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.

Histological evaluation of PAXgene tissue fixation in Barrett's esophagus and esophageal adenocarcinoma diagnostics.

The dysplasia grading of Barrett's esophagus (BE), based on the histomorphological assessment of formalin-fixed, paraffin-embedded (FFPE) tissue, suffers from high interobserver variability leading to an unsatisfactory prediction of cancer risk. Thus, pre-analytic preservation of biological molecules, which could improve risk prediction in BE enabling molecular and genetic analysis, is needed. We aimed to evaluate such a molecular pre-analytic fixation tool, PAXgene-fixed paraffin-embedded (PFPE) biopsies, and their suitability for histomorphological BE diagnostics in comparison to FFPE. In a ring trial, 9 GI pathologists evaluated 116 digital BE slides of non-dysplastic BE (NDBE), low-grade dysplasia (LGD), high-grade dysplasia (HGD), and esophageal adenocarcinomas (EAC) using virtual microscopy. Overall quality, cytological and histomorphological parameters, dysplasia criteria, and diagnosis were analyzed. PFPE showed better preservation of nuclear details as chromatin and nucleoli, whereas overall quality and histomorphologic parameters as visibility of basal lamina, goblet cells, and presence of artifacts were scored as equal to FFPE. The interobserver reproducibility with regard to the diagnosis was best for NDBE and EAC (κF = 0.72-0.75) and poor for LGD and HGD (κF = 0.13-0.3) in both. In conclusion, our data suggest that PFPE allows equally confident histomorphological diagnosis of BE and EAC, introducing a novel tool for molecular analysis and parallel histomorphological evaluation.

Stakeholder engagement to ensure the sustainability of biobanks: a survey of potential users of biobank services.

Biobanks are important infrastructures facilitating biomedical research. After a decade of rolling out such infrastructures, a shift in attention to the sustainability of biobanks could be observed in recent years. In this regard, an increase in the as yet relatively low utilisation rates of biobanks has been formulated as a goal. Higher utilisation rates can only be achieved if the perspectives of potential users of biobanks-particularly researchers not yet collaborating with biobanks-are adequately considered. To better understand their perspectives, a survey was conducted at ten different research institutions in Germany hosting a centralised biobank. The survey targeted potential users of biobank services, i.e. researchers working with biosamples. It addressed the general demand for biosamples, strategies for biosample acquisition/storage and reasons for/against collaborating with biobanks. In total, 354 researchers filled out the survey. Most interestingly, only a minority of researchers (12%) acquired their biosamples via biobanks. Of the respondents not collaborating with biobanks on sample acquisition, around half were not aware of the (services of the) respective local biobank. Those who actively decided against acquiring biosamples via a biobank provided different reasons. Most commonly, respondents stated that the biosamples required were not available, the costs were too high and information about the available biosamples was not readily accessible. Biobanks can draw many lessons from the results of the survey. Particularly, external communication and outreach should be improved. Additionally, biobanks might have to reassess whether their particular collection strategies are adequately aligned with local researchers' needs.

Morphology Matters: A Critical Reappraisal of the Clinical Relevance of Morphologic Criteria From the 2019 WHO Classification in a Large Colorectal Cancer Cohort Comprising 1004 Cases.

The 2019 World Health Organization (WHO) classification of colorectal carcinoma (CRC) profoundly reclassified CRC subtypes and introduces tumor budding as a second major grading criterion, while condensing conventional grade into a 2-tiered system. So far it remains largely unexplored how these parameters interact with each other and whether they truly have an independent impact on patient prognosis. We reclassified a large single-center cohort of 1004 CRCs spanning 2 decades for adjusted WHO grade (low vs. high), tumor budding (Bd1/Bd2/Bd3), and CRC subtype (adenocarcinoma not otherwise specified, micropapillary, mucinous, serrated, medullary, adenoma-like, signet-ring cell, mixed adenoneuroendocrine carcinoma/neuroendocrine carcinoma, undifferentiated) according to the criteria of the 2019 WHO classification. We investigated the interaction of these parameters, their connection to stage/microsatellite status, and their significance for patient survival in the different subgroups. Specific subtypes other than adenocarcinoma not otherwise specified represented one third of all CRCs and were unevenly distributed throughout stage and microsatellite subgroups. Subtypes, WHO grade and tumor budding profoundly impacted all survival parameters (P<0.001 for all analyses), with CRC subtypes and tumor budding-but not WHO grade-being stage-independent prognosticators for all survival comparisons. WHO grade had very limited prognostic value in CRC subtypes, while tumor budding retained its strong prognostic impact in most scenarios. Accurate delineation of CRC subtypes introduced in the 2019 WHO classification provides strong stage-independent prognostic information, arguing that they should be considered in pathology reports and in clinical trials. Of the morphology-based grading schemes included in the 2019 WHO, tumor budding outperforms WHO grade.

