Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.This study was financially supported by a grant from the Mason Medical Research Foundation.This is the author accepted manuscript. The final version is available via Wiley at http://onlinelibrary.wiley.com/doi/10.1111/ajt.13303/abstract

Crowdsourcing for translational research: analysis of biomarker expression using cancer microarrays

Background: Academic pathology suffers from an acute and growing lack of workforce resource. This especially impacts on translational elements of clinical trials, which can require detailed analysis of thousands of tissue samples. We tested whether crowdsourcing – enlisting help from the public – is a sufficiently accurate method to score such samples. Methods: We developed a novel online interface to train and test lay participants on cancer detection and immunohistochemistry scoring in tissue microarrays. Lay participants initially performed cancer detection on lung cancer images stained for CD8, and we measured how extending a basic tutorial by annotated example images and feedback-based training affected cancer detection accuracy. We then applied this tutorial to additional cancer types and immunohistochemistry markers – bladder/ki67, lung/EGFR, and oesophageal/CD8 – to establish accuracy compared with experts. Using this optimised tutorial, we then tested lay participants’ accuracy on immunohistochemistry scoring of lung/EGFR and bladder/p53 samples. Results: We observed that for cancer detection, annotated example images and feedback-based training both improved accuracy compared with a basic tutorial only. Using this optimised tutorial, we demonstrate highly accurate (>0.90 area under curve) detection of cancer in samples stained with nuclear, cytoplasmic and membrane cell markers. We also observed high Spearman correlations between lay participants and experts for immunohistochemistry scoring (0.91 (0.78, 0.96) and 0.97 (0.91, 0.99) for lung/EGFR and bladder/p53 samples, respectively). Conclusions: These results establish crowdsourcing as a promising method to screen large data sets for biomarkers in cancer pathology research across a range of cancers and immunohistochemical stains

Rapid Imaging of Tumor Cell Death in vivo using the C2A domain of Synaptotagmin-I

Cell death is an important target for imaging the early response of tumors to treatment. We describe here validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of Synaptotagmin-I. Methods: The capability of near infrared fluorophore-labeled and 99mTechnetium- and 111Indium-labeled derivatives of C2Am for imaging tumor cell death, using planar near infrared fluorescence (NIRF) imaging and single photon computed tomography (SPECT) respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2-5%). There was a significant correlation (R>0.9, P<0.05) between fluorescently-labeled C2Am binding and histological markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99mTc-C2Am and 111In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low non-specific retention in liver and spleen at 24 h after probe administration. 99mTc-C2Am and 111In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2× respectively at two hours and 7.3× and 4.1× respectively at twenty-four hours after administration. Conclusion: Given the favorable biodistribution profile of 99mTc- and 111In-labelled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation.This work was supported by a Cancer Research UK programme grant to K.M.B. S.F. was the recipient of a Ph.D. studentship from the Cambridge Biomedical Research Centre of the National Institute of Health Research with financial support from GlaxoSmithKline UK. T.B.R. was in receipt of Intra-European Marie Curie (FP7-PEOPLE-2009-IEF, Imaging Lymphoma) and Long-term EMBO (EMBO-ALT-1145-2009) fellowships

Physical activity and nutrition program for seniors (PANS): protocol of a randomized controlled trial

Background Along with reduced levels of physical activity, older Australian's mean energy consumption has increased. Now over 60% of older Australians are considered overweight or obese. This study aims to confirm if a low-cost, accessible physical activity and nutrition program can improve levels of physical activity and diet of insufficiently active 60-70 year-olds. Methods/Design This 12-month home-based randomised controlled trial (RCT) will consist of a nutrition and physical activity intervention for insufficiently active people aged 60 to 70 years from low to medium socio-economic areas. Six-hundred participants will be recruited from the Australian Federal Electoral Role and randomly assigned to the intervention (n = 300) and control (n = 300) groups. The study is based on the Social Cognitive Theory and Precede-Proceed Model, incorporating voluntary cooperation and self-efficacy. The intervention includes a specially designed booklet that provides participants with information and encourages dietary and physical activity goal setting. The booklet will be supported by an exercise chart, calendar, bi-monthly newsletters, resistance bands and pedometers, along with phone and email contact. Data will be collected over three time points: pre-intervention, immediately post-intervention and 6-months post-study. Discussion This trial will provide valuable information for community-based strategies to improve older adults' physical activity and dietary intake. The project will provide guidelines for appropriate sample recruitment, and the development, implementation and evaluation of a minimal intervention program, as well as information on minimising barriers to participation in similar programs

Utility of stages of change construct in the planning of physical activity interventions among playgroup mothers

Background: The objective of this research was to assess the physical activity levels among a unique cohort of Western Australian (WA) mothers with young children who attend a WA Playgroup. Associated factors were also investigated, including self-efficacy for physical activity, social support for exercise, relevant socio-demographic correlates, as well as the stages of change construct within the Transtheoretical Model (TTM).Results: 421 women completed a questionnaire assessing physical activity behaviours. Of these, 368 participants completed the relevant physical activity evaluation items. 82.5% and 17.5% of the sample were classified as active and inactive, respectively. Associations between physical activity status and exercise stage of change were found. Additional associations were established for partner support and self-efficacy for physical activity. Conclusion: The majority of the sample was classified as active. Despite the high percentage of active participants, this study confirms the usefulness of the stages of change measure in that it can be utilised by health promotion practitioners to report physical activity behaviour and develop appropriate intervention strategies among a time poor and hard to reach population. Specifically the results are relevant to mothers in over 16,000 WA families who are involved with Playgroup WA programs. Interventions aimed at improving physical activity levels in mothers with young children should also consider the need to improve self-efficacy and social support

High-throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium.

Automated methods are needed to facilitate high-throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large-scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37-0.87) and study (kappa range = 0.39-0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p-value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000-4,500 cells: kappa = 0.78) than those with lower counts (50-500 cells: kappa = 0.41; p-value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre- and post-analytical quality control procedures are necessary in order to ensure satisfactory performance

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

BACKGROUND: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. PATIENTS AND METHODS: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. RESULTS: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. CONCLUSION: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors