PDF file - 54K, Antibodies used for Array and Western Blot Immunostaining

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.

PDF file - 103K, Validation of the reproducibility of the RPMA for HER2 and pHER2 analysis from FFPE-tissues

Supplementary Figure 1 from Snail and Slug Play Distinct Roles during Breast Carcinoma Progression

Supplementary Figures 2-3 from Snail and Slug Play Distinct Roles during Breast Carcinoma Progression

Supplementary Tables S1-S3 from Enhanced Activation of Epidermal Growth Factor Receptor Caused by Tumor-Derived E-Cadherin Mutations

Supplemental Table 1: Comparison of epidemiological risk factors in Barrett patients (n= 587) and population controls (n= 587) matched by gender and age

Supplementary Results, Figure 1, Methods and Materials from Enhanced Activation of Epidermal Growth Factor Receptor Caused by Tumor-Derived E-Cadherin Mutations

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 ( κ F = 0.72–0.75) and poor for LGD and HGD ( κ F = 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.

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.

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.

The constant drive to further miniaturization and heterogeneous system integration leads to a need for new packaging technologies that also allow large area processing and 3D integration with strong potential for low cost applications. Here, Fan-Out Wafer Level Packaging [FOWLP] is one of the latest packaging trends in microelectronics. The technology can be also used for multi-chip packages or System in Package (SiP). 3D integration is typically done by package on package (PoP) stacking where the electrical 3D routing is done by through mold (TMV) or through package vias (TPV) and a redistribution layer on both sides of the FOWLP. In summary the paper will give a review of the different technology approaches for through mold vias in a Fan-out Wafer or Panel Level Package.

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.

4018 Background: We evaluated a PET-guided treatment stratification for improvement in obtaining negative surgical margins (R0) in resectable gastroesophageal junction (GEJ) adenocarcinoma. According to sequential 18F-FDG PET, only 40–50% of patients (pts) respond to neoadjuvant chemotherapy (CTX). Early PET non-responders (P-NR) after induction CTX might benefit from changing to chemoradiation (CRT). Methods: 75 pts with resectable GEJ adenocarcinomas were enrolled in this interventional, prospective, non-randomized multicenter trial. Pts underwent baseline 18F-FDG PET scan followed by 1 cycle of CTX (physicians’ choice, e.g. EOX, XP, mFOLFOX6). PET was repeated at day 14-21 and responders (P-R), defined as ≥ 35% decrease in SUVmax from baseline, continued with CTX. P-NR switched to CRT (41.4 Gy/23 fractions with weekly carboplatin/paclitaxel). Pts underwent surgery 4-6 weeks post-CTX/CRT. Primary objective was an improvement of R0 resection rates in P-NR above a proportion of 70% based on results from the MUNICON1/2 trials. Secondary endpoints include disease-free survival (DFS), overall survival (OS), measured from randomization to death from any cause, and translational endpoints. Results: Between 12/2014 and 07/ 2018 160 pts with resectable GEJ adenocarcinomas were prospectively screened with PET in three German university centers. Overall, 85 pts (53%) could not be included due to previously undetectable metastases (40/25%), no or too low FDG uptake of the primary tumor (21/13%), other reasons (24/15%). 75 eligible pts were enrolled in the study and 69 were evaluable. Based on PET criteria, 47 (68%) and 22 (32%) were P-R and P-NR, respectively. R0 resection rates were 94% (44/47) for P-R and 91% (20/22) for P-NR. Pathologic complete remission (pCR; < 10% vital tumor cells), was 33% (15/46) in P-R and 55% (12/22) in P-NR. With a median follow-up time of 19 months (mo), estimated 18 mo DFS was 71%/61% for P-R/P-NR, respectively. Observed median 18 mo OS was 95% for P-R and 75% for P-NR. Conclusions: Alternative CRT for GEJ adenocarcinoma improved R0- and pCR rates among pts who were P-NR after induction CTX. PET response was prognostic for a prolonged OS and DFS. Clinical trial information: 2014-000860-16.

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.

Abstract 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.

Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.