Multi-colour FISH in oesophageal adenocarcinoma-predictors of prognosis independent of stage and grade.

BACKGROUND Oesophageal adenocarcinoma or Barrett's adenocarcinoma (EAC) is increasing in incidence and stratification of prognosis might improve disease management. Multi-colour fluorescence in situ hybridisation (FISH) investigating ERBB2, MYC, CDKN2A and ZNF217 has recently shown promising results for the diagnosis of dysplasia and cancer using cytological samples. METHODS To identify markers of prognosis we targeted four selected gene loci using multi-colour FISH applied to a tissue microarray containing 130 EAC samples. Prognostic predictors (P1, P2, P3) based on genomic copy numbers of the four loci were statistically assessed to stratify patients according to overall survival in combination with clinical data. RESULTS The best stratification into favourable and unfavourable prognoses was shown by P1, percentage of cells with less than two ZNF217 signals; P2, percentage of cells with fewer ERBB2- than ZNF217 signals; and P3, overall ratio of ERBB2-/ZNF217 signals. Median survival times for P1 were 32 vs 73 months, 28 vs 73 months for P2; and 27 vs 65 months for P3. Regarding each tumour grade P2 subdivided patients into distinct prognostic groups independently within each grade, with different median survival times of at least 35 months. CONCLUSIONS Cell signal number of the ERBB2 and ZNF217 loci showed independence from tumour stage and differentiation grade. The prognostic value of multi-colour FISH-assays is applicable to EAC and is superior to single markers.

Prognostic value of histopathological regression in 850 neoadjuvantly treated oesophagogastric adenocarcinomas.

BACKGROUND Recently, histopathological tumour regression, prevalence of signet ring cells, and localisation were reported as prognostic factors in neoadjuvantly treated oesophagogastric (junctional and gastric) cancer. This exploratory retrospective study analyses independent prognostic factors within a large patient cohort after preoperative chemotherapy including clinical and histopathological factors. METHODS In all, 850 patients presenting with oesophagogastric cancer staged cT3/4 Nany cM0/x were treated with neoadjuvant chemotherapy followed by resection in two academic centres. Patient data were documented in a prospective database and retrospectively analysed. RESULTS Of all factors prognostic on univariate analysis, only clinical response, complications, ypTNM stage, and R category were independently prognostic (P<0.01) on multivariate analysis. Tumour localisation and signet ring cells were independently prognostic only when investigator-dependent clinical response evaluation was excluded from the multivariate model. Histopathological tumour regression correlates with tumour grading, Laurén classification, clinical response, ypT, ypN, and R categories but was not identified as an independent prognostic factor. Within R0-resected patients only surgical complications and ypTNM stage were independent prognostic factors. CONCLUSIONS Only established prognostic factors like ypTNM stage, R category, and complications were identified as independent prognostic factors in resected patients after neoadjuvant chemotherapy. In contrast, histopathological tumour regression was not found as an independent prognostic marker.

„chöid ir mir säge was diir hüt anneheit?“ – die Hürden bei der Entwicklung eines Sprachkompetenz-Indikators

