Buccal swab as a reliable predictor for X inactivation ratio in inaccessible tissues.

BACKGROUND: As a result of the epigenetic phenomenon of X chromosome inactivation (XCI) every woman is a mosaic of cells with either an inactive paternal X chromosome or an inactive maternal X chromosome. The ratio between inactive paternal and maternal X chromosomes is different for every female individual, and can influence an X-encoded trait or disease. A multitude of X linked conditions is known, and for many of them it is recognised that the phenotype in affected female carriers of the causative mutation is modulated by the XCI ratio. To predict disease severity an XCI ratio is usually determined in peripheral blood samples. However, the correlation between XCI ratios in peripheral blood and disease affected tissues, that are often inaccessible, is poorly understood. Here, we tested several tissues obtained from autopsies of 12 female individuals for patch size and XCI ratio. METHODS: XCI ratios were analysed using methyl-sensitive PCR-based assays for the AR, PCSK1N and SLITRK4 loci. XCI patch size was analysed by testing the XCI ratio of tissue samples with decreasing size. RESULTS: XCI patch size was analysed for liver, muscle, ovary and brain samples and was found too small to confound testing for XCI ratio in these tissues. XCI ratios were determined in the easily accessible tissues, blood, buccal epithelium and hair follicle, and compared with ratios in several inaccessible tissues. CONCLUSIONS: Buccal epithelium is preferable over peripheral blood for predicting XCI ratios of inaccessible tissues. Ovary is the only inaccessible tissue showing a poor correlation to blood and buccal epithelium, but has a good correlation to hair follicle instead.

A reference image-based method for optimization of clinical immunohistochemistry.

AIMS: Cold ischaemic and formalin fixation time (CIT and FFT) are considered to be crucial parameters for intralaboratory variation in immunohistochemistry (IHC). Here we describe a new method to optimize IHC, by using control tissue blocks with known pre-analytical history and comparing the IHC outcome with digitized reference slides. METHODS AND RESULTS: Tissue specimens (two per tissue type) were divided into eight samples, which were subjected to different CIT and FFT. Immunohistochemistry was performed with 34 routinely used antibodies, following standard operating procedures. Relative staining intensity of four sections per slide was scored. Of the antibodies studied, seven were influenced by CIT, 13 by FFT and five by both parameters. IHC protocols were adapted until most sections on the slide showed the same intensity. Changing the antibody dilution for 10 protocols and the antigen retrieval method for six protocols improved the consistency of the IHC staining. Nine protocols could not be optimized. The optimized staining results were compared to reference slides and were found to be of adequate quality. CONCLUSIONS: It was possible to optimize most IHC protocols by adapting the analytical, rather than the pre-analytical, phase. If global references can be established, this method could decrease interlaboratory variation, preceding standardization of the pre-analytical workflow.

Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: Implications for research and diagnostics.

Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles and complicate molecular diagnostics. To investigate this effect, we characterized such pre-analytical effects in 143 non-malignant liver samples obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts treated either with the Pringle manoeuvre or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-PCR. We found 179 mutually deregulated genes which point to elevated cytokine signaling with NFκB as a dominant pathway in ischemia responses. In contrast to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFκB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were present in non-tumor tissue. The reported inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a set of 230 pre-analytically highly robust genes identified from histologically normal livers (<2% covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications.

Effects of intra- and post-operative ischemia on the metabolic profile of clinical liver tissue specimens monitored by NMR.

Metabolomic profiles of tissues could greatly contribute to advancements in personalized medicine but are influenced by differences in adopted preanalytical procedures; nonhomogeneous pre- and post-excision ischemia times are potential sources of variability. In this study, we monitored the impact of ischemia on the metabolic profiles, acquired with high-resolution magic-angle-spinning (1)H NMR, of 162 human liver samples collected during and up to 6 h after routine surgery. The profiles changed significantly as a function of intraoperative warm ischemia (WI) and postresection cold ischemia (CI) time, with significant variations in the concentration of the same 16 metabolites. Therefore, a tight control of the preanalytical phase is essential for reliable metabolomic analyses of liver diseases. The NMR profiles provide a reliable "fingerprint" of ischemia and have predictive value: the best-performing predictive models are found to discriminate extreme time points of CI (0' vs 360 ') in the training set with cross-validation accuracy of ~90%; samples in the validation cohort can discriminate short (≤60') from long (≥180') CI with an accuracy of ~80%. For WI, the corresponding figures are 95.6 and 92%, respectively. Therefore, ischemia NMR profiles might become a tool for tissue quality control in biobanks.