12. Review of diabetic papillitis.

Diabetic papillitis (used interchangeably with diabetic papillopathy) is a relatively rare ocular manifestation of systemic diabetic disease. Found in both type 1 and type 2 diabetic patients, it manifests as swelling of the optic nerve head in one or both eyes and is considered a diagnosis of exclusion. Signs and symptoms of optic nerve disease are not necessarily present, and there may be spontaneous recovery. The pathogenesis is poorly understood. The following discussion will cover the presentation, diagnosis, treatment, and prognosis of diabetic papillopathy.

On the Feasibility of Fan-Out Wafer-Level Packaging of Capacitive Micromachined Ultrasound Transducers (CMUT) by Using Inkjet-Printed Redistribution Layers.

Fan-out wafer-level packaging (FOWLP) is an interesting platform for Microelectromechanical systems (MEMS) sensor packaging. Employing FOWLP for MEMS sensor packaging has some unique challenges, while some originate merely from the fabrication of redistribution layers (RDL). For instance, it is crucial to protect the delicate structures and fragile membranes during RDL formation. Thus, additive manufacturing (AM) for RDL formation seems to be an auspicious approach, as those challenges are conquered by principle. In this study, by exploiting the benefits of AM, RDLs for fan-out packaging of capacitive micromachined ultrasound transducers (CMUT) were realized via drop-on-demand inkjet printing technology. The long-term reliability of the printed tracks was assessed via temperature cycling tests. The effects of multilayering and implementation of an insulating ramp on the reliability of the conductive tracks were identified. Packaging-induced stresses on CMUT dies were further investigated via laser-Doppler velocimetry (LDV) measurements and the corresponding resonance frequency shift. Conclusively, the bottlenecks of the inkjet-printed RDLs for FOWLP were discussed in detail.

Oncogenic Linear Collagen VI of Invasive Breast Cancer Is Induced by CCL5.

The triple-negative breast tumor boundary is made of aligned, linear collagen. The pro-oncogenic impact of linear collagen is well established; however, its mechanism of formation is unknown. An in vitro analogue of the tumor border is created by a co-culture of MDA-MB-231 cells, adipose derived stem cells, and dermal fibroblasts. Decellularization of this co-culture after seven days reveals an extracellular matrix that is linear in fashion, high in pro-oncogenic collagen type VI, and able to promote invasion of reseeded cells. Further investigation revealed linear collagen VI is produced by fibroblasts in response to a paracrine co-culture of adipose derived stem cells and MDA-MB-231, which together secrete high levels of the chemokine CCL5. The addition of monoclonal antibody against CCL5 to the co-culture results in an unorganized matrix with dramatically decreased collagen VI. Importantly, reseeded cells do not exhibit pro-oncogenic behavior. These data illustrate a cellular mechanism, which creates linear extracellular matrix (ECM) in vitro, and highlight a potential role of CCL5 for building striated tumor collagen in vivo.

Epidemiologic Risk Factors in a Comparison of a Barrett Esophagus Registry (BarrettNET) and a Case-Control Population in Germany.

