Identification of markers for human endometrial carcinoma stem cells

Cancer stem cells (CSCs) have recently been identified in many human solid cancers. It was hypothesised that CSCs are responsible for epithelial neoplasia associated with endometrial carcinoma (EC), the most common gynaecological malignancy in women. The aim of this study was to demonstrate that a rare population of EC cells possess CSC properties, and to commence identifying markers for these cells. Firstly, evidence for a subset of cells with CSC properties in human EC was required. Functional assays revealed that a small population (0.24%) of freshly isolated EC and endometrial hyperplasia cells exhibited the in vitro CSC characteristics of clonogenicty and self renewal. Further assays indicated that rare cells within EC were able to initiate tumourigenic growth, recapitulating the original tumour phenotype when transplanted subrenally in NOD/SCID mice. Importantly, this project discovered that not all cells within EC possessed the same clonogenic, tumour initiating, and self renewal properties, conforming to the CSC hypothesis in all grades of Type I EC, Type II EC and its precursor lesion, endometrial hyperplasia. However, these studies were retrospective and a cell surface marker or combination of markers for the isolation of endometrial cancer stem cells (ECSCs) is required so that they can be isolated, characterised and their role in the development of the disease studied. To identify potential ECSC markers, advantage was taken of the heterogeneity of EC cells. The hypothesis was that ECSCs would express different markers from their progenitor and more differentiated daughter cancer cells. A panel of 26 potential adult stem cell antibody supernatants was investigated. Since none of the antibodies showed a pattern of reactivity suggestive of CSCs i.e. strong reaction with a small number of EC epithelial cells, a strategy was devised to rank the antibodies into a priority list. This priority list was based on the percentage of cells expressing the marker, the marker’s robustness, and consistency of expression between samples. Based on this priority list, 2 antibodies were tested using functional assays to indicate if they selected for ECSCs. The CD133-1 antibody (W6B3) was identified as the top potential ECSC marker. Interestingly, the number 3 ranked antibody was the CD133-2 antibody (293-C3). Reports were appearing that indicated there were differences in the expression of these two CD133 epitopes, and thus both were concurrently investigated to determine if either were potential ECSC markers. Neither antibody enriched for epithelial EC cells with in vitro CSC properties. Similar percentages of clonogenic cells (0-0.5%) were observed in the CD133+ and CD133- (CD133-1 or CD133-2) sorted populations, indicating no enrichment of colony forming units (CFUs). Further, CFUs from both CD133+ and CD133- subpopulations were equally able to undergo self renewing divisions in vitro, with no observable difference between CD133-1 and CD133-2. This data indicated that CD133 did not enrich for ECSCs. This study lays the groundwork for future studies to explore further markers from the priority list that will enable the prospective isolation of ECSC required to confirm their existence and role in the development of human EC. Together with the identification of normal human endometrial epithelial stem cell markers, the development and progression of EC can be investigated, allowing the identification of potential drug targets selective for CSC, but sparing normal endometrial stem cells. Such treatments will be particularly useful for early stage EC and hyperplasia candidates where the uterus may be conserved, and for late stage cases where hysterectomy is not curative and current treatments target the bulk tumor cells rather than CSCs

