Quantification of HER2 heterogeneity in breast cancer-implications for identification of sub-dominant clones for personalised treatment

Breast cancer is a heterogeneous disease, at both an inter- and intra-tumoural level. Appreciating heterogeneity through the application of biomarkers and molecular signatures adds complexity to tumour taxonomy but is key to personalising diagnosis, treatment and prognosis. The extent to which heterogeneity exists, and its interpretation remains a challenge to pathologists. Using HER2 as an exemplar, we have developed a simple reproducible heterogeneity index. Cell-to-cell HER2 heterogeneity was extensive in a proportion of both reported 'amplified' and 'non-amplified' cases. The highest levels of heterogeneity objectively identified occurred in borderline categories and higher ratio non-amplified cases. A case with particularly striking heterogeneity was analysed further with an array of biomarkers in order to assign a molecular diagnosis. Broad biological complexity was evident. In essence, interpretation, depending on the area of tumour sampled, could have been one of three distinct phenotypes, each of which would infer different therapeutic interventions. Therefore, we recommend that heterogeneity is assessed and taken into account when determining treatment options.

Quantification of the smoking-associated cancer risk with rate advancement periods: meta-analysis of individual participant data from cohorts of the CHANCES consortium

BACKGROUND: Smoking is the most important individual risk factor for many cancer sites but its association with breast and prostate cancer is not entirely clear. Rate advancement periods (RAPs) may enhance communication of smoking related risk to the general population. Thus, we estimated RAPs for the association of smoking exposure (smoking status, time since smoking cessation, smoking intensity, and duration) with total and site-specific (lung, breast, colorectal, prostate, gastric, head and neck, and pancreatic) cancer incidence and mortality.

METHODS: This is a meta-analysis of 19 population-based prospective cohort studies with individual participant data for 897,021 European and American adults. For each cohort we calculated hazard ratios (HRs) for the association of smoking exposure with cancer outcomes using Cox regression adjusted for a common set of the most important potential confounding variables. RAPs (in years) were calculated as the ratio of the logarithms of the HRs for a given smoking exposure variable and age. Meta-analyses were employed to summarize cohort-specific HRs and RAPs.

RESULTS: Overall, 140,205 subjects had a first incident cancer, and 53,164 died from cancer, during an average follow-up of 12 years. Current smoking advanced the overall risk of developing and dying from cancer by eight and ten years, respectively, compared with never smokers. The greatest advancements in cancer risk and mortality were seen for lung cancer and the least for breast cancer. Smoking cessation was statistically significantly associated with delays in the risk of cancer development and mortality compared with continued smoking.

CONCLUSIONS: This investigation shows that smoking, even among older adults, considerably advances, and cessation delays, the risk of developing and dying from cancer. These findings may be helpful in more effectively communicating the harmful effects of smoking and the beneficial effect of smoking cessation.

Validation of a fast method for quantification of intra-abdominal and subcutaneous adipose tissue for large-scale human studies

Central obesity is the hallmark of a number of non-inheritable disorders. The advent of imaging techniques such asMRI has allowed for a fast and accurate assessment of body fat content and distribution. However, image analysis continues to be one of the major obstacles to the use of MRI in large-scale studies. In this study we assess the validity of the recently proposed fat–muscle quantitation system (AMRATM Profiler) for the quantification of intra-abdominal adipose tissue (IAAT) and abdominal subcutaneous adipose tissue (ASAT) from abdominal MR images. Abdominal MR images were acquired from 23 volunteers with a broad range of BMIs and analysed using sliceOmatic, the current gold-standard, and the AMRATM Profiler based on a non-rigid image registration of a library of segmented atlases. The results show that there was a highly significant correlation between the fat volumes generated by the two analysis methods, (Pearson correlation r = 0.97, p < 0.001), with the AMRATM Profiler analysis being significantly faster (~3 min) than the conventional sliceOmatic approach (~40 min). There was also excellent agreement between the methods for the quantification of IAAT (AMRA 4.73 ± 1.99 versus sliceOmatic 4.73 ± 1.75 l, p = 0.97). For the AMRATM Profiler analysis, the intra-observer coefficient of variation was 1.6% for IAAT and 1.1% for ASAT, the inter-observer coefficient of variationwas 1.4%for IAAT and 1.2%for ASAT, the intra-observer correlationwas 0.998 for IAAT and 0.999 for ASAT, and the inter-observer correlation was 0.999 for both IAAT and ASAT. These results indicate that precise and accurate measures of body fat content and distribution can be obtained in a fast and reliable form by the AMRATM Profiler, opening up the possibility of large-scale human phenotypic studies.

