96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

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(i.e. investment in equipment and expertise). Here we present simple pixel-algorithms that allow electronic merging and color-range conversion of traditional IHC stains. Methods. Single-label IHC-stains, performed on subsequent sections, are digitized and used as input data. After manual positioning, a merge algorithm compares the input images and selects pixels with higher values. A re-coloring algorithm reassigns color ranges. Algorithms are achieved via a customized link between software platforms (Aperio ImageScope10.0; Adobe PhotoshopCS3; ImageJ Version10.2) using AutoIT (v3.2.12.0), a freeware scripting language for automating the Microsoft Windows GUI. Results. The merge algorithm emulates double staining, which includes preservation of the traditional IHC-views (operative comfort zone). To allow distinction of similarly labeled stains in the merge, a re-coloring algorithm converts the color range of stained elements from each image (e.g. brown vs. red). As a specific re-coloring mode, stained elements can be extracted and converted into pseudo-immunofluorescence (IF), which includes all downstream assessments of co-localized elements (virtual IF-stain). The algorithms solve the common problem of species identity of primary antibodies because they allow co-visualization of markers without compromising staining specificity, while at the same time employing individual stains that remain available for traditional evaluation. When compared to investments in IF-equipment or validation of physical double-stains, the effort to learn and perform the conversion algorithms is negligible. Conclusions. The presented imaging tools emulate the principal advantages of multi-color fluorescence microscopy as well as multi-color IHC. These techniques represent a powerful expansion of one of the most versatile molecular tools in diagnostic pathology. Thereby, the combination of established methods (IHC) and imaging algorithms also exemplifies the potential of digital pathology. Translationale Forschung und AG Molekularpathologie FR-027 Delay to preservation does not induce a systematic phosphoprotein response during tissue processing S . Gündisch1 , C . Schott1 , K . Grundner-Culemann2 , M . Machatti2 , D . Groelz3 , C . Schaab2 , A . Tebbe 2 , K .-F . Becker1 1Technische Universität München, Institute of Pathology, München, 2 3 Evotec AG, Martinsried, PreAnalytiX GmbH, Hombrechtikon, Switzerland Aims. The quality of tissue samples can have a significant impact on analytical data sets for biomarker research. In particular, posttranslational modifications such as phosphorylation need to be systematically investigated in that phosphorylated protein levels indicate the activation status of signal transduction pathways controlled by kinases. However, little is known about the impact of pre-analytical factors on phosphoprotein stability. The aim of this study was to characterize the potential effects of delayed preservation and different preservation methods on the stability of phosphoproteins using targeted and non-targeted proteomic approaches. Methods. Murine and rat liver samples were exposed to different ischemic conditions before preservation and either cryopreserved, formalinfixed or fixed with the PAXgene Tissue System, a new non-crosslinking formalin-free fixative. The phosphoproteome was analyzed using quantitative tandem mass spectrometry (LC-MS/MS) and reverse phase protein array (RPPA) technology. Results. The phosphoproteomic analysis of ischemic mouse liver tissue samples by LC-MS/MS indicated no significant global alterations of more than 5000 phosphosite ratios analysed during 60 minutes of delayed cryopreservation. The analysis of ischemic rat liver tissue samples by RPPA revealed similar results as investigated phosphoproteins, including phospho-Akt, phospho-p38 MAPK or phospho-p44/42 MAPK, showed very stable profiles during the time-course experiment, independent of the preservation method applied. Conclusions. Since we could not detect significant global changes of the phosphoprotein profiles, neither with a targeted nor a non-targeted approach, we conclude that the phosphoproteome seems to be more stable than expected with regard to delayed preservation. This allows accurate quantitative measurements of the activation state of signalling pathways of tissue samples which had not been immediately preserved. This result is essential for the development of new targeted therapies involving kinase inhibitors which have recently been a focus in the field of personalized medicine. Studies are ongoing to validate our results in human tissue samples as inter-patient variability may occur which is absent in our well controlled model systems. This work has received funding from the Munich Biotech Cluster m4 (www . m4 .de) and the European project SPIDIA (www .spidia .eu) . FR-028 Validation of a novel DNA methylation based 2-gene biomarker panel for early detection of bladder cancer using urine