Identification of markers for human endometrial carcinoma stem cells

Cancer stem cells (CSCs) have recently been identified in many human solid cancers. It was hypothesised that CSCs are responsible for epithelial neoplasia associated with endometrial carcinoma (EC), the most common gynaecological malignancy in women. The aim of this study was to demonstrate that a rare population of EC cells possess CSC properties, and to commence identifying markers for these cells. Firstly, evidence for a subset of cells with CSC properties in human EC was required. Functional assays revealed that a small population (0.24%) of freshly isolated EC and endometrial hyperplasia cells exhibited the in vitro CSC characteristics of clonogenicty and self renewal. Further assays indicated that rare cells within EC were able to initiate tumourigenic growth, recapitulating the original tumour phenotype when transplanted subrenally in NOD/SCID mice. Importantly, this project discovered that not all cells within EC possessed the same clonogenic, tumour initiating, and self renewal properties, conforming to the CSC hypothesis in all grades of Type I EC, Type II EC and its precursor lesion, endometrial hyperplasia. However, these studies were retrospective and a cell surface marker or combination of markers for the isolation of endometrial cancer stem cells (ECSCs) is required so that they can be isolated, characterised and their role in the development of the disease studied. To identify potential ECSC markers, advantage was taken of the heterogeneity of EC cells. The hypothesis was that ECSCs would express different markers from their progenitor and more differentiated daughter cancer cells. A panel of 26 potential adult stem cell antibody supernatants was investigated. Since none of the antibodies showed a pattern of reactivity suggestive of CSCs i.e. strong reaction with a small number of EC epithelial cells, a strategy was devised to rank the antibodies into a priority list. This priority list was based on the percentage of cells expressing the marker, the marker’s robustness, and consistency of expression between samples. Based on this priority list, 2 antibodies were tested using functional assays to indicate if they selected for ECSCs. The CD133-1 antibody (W6B3) was identified as the top potential ECSC marker. Interestingly, the number 3 ranked antibody was the CD133-2 antibody (293-C3). Reports were appearing that indicated there were differences in the expression of these two CD133 epitopes, and thus both were concurrently investigated to determine if either were potential ECSC markers. Neither antibody enriched for epithelial EC cells with in vitro CSC properties. Similar percentages of clonogenic cells (0-0.5%) were observed in the CD133+ and CD133- (CD133-1 or CD133-2) sorted populations, indicating no enrichment of colony forming units (CFUs). Further, CFUs from both CD133+ and CD133- subpopulations were equally able to undergo self renewing divisions in vitro, with no observable difference between CD133-1 and CD133-2. This data indicated that CD133 did not enrich for ECSCs. This study lays the groundwork for future studies to explore further markers from the priority list that will enable the prospective isolation of ECSC required to confirm their existence and role in the development of human EC. Together with the identification of normal human endometrial epithelial stem cell markers, the development and progression of EC can be investigated, allowing the identification of potential drug targets selective for CSC, but sparing normal endometrial stem cells. Such treatments will be particularly useful for early stage EC and hyperplasia candidates where the uterus may be conserved, and for late stage cases where hysterectomy is not curative and current treatments target the bulk tumor cells rather than CSCs

Testing the reproducibility and robustness of the cancer biology literature by robot.

Scientific results should not just be 'repeatable' (replicable in the same laboratory under identical conditions), but also 'reproducible' (replicable in other laboratories under similar conditions). Results should also, if possible, be 'robust' (replicable under a wide range of conditions). The reproducibility and robustness of only a small fraction of published biomedical results has been tested; furthermore, when reproducibility is tested, it is often not found. This situation is termed 'the reproducibility crisis', and it is one the most important issues facing biomedicine. This crisis would be solved if it were possible to automate reproducibility testing. Here, we describe the semi-automated testing for reproducibility and robustness of simple statements (propositions) about cancer cell biology automatically extracted from the literature. From 12 260 papers, we automatically extracted statements predicted to describe experimental results regarding a change of gene expression in response to drug treatment in breast cancer, from these we selected 74 statements of high biomedical interest. To test the reproducibility of these statements, two different teams used the laboratory automation system Eve and two breast cancer cell lines (MCF7 and MDA-MB-231). Statistically significant evidence for repeatability was found for 43 statements, and significant evidence for reproducibility/robustness in 22 statements. In two cases, the automation made serendipitous discoveries. The reproduced/robust knowledge provides significant insight into cancer. We conclude that semi-automated reproducibility testing is currently achievable, that it could be scaled up to generate a substantive source of reliable knowledge and that automation has the potential to mitigate the reproducibility crisis

Implementation and evaluation of a standardized nurse‐administered assessment of functional and psychosocial issues for patients in acute care