Endoscopic screening for Barrett's esophagus as the major precursor lesion for esophageal adenocarcinoma is mostly offered to patients with symptoms of gastroesophageal reflux disease (GERD). However, other epidemiologic risk factors might affect the development of Barrett's esophagus and esophageal adenocarcinoma. Therefore, efforts to improve the efficiency of screening to find the Barrett's esophagus population "at risk" compared with the normal population are needed. In a cross-sectional analysis, we compared 587 patients with Barrett's esophagus from the multicenter German BarrettNET registry to 1976 healthy subjects from the population-based German KORA cohort, with and without GERD symptoms. Data on demographic and lifestyle factors, including age, gender, smoking, alcohol consumption, body mass index, physical activity, and symptoms were collected in a standardized epidemiologic survey. Increased age, male gender, smoking, heavy alcohol consumption, low physical activity, low health status, and GERD symptoms were significantly associated with Barrett's esophagus. Surprisingly, among patients stratified for GERD symptoms, these associations did not change. Demographic, lifestyle, and clinical factors as well as GERD symptoms were associated with Barrett's esophagus development in Germany, suggesting that a combination of risk factors could be useful in developing individualized screening efforts for patients with Barrett's esophagus and GERD in Germany.

Lysate Preparation for Reverse Phase Protein Arrays.

Protein lysates from a variety of sample materials, e.g., cell lines, serum, frozen tissues, formalin-fixed and paraffin-embedded (FFPE) tissues, and fine needle aspirates, are compatible with the analysis using reverse phase protein arrays (RPPAs). Due to this diversity of input material, lysate preparation is one of the critical steps for obtaining reliable RPPA results. The challenges include, but are not limited to, achieving complete solubilization, avoiding chemical or enzymatic protein modifications, and degradation. In this chapter, preparations of lysates for RPPA analysis are described, focusing on human tissue samples. Special emphasis is given to recently published ISO International Standards for the preanalytical phase.

Post-neoadjuvant cellular dissociation grading based on tumour budding and cell nest size is associated with therapy response and survival in oesophageal squamous cell carcinoma.

BACKGROUND: Cellular Dissociation Grade (CDG) composed of tumour budding and cell nest size has been shown to independently predict prognosis in pre-therapeutic biopsies and primary resections of oesophageal squamous cell carcinoma (ESCC). Here, we aimed to evaluate the prognostic impact of CDG in ESCC after neoadjuvant therapy. METHODS: We evaluated cell nest size and tumour budding activity in 122 post-neoadjuvant ESCC resections, correlated the results with tumour regression groups and patient survival and compared the results with data from primary resected cases as well as pre-therapeutic biopsies. RESULTS: CDG remained stable when results from pre-therapeutic biopsies and post-therapeutic resections from the same patient were compared. CDG was associated with therapy response and a strong predictor of overall, disease-specific (DSS) and disease-free (DFS) survival in univariate analysis and-besides metastasis-remained the only significant survival predictor for DSS and DFS in multivariate analysis. Multivariate DFS hazard ratios reached 3.3 for CDG-G2 and 4.9 for CDG-G3 neoplasms compared with CDG-G1 carcinomas (p = 0.016). CONCLUSIONS: CDG is the only morphology-based grading algorithm published to date, which in concert with regression grading, is able to contribute relevant prognostic information in the post-neoadjuvant setting of ESCC.

BarrettNET-a prospective registry for risk estimation of patients with Barrett's esophagus to progress to adenocarcinoma.

Risk stratification in patients with Barrett's esophagus (BE) to prevent the development of esophageal adenocarcinoma (EAC) is an unsolved task. The incidence of EAC and BE is increasing and patients are still at unknown risk. BarrettNET is an ongoing multicenter prospective cohort study initiated to identify and validate molecular and clinical biomarkers that allow a more personalized surveillance strategy for patients with BE. For BarrettNET participants are recruited in 20 study centers throughout Germany, to be followed for progression to dysplasia (low-grade dysplasia or high-grade dysplasia) or EAC for >10 years. The study instruments comprise self-administered epidemiological information (containing data on demographics, lifestyle factors, and health), as well as biological specimens, i.e., blood-based samples, esophageal tissue biopsies, and feces and saliva samples. In follow-up visits according to the individual surveillance plan of the participants, sample collection is repeated. The standardized collection and processing of the specimen guarantee the highest sample quality. Via a mobile accessible database, the documentation of inclusion, epidemiological data, and pathological disease status are recorded subsequently. Currently the BarrettNET registry includes 560 participants (23.1% women and 76.9% men, aged 22-92 years) with a median follow-up of 951 days. Both the design and the size of BarrettNET offer the advantage of answering research questions regarding potential causes of disease progression from BE to EAC. Here all the integrated methods and materials of BarrettNET are presented and reviewed to introduce this valuable German registry.