Ein soziolinguistisch angelegtes Projekt der Universität Bern greift die Idee der „language related major life events“ – die neue sprachliche und soziale Räume schaffen – auf. Das Forschungsprojekt besteht aus drei Modulen. Module 1 und 2 untersuchen Personen, die beim Übergang von der Grundausbildung in eine weiterführende Ausbildung mit einem Wechsel der Umgebungssprache konfrontiert sind: und zwar (1) frankofone Lernende in Biel/Bienne, die – im Kontext dieser zweisprachigen Stadt – neben ihrer Herkunftssprache mit Schweizerdeutsch konfrontiert sind, und (2) frankofone und italofone Studierende, die an Universitäten und Hochschulen der deutschen Schweiz studieren müssen, weil es das betreffende Fach weder in der Romandie noch im Tessin gibt. Im Modul 3 werden zwei Personengruppen untersucht (italienische MigrantInnen und Deutsch-schweizerInnen), die sich vor, im und nach dem Prozess der Pensionierung befinden, die bestehende berufliche Netzwerke verlieren, eventuell neue aufbauen und denen sich neue kommunikative Anforderungen stellen. Im Rahmen dieses Forschungsprojekts wurden wir mit der Frage konfrontiert, wie die Sprachkenntnisse erfasst und analysiert werden könnten. Die erste Hürde bestand darin, dass wir als Ausgangslage nur ein bis zwei Interviews in der Muttersprache der Probanden (Italienisch oder Französisch) führen würden und nicht klar war, wie wir in diesem Kontext zu zuverlässigen Sprachdaten auf Schweizerdeutsch kommen: - Sollten wir selbst oder eine zweite Person die Fragen stellen? oder - Wie standardisieren wir den Fragenkatalog? Nach langer Suche nach einem geeigneten Testinstrument entschieden wir uns eine eigene Methode zu entwickeln die auf SOPI/OPI basiert. Auch hier stellten sich mehrere Fragen, wie zum Beispiel: - Welchen Schweizerdeutsch Dialekt wird verwendet? - Welche Fragen stellen wir (unterschiedliche Probandengruppen)? - Wie stellen wir einen wachsenden Schwierigkeitsgrad her? Während der Durchführung der Tests begegneten wir neuen Problematiken. Aufgrund der negativ konnotierten Testsituation fühlten sich einzelne Probanden z.B. angegriffen. Weiter wurden wir mit dem Beobachterparadoxon konfrontiert und konnten bei wiederholten Tests Gewöhnungseffekte feststellen. Zusätzlich erwiesen sich einige Fragen als problematisch (z.B. in diesem Kontext nicht sinnvoll oder verschieden interpretierbar). Am Ende werden wir mit den verschieden Problemen der Analyse konfrontiert sein. Wir fragen uns aufgrund welcher Kriterien eine Analyse sinnvoll ist und ob unsere Daten überhaupt vergleichbar sind. In unserem Paper möchten wir die oben genannten methodischen Probleme darstellen und unsere Lösungsansätze diskutieren.

Optimal lange Fixationen? Eine experimentelle Studie zu überlangen "QuietEye"-Dauern

Einleitung: Im Zusammenhang mit der Leistungsdienlichkeit langer finaler Fixation (quiet eye, QE) wird vermutet, dass Leistungsverbesserungen nur für eine optimale Dauer zu beobachten sein sollten, also auch bei „überlangen“ QE-Dauern die Leistung wieder abnimmt (u.a. Janelle et al., 2000; Klostermann, 2014). Jedoch liegen zu dieser Vermutung bislang keine empirischen Befunde vor, so dass in der hier präsentierten Studie Präzisionsleistung in einer Wurfaufgabe in Abhängigkeit von (auch) sehr langen experimentell kontrollierten QE-Dauern untersucht wurde. Methode: In einem Within-subject-Design hatten 20 Sportstudierende unter acht verschiedenen QE-Bedingungen (Onset in 400-ms-Schritten von -3200 ms bis -400 ms vor Bewegungsbeginn; 16 Versuche pro Bedingung in randomisierter Abfolge) mit retro-reflektierenden Bällen auf eine Großleinwand projizierte Zielscheiben möglichst mittig zu treffen. Die QE-Manipulation erfolgte über eine an den Bewegungsbeginn gebundene Zielscheibeneinblendung samt Wurfrhythmisierung durch Tonvorgaben. Aus den mit einem Vicon-T20-System (200 Hz) sowie einem integrierten mobilen Eyetracker (EyeSeeCam, 220 Hz) erhobenen Daten wurden die QE-Dauer (ms) und die Wurfleistung (radialer Fehler, mm) als abhängige Variablen berechnet und varianzanalytisch auf Unterschiede untersucht. Resultate und Diskussion: Für die QE-Dauer wurde ein signifikanter Haupteffekt gefunden, F(7, 133) = 38.4, p < .01, ηp2 = .67, mit zumindest tendenziellen (-2000 ms vs. -2400 ms, -2800 ms vs. -3200 ms), größtenteils aber signifikanten QE-Anstiegen gemäß der experimentellen Manipulation (alle ps < .01), obgleich die jeweils angezielten QE-Dauern nicht erreicht und zum Teil deutlich unterschritten wurden (tatsächliche relativ zur angezielten Dauer im Mittel 59.95 %). Für den radialen Fehler ergab sich ein signifikanter Haupteffekt, F(7, 133) = 8.5, p < .01, ηp2 = .31, welcher durch signifikant schlechtere Leistungen bei den Onsets -400 ms und -800 ms gegenüber allen anderen Onsets erklärt wird (alle ps < .05; ausgenommen -400 ms vs. -800 ms und -800 ms vs. -2400 ms). Somit wurde der „klassische“ QE-Effekt schlechterer Leistungen infolge kurzer QE-Dauern repliziert; die Vermutung einer Leistungsverschlechterung bei überlangen QE-Dauern konnte jedoch – zumindest unter den infolge der Manipulation tatsächlich erzielten Werten – nicht untermauert werden. Literatur: Klostermann, A. (2014). Finale Fixationen, sportmotorische Leistung und eine Inhibitionshypothese: Mechanismen des „Quiet Eye“, Sportwissenschaft, 44, 49-59. Janelle, C. M., Hillman, C. H., Apparies, R. J., Murray, N. P., Meili, L., Fallon, E. A. & Hatfield, B. D. (2000). Expertise differences in cortical activation and gaze behavior during rifle shooting. Journal of Sport & Exercise Psychology, 22, 167-182.