Background Increasingly, adults presenting to healthcare facilities have multiple morbidities that impact medical management and require initial and ongoing assessment. The interRAI Acute Care (AC), one of a suite of instruments used for integrated care, is a nurse-administered standardized assessment of functional and psychosocial domains that contribute to complexity of patients admitted to acute care. Aim This study aimed to implement and evaluate the interRAI AC assessment system using a multi-strategy approach based on the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) framework. Methods This nurse-led quality improvement study was piloted in a 200-bed public hospital in Brisbane, Australia, over the period 2017 to 2018. The interRAI AC is a set of clinical observations of functional and psychosocial domains, supported by software to derive diagnostic and risk screeners, scales to measure and monitor severity, and alerts to assist in care planning. Empirical data, surveys, and qualitative feedback were used to measure process and impact outcomes using the RE-AIM evaluation framework (Reach, Efficacy, Adoption, Implementation, and Maintenance). Results In comparison to usual practice, the interRAI assessment system and supporting software was able to improve the integrity and compliance of nurse assessments, identifying key risk domains to facilitate management of care. Pre-implementation documentation (630 items in 45 patient admissions) had 39% missing data compared with 1% missing data during the interRAI implementation phase (9,030 items in 645 patient admissions). Qualitative feedback from nurses in relation to staff engagement and behavioral intention to use the new technology was mixed. Linking Evidence to Action Despite challenges to implementing a system-wide change, evaluation results demonstrated considerable efficiency gains in the nursing assessment system. For successful implementation of the interRAI AC, study findings suggest the need for interoperability with other information systems, access to training, and continued leadership support

Implementation and Evaluation of a Standardized Nurse‐Administered Assessment of Functional and Psychosocial Issues for Patients in Acute Care

Background: Increasingly, adults presenting to healthcare facilities have multiple morbidities that impact medical management and require initial and ongoing assessment. The interRAI Acute Care (AC), one of a suite of instruments used for integrated care, is a nurse-administered standardized assessment of functional and psychosocial domains that contribute to complexity of patients admitted to acute care. Aim: This study aimed to implement and evaluate the interRAI AC assessment system using a multi-strategy approach based on the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) framework. Methods: This nurse-led quality improvement study was piloted in a 200-bed public hospital in Brisbane, Australia, over the period 2017 to 2018. The interRAI AC is a set of clinical observations of functional and psychosocial domains, supported by software to derive diagnostic and risk screeners, scales to measure and monitor severity, and alerts to assist in care planning. Empirical data, surveys, and qualitative feedback were used to measure process and impact outcomes using the RE-AIM evaluation framework (Reach, Efficacy, Adoption, Implementation, and Maintenance). Results: In comparison to usual practice, the interRAI assessment system and supporting software was able to improve the integrity and compliance of nurse assessments, identifying key risk domains to facilitate management of care. Pre-implementation documentation (630 items in 45 patient admissions) had 39% missing data compared with 1% missing data during the interRAI implementation phase (9,030 items in 645 patient admissions). Qualitative feedback from nurses in relation to staff engagement and behavioral intention to use the new technology was mixed. Linking Evidence to Action: Despite challenges to implementing a system-wide change, evaluation results demonstrated considerable efficiency gains in the nursing assessment system. For successful implementation of the interRAI AC, study findings suggest the need for interoperability with other information systems, access to training, and continued leadership support

Implementation and evaluation of a standardized nurse-administered assessment of functional and psychosocial issues for patients in acute care

