Virtual Microscopy New, Old, Out, Or Just A Part Of Education

Introduction/ Background More than six years ago – accompanying the start of the new study program “Modellstudiengang” – we began a virtual microscopy program for our students. This started with slides to accompany the course to use during the lesson and for review of the slides at home (or in the library). But we wanted to go further. How far have we come?   Aims Our aim was to provide our students with more benefi- cial information and to increase the amount of material available - in the form of slides and accompanying exercises. A secondary goal was streamlining the education of our students (fewer slides to manage and better opportuni- ties for students to prepare for lessons).   Methods The slides were scanned using NanoZoomer 2.0-HT slide scanner. The virtual slides were made available to students using Slidebox system (version 4.4.3.) in three different ways:We provided annotated slides with healthy (physio- logical) and diseased (pathological) samples to ac- company the entire course. Some of the slides were for use during lessons, others as supplementary material. • The virtual slides were integrated into a new style of lecture (“blended learning”) mixing learning opportu- nities from case reports, including clinical information; radiological images; and virtual slides. To review acquired knowledge memory-quizzes were used. • We complement the cases seen in the practical course “Autopsy – How, why, to what end?” with histology. Thus students are able to have a complete overview of the case from clinical history, to macroscopic findings and their correlation with the microscopic findings, to the final report.   Results Usually the students were suprised, when they first come in contact with the virtual microscopy. But the Initial suprise yields to experimentation and getting used to it.   Virtual microscopy is not only just a part, but an import- ant part of our education.

Immunomodulatory molecules in renal cell cancer: CD80 and CD86 are expressed on tumor cells

Despite modern therapies with tyrosine kinase inhibitors (TKI), the management of patients with metastatic renal cell carcinoma (mRCC) remains a challenge. Significant immunosuppression has been described in patients with mRCC. Therefore, immunotherapeutic strategies such as checkpoint inhibitors have been developed. To further elucidate the underlying mechanisms of immunosuppression and response by therapy, different features of the immune microenvironment (expression of HIF-1-{alpha}, VEGFR-1, FOXP3, TGF-{beta}1, CD80, CD86, PD-1, and PD-L1) were analyzed in tumor tissues within different subgroups of mRCC patients (responders vs. non-responders to therapy). Results: The most interesting finding was low level CD80 and CD86-expression on tumor tissue samples (n = 18) of nearly all mRCC patients. This finding was in line with CD86 expression, which could also be found in renal carcinoma cell lines. To the best of our knowledge, this is the first report on CD820/CD86 expression in human renal cell carcinoma-possibility reflecting an immunomodulatory mechanism of the tumor

Dynamics of the intratumoral immune response during progression of high-grade serous ovarian cancer

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC. EXPERIMENTAL DESIGN: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project. RESULTS: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; P<.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012). CONCLUSIONS: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics

Molecular subtyping of head and neck cancer - Clinical applicability and correlations with morphological characteristics

AIM: We aimed to evaluate the applicability of a customized NanoString panel for molecular subtyping of recurrent or metastatic head and neck squamous cell carcinoma (R/M-HNSCC). Additionally, histological analyses were conducted, correlated with the molecular subtypes and tested for their prognostic value. MATERIAL AND METHODS: We conducted molecular subtyping of R/M-HNSCC according to the molecular subtypes defined by Keck et al. For molecular analyses a 231 gene customized NanoString panel (the most accurately subtype defining genes, based on previous analyses) was applied to tumor samples from R/M-HNSCC patients that were treated in the CeFCiD trial (AIO/IAG-KHT trial 1108). A total of 130 samples from 95 patients were available for sequencing, of which 80 samples from 67 patients passed quality controls and were included in histological analyses. H&E stained slides were evaluated regarding distinct morphological patterns (e.g. tumor budding, nuclear size, stroma content). RESULTS: Determination of molecular subtypes led to classification of tumor samples as basal (n = 46, 45 %), inflamed/mesenchymal (n = 31, 30 %) and classical (n = 26, 25 %). Expression levels of Amphiregulin (AREG) were significantly higher for the basal and classical subtypes compared to the mesenchymal subtype. While molecular subtypes did not have an impact on survival, high levels of tumor budding were associated with poor outcomes. No correlation was found between molecular subtypes and histological characteristics. CONCLUSIONS: Utilizing the 231-gene NanoString panel we were able to determine the molecular subtype of R/M-HNSCC samples by the use of FFPE material. The value to stratify for different treatment options remains to be explored in the future. The prognostic value of tumor budding was underscored in this clinically well annotated cohort

Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid dendritic cell neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare haematopoietic malignancy characterized by dismal prognosis and overall poor therapeutic response. Since the biology of BPDCN is barely understood, our study aims to shed light on the genetic make-up of these highly malignant tumors. Using targeted high-coverage massive parallel sequencing, we investigated 50 common cancer genes in 33 BPDCN samples. We detected point mutations in NRAS (27.3% of cases), ATM (21.2%), MET, KRAS, IDH2, KIT (9.1% each), APC and RB1 (6.1% each), as well as in VHL, BRAF, MLH1, TP53 and RET (3% each). Moreover, NRAS, KRAS and ATM mutations were found to be mutually exclusive and we observed recurrent mutations in NRAS, IDH2, APC and ATM. CDKN2A deletions were detected in 27.3% of the cases followed by deletions of RB1 (9.1%), PTEN and TP53 (3% each). The mutual exclusive distribution of some mutations may point to different subgroups of BPDCN whose biological significance remains to be explored