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.

Explaining car drivers' intention to prevent road-traffic noise: An application of the Norm Activation Model

Strengthening car drivers’ intention to prevent road-traffic noise is a first step toward noise abatement through voluntary change of behavior. We analyzed predictors of this intention based on the norm activation model (i.e., personal norm, problem awareness, awareness of consequences, social norm, and value orientations). Moreover, we studied the effects of noise exposure, noise sensitivity, and noise annoyance on problem awareness. Data came from 1,002 car drivers who participated in a two-wave longitudinal survey over 4 months. Personal norm had a large prospective effect on intention, even when the previous level of intention was controlled for, and mediated the effect of all other variables on intention. Almost 60% of variance in personal norm was explained by problem awareness, social norm, and biospheric value orientation. The effects of noise sensitivity and noise exposure on problem awareness were small and mediated by noise annoyance. We propose four communication strategies for strengthening the intention to prevent road-traffic noise in car drivers.

K-t GRAPPA-accelerated 4D flow MRI of liver hemodynamics: influence of different acceleration factors on qualitative and quantitative assessment of blood flow.

OBJECTIVE We sought to evaluate the feasibility of k-t parallel imaging for accelerated 4D flow MRI in the hepatic vascular system by investigating the impact of different acceleration factors. MATERIALS AND METHODS k-t GRAPPA accelerated 4D flow MRI of the liver vasculature was evaluated in 16 healthy volunteers at 3T with acceleration factors R = 3, R = 5, and R = 8 (2.0 × 2.5 × 2.4 mm(3), TR = 82 ms), and R = 5 (TR = 41 ms); GRAPPA R = 2 was used as the reference standard. Qualitative flow analysis included grading of 3D streamlines and time-resolved particle traces. Quantitative evaluation assessed velocities, net flow, and wall shear stress (WSS). RESULTS Significant scan time savings were realized for all acceleration factors compared to standard GRAPPA R = 2 (21-71 %) (p < 0.001). Quantification of velocities and net flow offered similar results between k-t GRAPPA R = 3 and R = 5 compared to standard GRAPPA R = 2. Significantly increased leakage artifacts and noise were seen between standard GRAPPA R = 2 and k-t GRAPPA R = 8 (p < 0.001) with significant underestimation of peak velocities and WSS of up to 31 % in the hepatic arterial system (p <0.05). WSS was significantly underestimated up to 13 % in all vessels of the portal venous system for k-t GRAPPA R = 5, while significantly higher values were observed for the same acceleration with higher temporal resolution in two veins (p < 0.05). CONCLUSION k-t acceleration of 4D flow MRI is feasible for liver hemodynamic assessment with acceleration factors R = 3 and R = 5 resulting in a scan time reduction of at least 40 % with similar quantitation of liver hemodynamics compared with GRAPPA R = 2.

Tiefengrundwasser – Vorkommen, Nutzungspotenzial und Schutzwürdigkeit

Tiefengrundwasser findet sich in allen grosstektonischen Einheiten der Schweiz. Die bekannten, teilweise genutzten Vorkommen von Tiefengrundwas-ser und insbesondere die chemische Zusammensetzung dieser Wässer wird für die verschiedenen Einheiten aufgezeigt. Im Hinblick auf mögliche Nutzungskon-flikte werden die gesetzlichen Grundlagen und Massnahmen zum Schutz dieser Ressource diskutiert.