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi- walled carbon nanotubes(MWCNTs)paste electrode modified by dispersion of laccase(3%,w/w) within the optimum composite matrix(60:40%,w/w,MWCNTs and paraffin binder)showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate4- aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 ×10- 7 to 1.15 ×10- 5 molL 1 using 4- aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%).The limit of detection obtained was 1.8 × 10-7 molL 1 (0.04 mgkg 1 on a fresh weight vegetable basis).The high activity and catalytic properties of the laccase- based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0±0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electro- analysis were observed by the presence of pro-vitamin A, vitamins B1 and C,and glucose in the vegetable extracts. The proposed biosensor- based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

A case study on the eco-efficiency performance of a composite processing industry: evaluation and quantification of potential improvements

In this study, an attempt was made in order to measure and evaluate the eco-efficiency performance of a pultruded composite processing company. For this purpose the recommendations of World Business Council for Sustainable Development (WCSD) and the directives of ISO 14301 standard were followed and applied. The main general indicators of eco-efficiency, as well as the specific indicators, were defined and determined. With basis on indicators’ figures, the value profile, the environmental profile, and the pertinent eco-efficiency ratios were established and analyzed. In order to evaluate potential improvements on company eco-performance, new indicators values and eco-efficiency ratios were estimated taking into account the implementation of new proceedings and procedures, at both upstream and downstream of the production process, namely: i) Adoption of a new heating system for pultrusion die-tool in the manufacturing process, more effective and with minor heat losses; ii) Recycling approach, with partial waste reuse of scrap material derived from manufacturing, cutting and assembly processes of GFRP profiles. These features lead to significant improvements on the sequent assessed eco-efficiency ratios of the present case study, yielding to a more sustainable product and manufacturing process of pultruded GFRP profiles.

Assessing the eco-efficiency of a composite processing industry: evaluation and quantification of potential improvements’

In this study, an attempt was made in order to measure and evaluate the eco-efficiency performance of a pultruded composite processing company. For this purpose the recommendations of World Business Council for Sustainable Development (WCSD) and the directives of ISO 14301 standard were followed and applied. The main general indicators of eco-efficiency, as well as the specific indicators, were defined and determined. With basis on indicators’ figures, the value profile, the environmental profile, and the pertinent ecoefficiency’s ratios were established and analyzed. In order to evaluate potential improvements on company eco-performance, new indicators values and eco-efficiency ratios were estimated taking into account the implementation of new proceedings and procedures, both in upstream and downstream of the production process, namely: a) Adoption of new heating system for pultrusion die in the manufacturing process, more effective and with minor heat losses; c) Recycling approach, with partial waste reuse of scrap material derived from manufacturing, cutting and assembly processes of GFRP profiles. These features lead to significant improvements on the sequent assessed eco-efficiency ratios of the present case study, yielding to a more sustainable product and manufacturing process of pultruded GFRP profiles.

Quantification and viability analyses of Pseudokirchneriella subcapitata algal cells using image-based cytometry

This work aims to evaluate the feasibility of using image-based cytometry (IBC) in the analysis of algal cell quantification and viability, using Pseudokirchneriella subcapitata as a cell model. Cell concentration was determined by IBC to be in a linear range between 1 × 105 and 8 × 106 cells mL−1. Algal viability was defined on the basis that the intact membrane of viable cells excludes the SYTOX Green (SG) probe. The disruption of membrane integrity represents irreversible damage and consequently results in cell death. Using IBC, we were able to successfully discriminate between live (SG-negative cells) and dead algal cells (heat-treated at 65 °C for 60 min; SG-positive cells). The observed viability of algal populations containing different proportions of killed cells was well correlated (R 2 = 0.994) with the theoretical viability. The validation of the use of this technology was carried out by exposing algal cells of P. subcapitata to a copper stress test for 96 h. IBC allowed us to follow the evolution of cell concentration and the viability of copper-exposed algal populations. This technology overcomes several main drawbacks usually associated with microscopy counting, such as labour-intensive experiments, tedious work and lack of the representativeness of the cell counting. In conclusion, IBC allowed a fast and automated determination of the total number of algal cells and allowed us to analyse viability. This technology can provide a useful tool for a wide variety of fields that utilise microalgae, such as the aquatic toxicology and biotechnology fields.