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.

Fan-Out Wafer and Panel Level Packaging as Packaging Platform for Heterogeneous Integration.

Fan-out wafer level packaging (FOWLP) is one of the latest packaging trends in microelectronics. Besides technology developments towards heterogeneous integration, including multiple die packaging, passive component integration in packages and redistribution layers or package-on-package approaches, larger substrate formats are also targeted. Manufacturing is currently done on a wafer level of up to 12"/300 mm and 330 mm respectively. For a higher productivity and, consequently, lower costs, larger form factors are introduced. Instead of following the wafer level roadmaps to 450 mm, panel level packaging (PLP) might be the next big step. Both technology approaches offer a lot of opportunities as high miniaturization and are well suited for heterogeneous integration. Hence, FOWLP and PLP are well suited for the packaging of a highly miniaturized energy harvester system consisting of a piezo-based harvester, a power management unit and a supercapacitor for energy storage. In this study, the FOWLP and PLP approaches have been chosen for an application-specific integrated circuit (ASIC) package development with integrated SMD (surface mount device) capacitors. The process developments and the successful overall proof of concept for the packaging approach have been done on a 200 mm wafer size. In a second step, the technology was scaled up to a 457 × 305 mm2 panel size using the same materials, equipment and process flow, demonstrating the low cost and large area capabilities of the approach.

Genetic Biopsy for Prediction of Surveillance Intervals after Endoscopic Resection of Colonic Polyps: Results of the GENESIS Study.

BACKGROUND AND OBJECTIVE: Current surveillance strategies for colorectal cancer following polypectomy are determined by endoscopic and histopathological factors. Such a distinction has been challenged. The present study was designed to identify molecular parameters in colonic polyps potentially defining new sub-groups at risk. METHODS: One hundred patients were enrolled in this multicentre study. Polyps biopsies underwent formalin-free processing (PAXgene, PreAnalytiX) and targeted next generation sequencing (38 genes (QIAGEN), NextSeq 500 platform (Illumina)). Genetic and histopathological analyses were done blinded to other data. RESULTS: In 100 patients, 224 polyps were removed. Significant associations of genetic alterations with endoscopic or histological polyp characteristics were observed for BRAF, KRAS, TCF7L2, FBXW7 and CTNNB1 mutations. Multivariate analysis revealed that polyps ≥ 10 mm have a significant higher relative risk for harbouring oncogene mutations (relative risk 3.467 (1.742-6.933)). Adenomas and right-sided polyps are independent risk factors for CTNNB1 mutations (relative risk 18.559 (2.371-145.245) and 12.987 (1.637-100.00)). CONCLUSIONS: Assessment of the mutational landscape of polyps can be integrated in the workflow of current colonoscopy practice. There are distinct genetic patterns related to polyp size and location. These results suffice to optimise individual risk calculation and may help to better define surveillance intervals.

Increased PDGFR-beta and VEGFR-2 protein levels are associated with resistance to platinum-based chemotherapy and adverse outcome of ovarian cancer patients.