Variability of protein and phosphoprotein levels in clinical tissue specimens during the preanalytical phase.

The quality of human tissue specimens can have a significant impact on analytical data sets for biomarker research. The aim of this study was to characterize fluctuations of protein and phosphoprotein levels in human tissue samples during the preanalytical phase. Eleven intestine and 17 liver specimens were surgically resected, aliquoted, and either snap-frozen or fixed in formalin immediately or exposed to different ischemic conditions before preservation. Protein levels in the resultant samples were investigated by reverse phase protein array, Western blot analysis, and liquid chromatography-tandem mass spectrometry. Our data revealed that the degree of sensitivity of proteins and phosphoproteins to delayed preservation varied between different patients and tissue types. For example, up-regulation of phospho-p42/44 MAPK in intestine samples was seen in some patients but not in others. General trends toward up- or down-regulation of most proteins were not evident due to pronounced interpatient variability but signal intensities of only a few proteins, such as cytokeratin 18, were altered from baseline in postresection samples. In contrast, glyceraldehyde 3-phosphate dehydrogenase was found to be stable during periods of cold ischemia. Our study represents a proper approach for studying potential protein fluctuations in tissue specimens for future biomarker development programs.

Biospecimen reporting for improved study quality (BRISQ).

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

Biobanking residual tissues.

Health-care research relies largely on human materials stored in highly specialised biorepositories. Medical translational research on tissues can be performed using a variety of resources in distinct situations. The best known is the secondary use of pathology archives where paraffin-embedded tissues are stored for diagnostic reasons. Another is collecting and storing frozen material obtained from leftover surgical diagnosis. Such residual tissues can either be used directly in research projects or used in the context of a clinical trial with new interventional medicinal products. The latter can make the regulations governing the use of these materials for medical research much more complicated. The use of residual materials is very distinct from biobanking projects for which tissue is specifically collected. This article describes the consequences of using residual human material from different sources in distinct situations and why signed informed consent is not always the preferred choice of individual countries regarding the use of residual material. In addition, signed informed consent is overdone when using residual tissues in medical research. We maintain that the opt-out system is a balanced choice if certain requirements are met, relating to sufficient transparency about using residual tissue for research, the purpose of such research and to the confidentiality of the data used in that research. Finally, the international exchange of samples can be based on the laws and regulations of the countries of origin. Respecting these form the basis of what can and cannot be done in the country where the research on the samples is being performed.

Bringing Onco-Innovation to Europe's Healthcare Systems: The Potential of Biomarker Testing, Real World Evidence, Tumour Agnostic Therapies to Empower Personalised Medicine.

Rapid and continuing advances in biomarker testing are not being matched by uptake in health systems, and this is hampering both patient care and innovation. It also risks costing health systems the opportunity to make their services more efficient and, over time, more economical. The potential that genomics has brought to biomarker testing in diagnosis, prediction and research is being realised, pre-eminently in many cancers, but also in an ever-wider range of conditions-notably BRCA1/2 testing in ovarian, breast, pancreatic and prostate cancers. Nevertheless, the implementation of genetic testing in clinical routine setting is still challenging. Development is impeded by country-related heterogeneity, data deficiencies, and lack of policy alignment on standards, approval-and the role of real-world evidence in the process-and reimbursement. The acute nature of the problem is compellingly illustrated by the particular challenges facing the development and use of tumour agnostic therapies, where the gaps in preparedness for taking advantage of this innovative approach to cancer therapy are sharply exposed. Europe should already have in place a guarantee of universal access to a minimum suite of biomarker tests and should be planning for an optimum testing scenario with a wider range of biomarker tests integrated into a more sophisticated health system articulated around personalised medicine. Improving healthcare and winning advantages for Europe's industrial competitiveness and innovation require an appropriate policy framework-starting with an update to outdated recommendations. We show herein the main issues and proposals that emerged during the previous advisory boards organised by the European Alliance for Personalized Medicine which mainly focus on possible scenarios of harmonisation of both oncogenetic testing and management of cancer patients.

