlorambucil treatment. Correlation of dox retention with the monoclonal antibodies was mild to moderate and became stronger following chlorambucil treatment. Three patients who became resistant to VAD were found to express p-gp 170. We conclude that MDR can frequently be detected in patients with CLL. Furthermore, the expression of p-gp 170 increases with advancing stage, but not prior alkylating agent therapy. The functional expression of p-gp 170 increases with advancing stage and prior treatment and correlates well with monoclonal antibody detection (especially MRK-16). Patients who become resistant to VAD more frequently express p-gp 170 by a variety of techniques. PSC-833 is a more potent modulator of MDR than cyclosporin-A (CsA) ex vivo, and correlates better with stage of disease.

Fluoroderma.

Papulonodular eruptions from certain bromide and iodide preparations are widely recognized entities, but skin lesions following fluoride ingestion are rare. Modern prophylaxis for postirradiation dental caries includes the use of fluoride gel preparations applied to the teeth. In two patients receiving such therapy a papulonodular eruption developed, similar to the recognized halogenodermas. These cases may help to increase awareness of this entity.

Glucagonoma syndrome. Report of two cases and literature review.

The glucagonoma syndrome is characterized by dermatitis, stomatitis, elevated serum glucagon levels, abnormal glucose tolerance, weight loss, and anemia--all in association with a glucagon-secreting alpha-cell tumor of the pancreas. A review of 21 cases showed strikingly similar features. A generalized, symmetrical dermatitis initially appeared to be asteatotic or eczematous over the perineum, buttocks, and lower extremities. Gradually, a more characteristic migratory necrolytic erythema with transient bulla formation and erosions developed in intertriginous and dependent areas. Histologically, the most specific features included necrolysis of the upper epidermis, with liquefaction necrosis of the granular cell layer and subcorneal clefting or blister formation. The dermatologist is often first to examine such patients; early recognition of this syndrome with prompt surgical removal of the primary pancreatic lesion may afford cure of the neoplasm.

Therapeutic ionizing radiation and the incidence of basal cell carcinoma and squamous cell carcinoma. The New Hampshire Skin Cancer Study Group.

OBJECTIVE: To estimate the relative risk of developing basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) after receiving therapeutic ionizing radiation. DESIGN: Population-based case-control study. SETTING: New Hampshire. PATIENTS: A total of 592 cases of BCC and 289 cases of SCC identified through a statewide surveillance system and 536 age- and sex-matched controls selected from population lists. MAIN OUTCOME MEASURES: Histologically confirmed BCC and invasive SCC diagnosed between July 1, 1993, through June 30, 1995, among New Hampshire residents. RESULTS: Information regarding radiotherapy and other factors was obtained through personal interviews. An attempt was made to review the radiation treatment records of subjects who reported a history of radiotherapy. Overall, an increased risk of both BCC and SCC was found in relation to therapeutic ionizing radiation. Elevated risks were confined to the site of radiation exposure (BCC odds ratio, 3. 30; 95% confidence interval, 1.60-6.81; SCC odds ratio, 2.94; 95% confidence interval, 1.30-6.67) and were most pronounced for those irradiated for acne exposure. For SCC, an association with radiotherapy was observed only among those whose skin was likely to sunburn with sun exposure. CONCLUSIONS: These results largely agree with those of previous studies on the risk of BCC in relation to ionizing radiation exposure. In addition, they suggest that the risk of SCC may be increased by radiotherapy, especially in individuals prone to sunburn with sun exposure. Arch Dermatol. 2000;136:1007-1011

Human and bovine viruses in the Milwaukee River watershed: hydrologically relevant representation and relations with environmental variables.

To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56-2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n=63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing human exposure and disease transmission.

Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples.

Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, the method 1) employs a commercially available standard RNA control; 2) defines inhibition by the change in the quantification cycle (C(q)) of the standard RNA control when added to the sample concentrate; and 3) calculates a dilution factor using a mathematical formula applied to the change in C(q) to indicate the specific volume of nuclease-free water necessary to dilute the effect of inhibitors. The standardized inhibition method was applied to 3,193 large-volume water (surface, groundwater, drinking water, agricultural runoff, sewage) concentrates of which 1,074 (34%) were inhibited. Inhibition level was not related to sample volume. Samples collected from the same locations over a one to two year period had widely variable inhibition levels. The proportion of samples that could have been reported as false negatives if inhibition had not been mitigated was between 0.3% and 71%, depending on water source. These findings emphasize the importance of measuring and mitigating inhibition when reporting qPCR results for viral pathogens in environmental waters to minimize the likelihood of reporting false negatives and under-quantifying virus concentration.

New mathematical approaches to quantify human infectious viruses from environmental media using integrated cell culture-qPCR.

Quantifying infectious viruses by cell culture depends on visualizing cytopathic effect, or for integrated cell culture-PCR, attaining confidence a PCR-positive signal is the result of virus growth and not inoculum carryover. This study developed mathematical methods to calculate infectious virus numbers based on viral growth kinetics in cell culture. Poliovirus was inoculated into BGM cell monolayers at 10 concentrations from 0.001 to 1000 PFU/ml. Copy numbers of negative-strand RNA, a marker of infectivity for single-stranded positive RNA viruses, were measured over time by qRT-PCR. Growth data were analyzed by two approaches. First, data were fit with a continuous function to estimate directly the initial virus number, expressed as genomic copies. Such estimates correlated with actual inoculum numbers across all concentrations (R(2)=0.62, n=17). Second, the length of lag phase appeared to vary inversely with inoculum titers; hence, standard curves to predict inoculum virus numbers were derived based on three definitions of lag time: (1) time of first detection of (-)RNA, (2) second derivative maximum of the fitted continuous function, and (3) time when the fitted curve crossed a threshold (-)RNA concentration. All three proxies yielded standard curves with R(2)=0.69-0.90 (n=17). The primary advantage of these growth kinetics approaches is being able to quantify virions that are unambiguously infectious, a particular advantage for viruses that do not produce CPE.

Concentration of Cryptosporidium, microsporidia and other water-borne pathogens by continuous separation channel centrifugation.

AIMS: The aim of this study was to determine the effectiveness of continuous separation channel centrifugation for concentrating water-borne pathogens of various taxa and sizes. METHODS AND RESULTS: Cryptosporidium parvum oocysts, Giardia lamblia cysts, Encephalitozoon intestinalis spores and Escherichia coli were seeded into different water matrices at densities ranging from 5 to 10 000 organisms l(-1) and recovered using continuous separation channel centrifugation. All pathogens were enumerated on membrane filters using microscopy. Recovery efficiencies were usually > 90%. Oocyst recovery did not vary with source water turbidity or with centrifuge flow rate up to 250 ml min(-1). Based on excystation, this concentration method did not alter oocyst viability. CONCLUSIONS: Continuous separation channel centrifugation is an effective means of concentrating water-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods are needed for detecting pathogens in drinking water to ensure public health. The first step for any pathogen detection procedure is concentration. However, this step has been problematic because recovery efficiencies of conventional methods, like filtration, are often low and variable, which may lead to false negatives. Continuous separation channel centrifugation can simultaneously concentrate multiple pathogens as small as 1 microm with high and reproducible efficiency in a variety of water matrices.