Background: Increasingly, adults presenting to healthcare facilities have multiple morbidities that impact medical management and require initial and ongoing assessment. The interRAI Acute Care (AC), one of a suite of instruments used for integrated care, is a nurse-administered standardized assessment of functional and psychosocial domains that contribute to complexity of patients admitted to acute care. Aim: This study aimed to implement and evaluate the interRAI AC assessment system using a multi-strategy approach based on the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) framework. Methods: This nurse-led quality improvement study was piloted in a 200-bed public hospital in Brisbane, Australia, over the period 2017 to 2018. The interRAI AC is a set of clinical observations of functional and psychosocial domains, supported by software to derive diagnostic and risk screeners, scales to measure and monitor severity, and alerts to assist in care planning. Empirical data, surveys, and qualitative feedback were used to measure process and impact outcomes using the RE-AIM evaluation framework (Reach, Efficacy, Adoption, Implementation, and Maintenance). Results: In comparison to usual practice, the interRAI assessment system and supporting software was able to improve the integrity and compliance of nurse assessments, identifying key risk domains to facilitate management of care. Pre-implementation documentation (630 items in 45 patient admissions) had 39% missing data compared with 1% missing data during the interRAI implementation phase (9,030 items in 645 patient admissions). Qualitative feedback from nurses in relation to staff engagement and behavioral intention to use the new technology was mixed. Linking Evidence to Action: Despite challenges to implementing a system-wide change, evaluation results demonstrated considerable efficiency gains in the nursing assessment system. For successful implementation of the interRAI AC, study findings suggest the need for interoperability with other information systems, access to training, and continued leadership support

Sensitivity and specificity of a novel classifier for the early diagnosis of dengue.

BACKGROUND:Dengue is the commonest arboviral disease of humans. An early and accurate diagnosis of dengue can support clinical management, surveillance and disease control and is central to achieving the World Health Organisation target of a 50% reduction in dengue case mortality by 2020. METHODS:5729 children with fever of <72 hrs duration were enrolled into this multicenter prospective study in southern Vietnam between 2010-2012. A composite of gold standard diagnostic tests identified 1692 dengue cases. Using statistical methods, a novel Early Dengue Classifier (EDC) was developed that used patient age, white blood cell count and platelet count to discriminate dengue cases from non-dengue cases. RESULTS:The EDC had a sensitivity of 74.8% (95%CI: 73.0-76.8%) and specificity of 76.3% (95%CI: 75.2-77.6%) for the diagnosis of dengue. As an adjunctive test alongside NS1 rapid testing, sensitivity of the composite test was 91.6% (95%CI: 90.4-92.9%). CONCLUSIONS:We demonstrate that the early diagnosis of dengue can be enhanced beyond the current standard of care using a simple evidence-based algorithm. The results should support patient management and clinical trials of specific therapies

Univariate and multivariate analysis of candidate predictors of laboratory-confirmed dengue.

<p>BMI: body mass index; WBC: white blood cell count; PLT: platelet count; HCT: hematocrit; ALB: albumin; AST: aspartate aminotransferase; CK: creatine kinase</p><p>Univariate and multivariate analysis of candidate predictors of laboratory-confirmed dengue.</p

Performance of the Early Dengue Classifier (EDC) in all subjects.

<p>Figure A displays possible sensitivity/specificity trade-offs for different cut-off values and the distance from the corresponding points on the ROC curve to the upper left corner (perfect model). Figure B displays the receiver operating characteristic (ROC) curve. Figure C is a calibration plot. It displays a scatterplot-smoother of predicted versus observed risks (dotted line), predicted versus observed risks for ten patient strata of equal size grouped according to predicted risks (triangles) and the ideal identity line (dashed line). The rugs at the bottom of the graphs characterize the distribution of predicted risks in true dengue and non-dengue cases, respectively.</p

Diagnostic performance of NS1 rapid test in enrolment plasma samples and odds of NS1 detection in relation to plasma viremia.

<p>^a Viremia measurement in the enrolment plasma sample (the same sample was also used for NS1 testing).</p><p>PPV: positive predictive value; NPV: negative predictive value; DENV: dengue virus; OR: odds ratios for detecting NS1 for each 10-fold higher DENV RNA concentration. There were 77 dengue cases where the infecting serotype was unknown.</p><p>Diagnostic performance of NS1 rapid test in enrolment plasma samples and odds of NS1 detection in relation to plasma viremia.</p