Semi-automatic identification of punching areas for tissue microarray building: the tubular breast cancer pilot study.

BACKGROUND: Tissue MicroArray technology aims to perform immunohistochemical staining on hundreds of different tissue samples simultaneously. It allows faster analysis, considerably reducing costs incurred in staining. A time consuming phase of the methodology is the selection of tissue areas within paraffin blocks: no utilities have been developed for the identification of areas to be punched from the donor block and assembled in the recipient block. RESULTS: The presented work supports, in the specific case of a primary subtype of breast cancer (tubular breast cancer), the semi-automatic discrimination and localization between normal and pathological regions within the tissues. The diagnosis is performed by analysing specific morphological features of the sample such as the absence of a double layer of cells around the lumen and the decay of a regular glands-and-lobules structure. These features are analysed using an algorithm which performs the extraction of morphological parameters from images and compares them to experimentally validated threshold values. Results are satisfactory since in most of the cases the automatic diagnosis matches the response of the pathologists. In particular, on a total of 1296 sub-images showing normal and pathological areas of breast specimens, algorithm accuracy, sensitivity and specificity are respectively 89%, 84% and 94%. CONCLUSIONS: The proposed work is a first attempt to demonstrate that automation in the Tissue MicroArray field is feasible and it can represent an important tool for scientists to cope with this high-throughput technique.

Proteomic analysis of PAXgene-fixed tissues.

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.

Propelling Health Care into the Twenties.

The scope and potential of personalised health care are underappreciated and underrealised, often because of resistance to change. The consequence is that many inadequacies of health care in Europe persist unnecessarily, and many opportunities for improvement are neglected. This article identifies the principal challenges, outlines possible approaches to resolving them, and highlights the benefits that could result from greater adoption of personalised health care. It locates the discussion in the context of European policy, focusing particularly on the most recent and authoritative reviews of health care in the EU Member States, and on the newly acquired spirit of readiness and pragmatism among European officials to embrace change and innovative technologies in a new decade. It highlights the attention now being given by policymakers to incentives, innovation, and investment as levers to improve European citizens' prospects in a rapidly evolving world, and how these distinct and disruptive themes contribute to a renaissance in thinking about delivering optimal health care in Europe. It explores the chances offered to patients by specific initiatives in health domains such as cancer and antimicrobial resistance, and by innovative science, novel therapies, earlier diagnosis tools, and deeper understanding of health promotion and prevention. And it reflects on how health care providers could benefit from a shift towards better primary care and towards deploying health data more effectively, including the use of artificial intelligence, coupled with a move to a smoother organisational/regulatory structure and realigned professional responsibilities. The conclusion is that preparing Europe's health care systems for the inevitable strains of the coming years is both possible and necessary. A more courageous approach to embracing personalised health care could guarantee the sustainability of Europe's health care systems before rising demands and exponential costs overwhelm them - an exercise in future-proofing, in ensuring that they are equipped to withstand whatever lies ahead. A focus on the potential and implementation of personalised care would permit more efficient use of resources and deliver better quality health-preserving care.

Bringing Greater Accuracy to Europe's Healthcare Systems: The Unexploited Potential of Biomarker Testing in Oncology.