Full-scale biological phosphorus removal: quantification of storage polymers, microbial performance and metabolic modelling

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Biodiversity and quantification of functional bacteria in deammonification process.

2015

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Quantification of aortic flow by phase-contrast magnetic resonance in patients with bicuspid aortic valve.

AIMS: Bicuspid aortic valve (BAV) causes complex flow patterns in the ascending aorta (AAo), which may compromise the accuracy of flow measurement by phase-contrast magnetic resonance (PC-MR). Therefore, we aimed to assess and compare the accuracy of forward flow measurement in the AAo, where complex flow is more dominant in BAV patients, with flow quantification in the left ventricular outflow tract (LVOT) and the aortic valve orifice (AV), where complex flow is less important, in BAV patients and controls. METHODS AND RESULTS: Flow was measured by PC-MR in 22 BAV patients and 20 controls at the following positions: (i) LVOT, (ii) AV, and (iii) AAo, and compared with the left ventricular stroke volume (LVSV). The correlation between the LVSV and the forward flow in the LVOT, the AV, and the AAo was good in BAV patients (r = 0.97/0.96/0.93; P < 0.01) and controls (r = 0.96/0.93/0.93; P < 0.01). However, in relation with the LVSV, the forward flow in the AAo was mildly underestimated in controls and much more in BAV patients [median (inter-quartile range): 9% (4%/15%) vs. 22% (8%/30%); P < 0.01]. This was not the case in the LVOT and the AV. The severity of flow underestimation in the AAo was associated with flow eccentricity. CONCLUSION: Flow measurement in the AAo leads to an underestimation of the forward flow in BAV patients. Measurement in the LVOT or the AV, where complex flow is less prominent, is an alternative means for quantifying the systolic forward flow in BAV patients.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Practical signal-to-noise ratio quantification for sensitivity encoding: Application to coronary MR angiography.

Purpose: To develop and evaluate a practical method for the quantification of signal-to-noise ratio (SNR) on coronary MR angiograms (MRA) acquired with parallel imaging.Materials and Methods: To quantify the spatially varying noise due to parallel imaging reconstruction, a new method has been implemented incorporating image data acquisition followed by a fast noise scan during which radio-frequency pulses, cardiac triggering and navigator gating are disabled. The performance of this method was evaluated in a phantom study where SNR measurements were compared with those of a reference standard (multiple repetitions). Subsequently, SNR of myocardium and posterior skeletal muscle was determined on in vivo human coronary MRA.Results: In a phantom, the SNR measured using the proposed method deviated less than 10.1% from the reference method for small geometry factors (<= 2). In vivo, the noise scan for a 10 min coronary MRA acquisition was acquired in 30 s. Higher signal and lower SNR, due to spatially varying noise, were found in myocardium compared with posterior skeletal muscle.Conclusion: SNR quantification based on a fast noise scan is a validated and easy-to-use method when applied to three-dimensional coronary MRA obtained with parallel imaging as long as the geometry factor remains low.

Quantification of the relation between surface morphodynamics and subsurface sedimentological product in sandy braided rivers

This study uses digital elevation models and ground-penetrating radar to quantify the relation between the surface morphodynamics and subsurface sedimentology in the sandy braided South Saskatchewan River, Canada. A unique aspect of the methodology is that both digital elevation model and ground-penetrating radar data were collected from the same locations in 2004, 2005, 2006 and 2007, thus enabling the surface morphodynamics to be tied explicitly to the associated evolving depositional product. The occurrence of a large flood in 2005 also allowed the influence of discharge to be assessed with respect to the processproduct relationship. The data demonstrate that the morphology of the study reach evolved even during modest discharges, but more extensive erosion was caused by the large flood. In addition, the study reach was dominated by compound bars before the flood, but switched to being dominated by unit bars during and after the flood. The extent to which the subsurface deposits (the product') were modified by the surface morphodynamics (the process') was quantified using the changes in radar-facies recorded in sequential ground-penetrating radar surveys. These surveys reveal that during the large flood there was an increase in the proportion of facies associated with bar margin accretion and larger dunes. In subsequent years, these facies became truncated and replaced with facies associated with smaller dune sets. This analysis shows that unit bars generally become truncated more laterally than vertically and, thus, they lose the high-angle bar margin deposits and smaller scale bar-top deposits. In general, the only fragments that remain of the unit bars are dune sets, thus making identification of the original unit barform problematic. This novel data set has implications for what may ultimately become preserved in the rock record.