Mutations in SLC1A4, encoding the brain serine transporter, are associated with developmental delay, microcephaly and hypomyelination

BACKGROUND L-serine plays an essential role in neuronal development and function. Although a non-essential amino acid, L-serine must be synthesised within the brain because of its poor permeability by the blood-brain barrier. Within the brain, its synthesis is confined to astrocytes, and its shuttle to neuronal cells is performed by a dedicated neutral amino acid transporter, ASCT1. METHODS AND RESULTS Using exome analysis we identified the recessive mutations, p.E256K, p.L315fs, and p.R457W, in SLC1A4, the gene encoding ASCT1, in patients with developmental delay, microcephaly and hypomyelination; seizure disorder was variably present. When expressed in a heterologous system, the mutations did not affect the protein level at the plasma membrane but abolished or markedly reduced L-serine transport for p.R457W and p.E256K mutations, respectively. Interestingly, p.E256K mutation displayed a lower L-serine and alanine affinity but the same substrate selectivity as wild-type ASCT1. CONCLUSIONS The clinical phenotype of ASCT1 deficiency is reminiscent of defects in L-serine biosynthesis. The data underscore that ASCT1 is essential in brain serine transport. The SLC1A4 p.E256K mutation has a carrier frequency of 0.7% in the Ashkenazi-Jewish population and should be added to the carrier screening panel in this community.

Tumour border configuration in colorectal cancer: proposal for an alternative scoring system based on the percentage of infiltrating margin.

AIMS Information on tumour border configuration (TBC) in colorectal cancer (CRC) is currently not included in most pathology reports, owing to lack of reproducibility and/or established evaluation systems. The aim of this study was to investigate whether an alternative scoring system based on the percentage of the infiltrating component may represent a reliable method for assessing TBC. METHODS AND RESULTS Two hundred and fifteen CRCs with complete clinicopathological data were evaluated by two independent observers, both 'traditionally' by assigning the tumours into pushing/infiltrating/mixed categories, and alternatively by scoring the percentage of infiltrating margin. With the pushing/infiltrating/mixed pattern method, interobserver agreement (IOA) was moderate (κ = 0.58), whereas with the percentage of infiltrating margins method, IOA was excellent (intraclass correlation coefficient of 0.86). A higher percentage of infiltrating margin correlated with adverse features such as higher grade (P = 0.0025), higher pT (P = 0.0007), pN (P = 0.0001) and pM classification (P = 0.0063), high-grade tumour budding (P < 0.0001), lymphatic invasion (P < 0.0001), vascular invasion (P = 0.0032), and shorter survival (P = 0.0008), and was significantly associated with an increased probability of lymph node metastasis (P < 0.001). CONCLUSIONS Information on TBC gives additional prognostic value to pathology reports on CRC. The novel proposed scoring system, by using the percentage of infiltrating margin, outperforms the 'traditional' way of reporting TBC. Additionally, it is reproducible and simple to apply, and can therefore be easily integrated into daily diagnostic practice.

Epstein-barr virus in gastro-esophageal adenocarcinomas - single center experiences in the context of current literature.

Epstein-Barr virus (EBV)-associated gastric carcinomas (GC) represent a distinct and well-recognized subtype of gastric cancer with a prevalence of around 10% of all GC. In contrast, EBV has not been reported to play a major role in esophageal adenocarcinomas (EAC) and adenocarcinomas of the gastro-esophageal junction (GEJ). We report our experiences on EBV in collections of gastro-esophageal adenocarcinomas from two surgical centers and discuss the current state of research in this field. Tumor samples from 465 primary resected gastro-esophageal adenocarcinomas (118 EAC, 73 GEJ, and 274 GC) were investigated. Presence of EBV was determined by EBV-encoded small RNAs (EBER) in situ hybridization. Results were correlated with pathologic parameters (UICC pTNM category, Her2 status, tumor grading) and survival. EBER positivity was observed in 14 cases. None of the EAC were positive for EBER. In contrast, we observed EBER positivity in 2/73 adenocarcinomas of the GEJ (2.7%) and 12/274 GC (4.4%). These were of intestinal type (seven cases) or unclassifiable (six cases), while only one case was of diffuse type according to the Lauren classification. No association between EBV and pT, pN, or tumor grading was found, neither was there a correlation with clinical outcome. None of the EBER positive cases were Her2 positive. In conclusion, EBV does not seem to play a role in the carcinogenesis of EAC. Moreover, adenocarcinomas of the GEJ show lower rates of EBV positivity compared to GC. Our data only partially correlate with previous reports from the literature. This highlights the need for further research on this distinct entity. Recent reports, however, have identified specific epigenetic and genetic alterations in EBV-associated GC, which might lead to a distinct treatment approach for this specific subtype of GC in the future.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.