Rapid and continuing advances in biomarker testing are not being matched by take-up in health systems, and this is hampering both patient care and innovation. It also risks costing health systems the opportunity to make their services more efficient and, over time, more economical. This paper sets out the potential of biomarker testing, the unfolding precision and range of possible diagnosis and prediction, and the many obstacles to adoption. It offers case studies of biomarker testing in breast, ovarian, prostate, lung, thyroid and colon cancers, and derives specific lessons as to the potential and actual use of each of them. It also draws lessons about how to improve access and alignment, and to remedy the data deficiencies that impede development. And it suggests solutions to outstanding issues - notably including funding and the tangled web of obtaining reimbursement or equivalent coverage that Europe's fragmented health system implies. It urges a European evolution towards an initial minimum testing scenario, which would guarantee universal access to a suite of biomarker tests for the currently most common conditions, and, further into the future, to an optimum testing scenario in which a much wider range of biomarker tests would be introduced and become part of a more sophisticated health system articulated around personalised medicine. For exploiting genomics to the full, it argues the need for a new policy framework for Europe. Biomarker testing is not an issue that can be treated in isolation, since the purpose of testing is to improve health. Its use is therefore always closely linked to specific health challenges and needs to be viewed in the broader policy context in the EU and more widely. The paper is the result of extensive engagement with experts and decision makers to develop the framework, and consequently represents a wide consensus of views on how healthcare systems should respond from push and pull factors at local, national and cross-border and EU level. It contains strong views and clear recommendations springing from the convictions of patients, clinicians, academics, medicines authorities, HTA bodies, payers, the diagnostic, pharmaceutical and ICT industries, and national policy makers.

Pre-analytical processes in medical diagnostics: New regulatory requirements and standards.

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.

Cryo-Gel embedding compound for renal biopsy biobanking.

Optimal preservation and biobanking of renal tissue is vital for good diagnostics and subsequent research. Optimal cutting temperature (OCT) compound is a commonly used embedding medium for freezing tissue samples. However, due to interfering polymers in OCT, analysis as mass spectrometry (MS) is difficult. We investigated if the replacement of OCT with Cryo-Gel as embedding compound for renal biopsies would enable proteomics and not disturb other common techniques used in tissue diagnostics and research. For the present study, fresh renal samples were snap-frozen using Cryo-Gel, OCT and without embedding compound and evaluated using different techniques. In addition, tissue samples from normal spleen, skin, liver and colon were analyzed. Cryo-Gel embedded tissues showed good morphological preservation and no interference in immunohistochemical or immunofluorescent investigations. The quality of extracted RNA and DNA was good. The number of proteins identified using MS was similar between Cryo-Gel embedded samples, samples without embedding compound and OCT embedded samples. However, polymers in the OCT disturbed the signal in the MS, while this was not observed in the Cryo-Gel embedded samples. We conclude that embedding of renal biopsies in Cryo-Gel is an excellent and preferable alternative for OCT compound for both diagnostic and research purposes, especially in those cases where proteomic analysis might be necessary.

Targeted disruption of the Mn1 oncogene results in severe defects in development of membranous bones of the cranial skeleton.

Fusion of the MN1 gene to TEL (ETV6) results in myeloid leukemia. The fusion protein combines the transcription activating domain of MN1 and the DNA binding domain of TEL and is thought to act as a deranged transcription factor. In addition, disruption of the large first exon of the MN1 gene is thought to inactivate MN1 function in a meningioma. To further investigate the role of MN1 in cancer, we generated Mn1 knockout mice. Mn1(+/-) animals were followed for 30 months, but they had no higher incidence of tumor formation than wild-type littermates. Mn1 null mice, however, were found to die at birth or shortly thereafter as the result of a cleft palate. Investigation of newborn or embryonic day 15.5 (E15.5) to E17.5 null mice revealed that the development of several bones in the skull was abnormal. The affected bones are almost exclusively formed by intramembranous ossification. They are either completely agenic at birth (alisphenoid and squamosal bones and vomer), hypoplastic, deformed (basisphenoid, pterygoid, and presphenoid), or substantially thinner (frontal, parietal, and interparietal bones). In heterozygous mice hypoplastic membranous bones and incomplete penetrance of the cleft palate were observed. We conclude that Mn1 is an important factor in development of membranous bones.

OECI TuBaFrost tumor biobanking.