samples M . Rose1 , D . Fiedler1 , N .T . Gaisa1 , C . Schubert 1 , P . Antony1 , R . Davtalab1 , D . Pfister2 , A . Heidenreich2 , R . Knüchel1 , E . Dahl1 1RWTH Aachen University/Institute of Pathology, Aachen, 2RWTH Aachen University/Clinic of Urology, Aachen Aims. The early detection of bladder cancer and its recurrent tumours is currently based on cystoscopy, which is highly sensitive and specific, but also invasive and expensive. Alternative methods still lack suitable sensitivity especially with regard to non-invasive bladder tumours. In the line with accumulating evidence suggesting that DNA methylation pattern could serve as sensitive and specific biomarkers, we aimed to identify novel DNA methylation loci potentially useful for early cancer detection and treatment stratification of bladder cancer patients, respectively. Methods. In order to discover potential biomarkers we analyzed the methylation levels of array-based candidate genes by using MSP in bladder cell lines (n=5), non-cancerous (n=20) and cancerous bladder tissues (n=60). Subsequently, we determined the methylation status of selected genes performing MSP assays on a so called “screening cohort” containing DNA extracted from urine sediments of bladder cancer patients (n=60). Age-matched non-urological-cancer patients (n=30) as well as prostate cancer patients (n=15) were included as controls to ensure specificity. The biomarker potential of the most frequently methylated gene loci were then discriminated by using quantitative pyrosequencing in a “validation urine sample cohort” (currently; n=30) of bladder cancer patients in comparison to controls. The performance of a 2-gene-panel was optimised using receiver operator characteristics (ROC) curve analysis. Results. MSP assays performed on bladder cells lines and bladder cancer tissue revealed novel candidate genes exhibiting frequently cancer specific promoter hypermethylation. In the urine screening samples of bladder cancer patients, these gene loci showed frequent methylation with an overall-panel sensitivity of 81.5%, i.e. 81.5% of samples presented promoter methylation in at least one of the two genes. Importantly, none of the age-matched controls exhibited methylation signals excluding rare and weak age-depended side effects. By using quantitative pyrosequencing technique we were able to increase the sensitivity of the 2-gene panel up to 83.3% (p≤0.001, AUC=0.940) with 100% specificity. Conclusions. We have identified a 2-gene putative biomarker panel based on detection of DNA promoter methylation. Applying this panel in urine sediments using pyrosequencing may provide a highly sensitive and specific, non-invasive approach for early detection of primary bladder tumours. Der Pathologe · Supplement 1 · 2012 | 55
Abstracts FR-029 Whole exome sequencing identifies potential therapeutic targets for castration resistant prostate cancer R . Menon 1 , M . Deng 1 , D . Boehm 1 , F . Fend 2 , D . Boehm 3 , S . Biskup 3 , S . Perner 1 1 Institute of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, 2 Institute of Pathology, University Hospital Tübingen, Tübingen, 3 Center for Genomics and Transcriptomics, Tübingen Aims. Castration resistant prostate cancer (CR-PCa) is the most aggressive form of prostate cancer (PCa) having a poor prognosis, and is a significant therapeutic challenge. The key to the development of novel therapeutic targets for CR-PCa is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to perform whole exome sequencing and gene copy number analysis on 5 CR-PCa/normal paired formalin fixed paraffin embedded (FFPE) samples using the SOLiD4 next generation sequencing platform. Methods. Genomic DNA was extracted from 5 CR-PCa/normal paired FFPE samples. The DNA was subjected to targeted exon capture using the Agilent Sure Select kit. The captured DNA was sequenced using the SOLiD4 next generation sequencing platform. The sequencing output was mapped, sorted, filtered and annotated using well-known human genome databases. The results were further analyzed for SNPs and copy number variations. A set of amplified/deleted genes were validated using fluorescence in-situ hybridization (FISH) assays with a PCa progression cohort. The cohort consisted of 138 cases for localized cancer, 105 patients with primary PCa and corresponding LN metastasis, and 39 samples for castration resistant tumors. Results. Whole exome sequencing analysis identified focal regions of deletion, which included well-known tumor suppressors such as NKX3.1 and PTEN. Focal regions of amplification included well-known genes such as cmYC and AR that are known to play a role in PCa. Furthermore, we identified several amplified genes as druggable targets e.g. HDAC6, NTRK1, PLD1, SPHK1, and SIRT7. NTRK1 is a kinase that plays an active role in cell proliferation. HDAC6, PLD1, SPHK1 and SIRT7 regulate numerous complex cellular processes including signal transduction, transcription and apoptosis. Conclusions. This is the first study to use whole exome sequencing approaches on FFPE CR-PCa material to