Despite frequent initial response rates of epithelial ovarian cancer to platinum-based chemotherapy the majority of patients develop drug resistance. Our aim was to evaluate differential expression of signaling-pathway proteins in platinum-sensitive versus platinum-resistant primary epithelial ovarian cancer specimens to identify predictive biomarkers for treatment response. 192 patients were studied comprising of independent training (n = 89) and validation (n = 103) cohorts. Full-length proteins were extracted from paraffin-embedded samples including multiple regions per tumor to account for intratumoral heterogeneity. Quantitative reverse-phase-protein-arrays were used to analyze protein and phospho-protein levels of 41 signaling molecules including growth-factor receptors, AKT and MAPK signaling pathways as well as angiogenesis and cell-adhesion. Platinum-resistant ovarian cancers (56/192) demonstrated significantly higher intratumoral levels of the angiogenesis-associated growth-factor receptors PDGFR-beta and VEGFR2 compared to platinum-sensitive tumors. In addition, patients with high PDGFR-beta expression had significantly shorter overall and progression-free survival (HR 3.6 and 2.4; p < 0.001). The prognostic value of PDGFR-beta and VEGFR2 was confirmed in publicly available microarray-datasets. High intratumoral levels of the angiogenesis-related growth-factor receptors PDGFR-beta and VEGFR2 might serve as novel predictive biomarkers to identify primary resistance to platinum-based chemotherapy. Those ovarian cancer patients might particularly benefit from additional anti-vascular therapy including anti-VEGF antibody or receptor tyrosine-kinase-inhibitor therapy.

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer.

Autophagy is a cellular degrading process that promotes tumor cell survival or cell death in cancer, depending on the progress of oncogenesis. Protein light chain 3 (LC3) and p62/SQSTM1 (p62) are associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation. We studied LC3 and p62 expression using immunohistochemistry in a large cohort of 466 stage I/II non-small cell lung cancer (NSCLC) using a tissue microarray. We evaluated dot-like cytoplasmic expression of LC3 and dot-like, cytoplasmic and nuclear staining for p62 in relation to clinico-pathological parameters.LC3 expression correlated with all p62 patterns, as those correlated among each other (p < 0.001 each). There was no correlation with stage, age or gender. A combination of high LC3/high p62 dot-like staining (suggesting impaired autophagy) showed a trend for better outcome (p = 0.11). Interestingly, a combined low cytoplasmic/low nuclear p62 expression regardless of dot-like staining was an independent prognostic factor for longer survival (p = 0.006; HR=1.96), in addition to tumor stage (p = 0.004; HR=1.4).The autophagy markers LC3 and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role. High LC3 levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior.

Recent progress in protein profiling of clinical tissues for next-generation molecular diagnostics.

Differences in protein levels and the activation of signaling pathways have been extensively studied in tumor tissues, but the implementation of protein profiling methods in routine hospital workflows lags far behind that of nucleic acid-based approaches. In this review, major technologies that are currently used for measuring protein abundances in human tissues are highlighted, and for each method several examples are provided. We differentiate between extract-based and section-based methods that are each further divided into targeted- and discovery-based approaches (i.e., when the proteins to be analyzed are known or for finding promising new biomarker candidates, respectively). Current problems in protein profiling are addressed and ways in which protein profiling can successfully be implemented in routine clinical workflows are shown.

Quantitative and integrated proteome and microRNA analysis of endothelial replicative senescence.

Age-related changes in vascular functioning are a harbinger of cardiovascular disease but the biological mechanisms during the progression of endothelial senescence have not been studied. We investigated alterations in the proteome and miRNA profiles in the course of replicative senescence using primary human umbilical vein endothelial cells as an in vitro vascular model. Quantitative proteomic profiling from early growth stage to senescence was performed by isotope-coded protein label coupled to LC-ESI-MS/MS analysis. Some proteins consistently changed their expression during the senescence whereas others appeared as deregulated only during the late senescence. The latter was accompanied by alterations in morphology of senescent endothelial cells. MicroRNA expression profiling revealed transient changes in the level of miR-16-5p, miR-28-3p and miR-886-5p in the early senescence, decrease in the level of miR-106b-3p at the late stage, and continuous changes in the expression of miR-181a-5p and miR-376a-3p during the whole senescence process. Integrating data on proteomic and microRNA changes indicated potential crosstalk between specific proteins and non-coding RNAs in the regulation of metabolism, cell cycle progression and cytoskeletal organization in the endothelial senescence. The knowledge of molecular targets that change during the senescence can ultimately contribute to a better understanding and prevention of age-related vascular diseases.