OECI TuBaFrost harbors a complete infrastructure for the exchange of frozen tumor samples between European countries. OECI TuBaFrost consists of: * A code of conduct on how to exchange human residual samples in Europe, * A central database application accessible over the Internet (www.tubafrost.org) where data can be uploaded and searched from samples that can be selected and ordered, * Access rules with incentives for collectors, * Standardization needed to enable the analysis of high quality samples derived from different centers, * Virtual Microscopy to support sample selection with difficult pathology. The entire infrastructure was, after completion, which was entirely financed by the European Commission, implemented in the OECI. But so far it has not been used to its capacity. A recent survey held amongst the OECI members shed light on the causes. The main conclusion is that all responders see OECI TuBaFrost as a good platform for exchange of samples, however, the biggest bottleneck found was that potential users are too unfamiliar with the communication between their own biobank tracking system and the TuBaFrost central database application. Therefore, new future plans are drawn. In addition, new infrastructure plans have been developed and the first preparatory steps have been set. For biobanks the BBMRI project has started aiming for Pan-European Biobanking and Biomolecular Resources Research Infrastructure.

Improvement of European translational cancer research. Collaboration between comprehensive cancer centers.

Even though the increasing incidence of cancer is mainly a consequence of a population with a longer life span, part of this augmentation is related to the increasing prevalence of patients living with a chronic cancer disease. To fight the problem, improved preventive strategies are mandatory in combination with an innovative health care provision that is driven by research. To overcome the weakness of translational research the OECI is proposing a practical approach as part of a strategy foreseen by the EUROCAN+PLUS feasibility study, which was launched by the EC in order to identify mechanisms for the coordination of cancer research in Europe.

Biospecimens and Biobanking in Global Health.

Biobanks provide a critical infrastructure to support research in human health. Biospecimens and their accompanying data are increasingly needed to support biomedical research and clinical care. The original text was initially published in the Handbook for Cancer Research in Africa. The value of this publication is great as it underlines the importance of biobanks in Africa as a key resource to increase quality scientific research and participate in global health research. Therefore, a revision to extend these principles to other low resource contexts, to include updated material and references and add the topic of biobank sustainability were relevant.

Personalised Medicine - Bringing Innovation to the Healthcare System.

Healthcare innovation has never been more prevalent than it is today. But these innovations are only very slowly being embedded into Europe's healthcare systems. There is a huge capacity here in the EU to improve the health and quality of life of all citizens, but the extent to which it is happening is far from optimal. What is ringing out like a bell is that there is a clear need for better focus from policy makers, as this article explains. A policy bridge is required and a conscious decision among the powers-that-be in Europe needs to find a way to harmonise multiple strands of activity and responsibility in the health arena. The end goal will be for the EU to more effectively integrate the incredible advances in science into healthcare systems, for the benefit of all patients.

Allelic imbalance of 7q32.3-q36.1 during tumorigenesis in Barrett's esophagus.

Malignant transformation of Barrett's esophagus is characterized by three distinct premalignant stages: intestinal metaplasia (MET), low- (LGD), and high-grade dysplasia (HGD). We reported recently an increase in the frequency of loss of 7q33-q35 between LGD and HGD as determined by comparative genomic hybridization (P. H. J. Riegman et al., Cancer Res., 61: 3164-3170, 2001). Now the 7q32.3-q36.1 region was additionally characterized by allelotype analysis with 11 polymorphic markers in 15 METs, 20 LGDs, 20 HGDs, and 20 Barrett's adenocarcinomas from different patients. Low percentages of imbalance were determined in METs and LGDs, 7% and 10%, respectively, whereas HGDs and Barrett's adenocarcinomas revealed high percentages of loss, 75% and 65%, respectively. This difference in frequency between LGDs and HGDs appeared highly significant: P = 0.00007. The majority of imbalances were found at D7S2439 and D7S483, located on 7q36.1. These data suggest that markers from this area can be used as a diagnostic tool in Barrett's esophagus, i.e., to distinguish between watchful waiting and active treatment.