identity novel therapeutic targets. Validation studies would further shed light into the biological understanding of the disease and its plausible treatment options. FR-030 Cut-offFinder – a web application for cut-off optimization for molecular markers J . Budczies1 , F . Klauschen1 , W . Schmitt1 , C . Denkert1 1Charité Hospital, Institute of Pathology, Berlin Aims. In order to translate a continuous diagnostic variable into a clinical decision, it is necessary to determine a cut-off point and to stratify patients into two groups, each of which requires a different kind of treatment. Methods. Cut-offFinder is implemented as Java Server Pages (JSPs) that connect to the statistical engine R. Using a web browser, the user can upload a molecular data set, assign biomarker and outcome variables and determine an optimal cut-off point for the biomarker. The web application offers three different methods for cut-off determination: The first method fits a mixture model of two Gaussian distribution to the distribution of the variable. The optimal cut-off is determined as the value where both probability density functions coincide. For the two other methods, all possible cut-off points are scanned. The second method correlates the dichotomized biomarker with a binary outcome variable using logistic regression. The optimal cut-off is defined as the point with the most significant (Fisher’s exact test) split. The third method fits Cox proportional hazard models to the dichotomized variable and the sur- 56 | Der Pathologe · Supplement 1 · 2012 vival variable. Then, the optimal cut-off is defined as the point with the most significant (log rank test) split. Results. As example, we have analyzed gene expression data of estrogen receptor (ESR1) and progesterone receptor (PGR) from a publicly available microarray data set of 286 breast cancer samples (GSE2034 at www. ncbi.nlm.nih.gov/geo) using Cut-offFinder. Histograms of the distribution of ESR1 and PGR showed a clear bimodal shape. Distribution derived cut-offs were located at 10.6 (ESR1) and 5.0 (PGR). The dependence on the cut-off of the odds ratio (OR) for correlation ERS1 expression with ER status (determined by immunohistochemistry) was analyzed and visualized. The optimal cut-off was determined as 10.1 with OR=67.8 (30.2–152.1). Using ERS1 expression measured by the microarray, determination of ER status was feasible with a sensitivity of 85.7% and a specificity of 91.9%. The dependence on the cut-off of the hazard ratio (HR) for correlation of PGR expression with distance-metastasis-free survival was analyzed and visualized. The optimal cut-off was determined as 2.5 with HR=0.46 (0.30–0.71). Kaplan Meier analysis showed a significantly better outcome for patients with high PGR expression (p=0.00028). Conclusions. In summary, Cut-offFinder is a comprehensive and easyto-use web application for cut-off determination for molecular markers. FR-031 BRAF-testing with pyrosequencing: a reliable alternative for the analysis of highly pigmented and degraded FFPE material A . Lehmann1 , C . Schewe1 , K . Jöhrens1 , C . Denkert1 , J . Budczies1 , M . Dietel1 1Charité Universitätsmedizin Berlin, Institute of Pathology, Berlin Aims. The approval of new BRAF inhibitors for the treatment of metastasized melanoma has led to a great demand for BRAF testing in molecular pathology laboratories. However, molecular analysis of formalin-fixed, paraffin-embedded (FFPE) melanoma tissue is challenging. Sanger sequencing often fails due to high amplification lengths and the influence of melanin. In this study we tested the applicability of a new pyrosequencing assay for BRAF analysis on 118 FFPE melanoma tissues and compared the results with those of Sanger sequencing. Methods. The study comprised 118 formalin-fixed, paraffin-embedded tissues of malignant melanoma which were referred to our department of Molecular Pathology for routine BRAF testing. DNA was extracted (QIAamp® DNA Mini Kit, Qiagen) and samples were subjected to both, Sanger sequencing (in-house method) and pyrosequencing (therascreen BRAF Pyro Kit®, Qiagen) of BRAF exon 15, codons 599 and 600. Results. BRAF sequences of 102 of 118 samples (86.4%) were evaluable by both methods pyrosequencing and Sanger sequencing. Mutational status of these samples was consistent in 98.0% (Cohen’s kappa coefficient =0.96, p=0.000). Sanger sequencing failed for 15 samples, which were mostly highly pigmented. Interestingly, 11 of these samples were still evaluable with pyrosequencing. With a success rate of 95.8% (CI95 [90.5–98.2%]), significantly more cases could be evaluated by pyrosequencing than by Sanger sequencing [success rate Sanger =87.3%, CI95 (80.1–92.1%); p=0.035]. Conclusions. Pyrosequencing requires comparably short target sequences for amplification which makes this method feasible even for problematic material for which standard methods like Sanger sequencing often fail. Particularly with regard to the high impact of BRAF-testing for therapy decision, pyrosequencing is a fast and reliable alternative for BRAF-testing.