Better prognosis of gastric cancer patients with high levels of tumor infiltrating lymphocytes is counteracted by PD-1 expression

The prognostic potential of anti-tumor immune responses is becoming increasingly important in adenocarcinoma of the gastroesophageal junction and stomach (AGE/S) especially regarding the use of immune checkpoint inhibitors. This study analyzes for the first time the prognostic impact of tumor-infiltrating lymphocytes (TILs) and checkpoint inhibitors in a large Caucasian cohort in patients with AGE/S. We screened tissue samples from 438 therapy-naïve patients with AGE/S undergoing surgery between 1992 and 2005, examined in a tissue microarray (TMA) and stained against human CD3, CD4, CD8, PD-1, and PD-L1. Out of 438 tissue samples, 210 were eligible for multivariate analysis. This revealed that high infiltration with CD3(+), CD4(+), or CD8(+) TILs was associated with an increased overall survival in AGE/S patients, which could only be confirmed in multivariate analysis for CD3 (HR: 0.326; p = .023). Independent improved survival was limited to gastric cancer patients and to early tumor stages as long as TILs did not express PD-1 (HR: 1.522; p = .021). Subgroup analyses indicate that TIL-dependent anti-tumor immune response is only effective in gastric cancer patients in early stages of disease in PD-1 negative TILs. Combined analysis of PD-1 and CD3 could serve as a prognostic marker for the clinical outcome of gastric cancer patients and could also be of interest for immunotherapy

B-cell receptor-driven MALT1 activity regulates MYC signaling in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients

Atypische Lage eines B-Zell Lymphoms am peripheren Nerven mit perineuralem Wachstum bei Lymphadenitis colli

Primäre Lymphome des zentralen Nervensystems, stellen etwa 1-3% aller primären Hirntumore dar. Eine Neurolymphomatose des peripheren Nervensystems durch Infiltration von Lymphomen ist eine Seltenheit. Wenn eine Beteiligung vorliegt ist diese in der Regel sekundär bedingt. Bislang wurde von sehr wenigen Fällen einer Erstmanifestation eines B-Zell Lymphoms im peripheren Nervensystem berichtet. In den meisten Fällen jedoch zeigt sich eine zeitgleiche Beteiligung des zentralen Nervensystems mit positiver Liquor Analyse, leptomeningealer Beteiligung, Infiltration von Nervenwurzeln, den Plexus oder der Hirnnerven. Dies könnte darauf hindeuten, dass in diesen Fällen die Infiltration des peripheren Nervensystems sekundär bei primärer Manifestation des zentralen Nervensystems erfolgte. Wir berichten über einen 57-jährigen Patienten, welcher sich mit einer asymptomatischen Lymphadenitis colli links, als Zufallsbefund, ohne nervale Ausfälle oder B-Symptome vorstellte. Bildmorphologisch zeigte sich im MRT des Halses eine langstreckige, glattbegrenzte atypisch enhancende Raumforderung links zervikal. Eine anatomische Zuordnung war hierdurch, als auch sonographisch nicht möglich. Intraoperativ zeigte sich an den aus dem Erb'schen Punkt entspringenden sensiblen Nerven eine ummantelnde weiche und helle Raumforderung Nach histologischer und immunhistochemischer Aufarbeitung der Biopsie des N. transversus zeigte sich ein CD-30 exprimierendes diffus großzelliges B-Zell-Lymphom. Der Patient wurde in der Klinik für Onkologie mit einer Chemotherapie nach dem R-CHOP Schema behandelt. In der aktuellen Literatur liegen nach unseren Recherchen bisher keine Fallbeschreibungen eines isolierten Befalls eines peripheren, sensiblen Nervens vor.Der Erstautor gibt keinen Interessenkonflikt an

Untersuchungen zur Expression von CD168 (RHAMM) bei Kopf-Hals-Karzinomen

Einleitung: Bislang konnte bei Kopf-Hals-Karzinomen kein Biomarker zum Screening oder zur Verlaufsbeurteilung etabliert werden. In dieser Studie untersuchten wir den bereits bei colorectalen Malignomen, malignen Gliomen, Blasenkarzinom, Brustkrebs und Endometriumkarzinom bekannten RHAMM Rezeptor in Kopf- Hals Karzinomen. Da der RHAMM Rezeptor eng mit der Migrationsfähigkeit des Tumors korreliert, müsste eine Aussage zur Metastasierungswahrscheinlichkeit gemacht werden können.Methoden: 60 Patienten unserer Nachsorgesprechstunde haben wir nach der Tumorlokalisation (Orophaynx, Hypopharynx und Larynx) getrennt beurteilt. Anamnese, Ausdehnung des Primärtumors und Metastasen-Status wurden mit Vorhandensein von CD168 im histologischen Schnitt des Tumors bei Erstdiagnose verglichen.Ergebnisse: In den ersten Ergebnissen konnten wir keine Korrelation feststellen. Allerdings wurde hierbei das Vorhandensein des extrakapsulären Wachstums der cervikalen Lymphknotenmetastasen nicht mit in die Untersuchung einbezogen.Schlussfolgerungen: Auf dem ersten Blick scheint die Expression von CD168 nicht mit der Tumorausdehnung und dem Metastasierungsverhalten zu korrelieren. Weitere detailiertere Untersuchungen sind nötig um eine mögliche Anwendung von CD168 als Biomarker zu evaluieren.Der Erstautor gibt keinen Interessenkonflikt an