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Inhalt Der Pathologe · Supplement
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Inhalt Der Pathologe · Supplement
Page 6 and 7: Editorial Liebe Kolleginnen und Kol
Page 8 and 9: Kolorektales Karzinom 2 VO-005 Tran
Page 10 and 11: Keynote Lecture VO-014 Genetic dete
Page 12 and 13: (AMACR, FASN, GOLM1, GSP-pi, ERG) t
Page 14 and 15: to assess the correct rate of R1 re
Page 16 and 17: DNA damage, and cytotoxic drugs. Au
Page 18 and 19: HCV-positive formalin-fixed and par
Page 20 and 21: well as MET activation were examine
Page 22 and 23: or BRAF mutation, c-MYC and SIRT1 e
Page 24 and 25: AG Pneumopathologie III DO-032 Remo
Page 26 and 27: immature granulopoiesis showed a st
Page 28 and 29: ned, if well-defined mantle zones,
Page 30 and 31: phomas). Most prominent gains or am
Page 32 and 33: DO-062 Tumor-associated macrophages
Page 34 and 35: DO-080 DOG1: an immunohistochemical
Page 36 and 37: AG Oralpathologie DO-087 Detection
Page 38 and 39: DO-094 Do activated fibroblasts inf
Page 40 and 41: DO-101 Histopathological analysis o
Page 42 and 43: DO-108 Identification of potential
Page 44 and 45: Conclusions. In conclusion, 454 par
Page 46 and 47: subgroup of patients with B-Raf mut
Page 48 and 49: DO-002b Impact of terminologies in
Page 50 and 51: Methods. We performed MCPyV-FISH of
Page 52 and 53: FR-013 HPV-genotype distribution in
Page 54 and 55: Methods. Three different techniques
Page 58 and 59: FR-032 COLD-PCR: a powerful tool in
Page 60 and 61: dissection (MBLND) technique to imp
Page 62 and 63: SO-005 Prevalence of mutations in s
Page 64 and 65: SO-011 Methylation profiling and in
Page 66 and 67: more effective than SAHA alone and
Page 68 and 69: SO-022 Specialized pathology review
Page 70 and 71: SO-027 Ki-67 in mitotic score group
Page 72 and 73: nificantly associated with advanced
Page 74 and 75: liver did not correlate with the mu
Page 76 and 77: AG Paidopathologie II SO-046 Overex
Page 78 and 79: Results. Histomorphology of the ova
Page 80 and 81: p42 and p27 have not yet been inves
Page 82 and 83: SO-068 Recent advances in understan
Page 84 and 85: SO-075 A novel, dual role of CCN3 i
Page 86 and 87: Pancreatic Adenocarcinoma SG-137 Se
Page 88 and 89: sease and 30 colon cancer serum sam
Page 90 and 91: Conclusions. Although CRC is charac
Page 92 and 93: differentiated and TNM stage III or
Page 94 and 95: pressed moderate levels of the prot
Page 96 and 97: FR-P-037 MALDI imaging reveals COX7
Page 98 and 99: in the context of therapy response
Page 100 and 101: on primary cells of wild-type and c
Page 102 and 103: chemotherapy was recommended and co
Page 104 and 105: aims are twofold: 1) to verify the
Page 106 and 107:
Results. Her2/neu status in TMAs an
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prognostic impact was found in rect
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provided information on survival ti
Page 112 and 113:
Methods. Biopsy specimens from 430
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the OS rate was 53% for patients wi
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FR-P-098 Immunohistological stainin
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FR-P-104 Histopathological evaluati
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within the earliest morphologic det
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Conclusions. In primary imaging a g
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fibrotic neointma development as we
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FR-P-129 Alpha-1-antitrypsin-PiZ-an
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FR-P-136 The expression of central
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cosa and in the periarterial tissue
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FR-P-149 Combination of Castleman d
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or without different concentrations
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Results. In 18 cases (79%) the caus
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FR-P-168 Autopsy findings in a 2-ye
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FR-P-174 MPGN-like glomerulonephrit
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Poster: Gynäkopathologie und Mamma
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SA-P-011 Genetic aberrations of pre
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Mechanismen, die eine Progression d
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internet server. A network of inter
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Methods. HE-gefärbte Schnittpräpa
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Conclusions. The newly released 450
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and desmoplastic reaction are diffe
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one patient revealed more than 9 mi
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situation, the V600E mutation in th
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SA-P-064 Evolution of molecular pat
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nic markers such as myf4 and MyoD1
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Conclusions. To our knowledge, this
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SA-P-085 Expression of the eukaryot
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Results. The papillary RCC type II
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SA-P-098 Male infertility: assessme
Page 172 and 173:
SA-P-104 Process oriented scientifi
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Conclusions. The IBDW offers a uniq
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L Lasitschka F. DO-046 Lehmann A. F
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