FR-032 COLD-PCR: a powerful tool in routine-diagnostic for cost-neutral detection of minor clones using real-time PCR or pyrosequencing quoted by the example of EGFR mutation analysis F . Mairinger 1 , A . Streubel 2 , A . Roth 2 , O . Landt 3 , W . Grüning 4 , J . Kohlmeier 4 , T . Mairinger 2 1 University Hospital Essen, Department of Pathology und Neuropathology, Essen, 2 Helios Klinikum Emil von Behring, Department of Pathology, Berlin, 3 TIB MOlBIOL Gmbh, Berlin, 4 Helios Klinikum Emil von Behring, Department of Pneumology, Berlin Aims. COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a novel method to enrich minority alleles from mixtures of wild-type (wt) and mutation containing (mt) sequences, irrespective of localization and property within the analyzed sequence. The heteroduplexes generated after an initial denaturation step will be preferentially melted at the critical denaturation temperature resulting in a radical enrichment of minor variants, enabling their detection with conventional methods which are intended to analyze normal bi-allelic variations. Molecular testing of tissues is faced with samples containing mixtures of tissues frequently containing only small fractions of mutations. A study was designed to overcome this problem of the too low sensitivity of nowadays routinely used molecular methods. For this, the effect of COLD-PCR in probe-based real-time PCR analysis or as initiating step for pyrosequencing to the sensitivity was tested. Methods. Different dilution steps of artificial EGFR T790M mutated and wild-type EGFR exon20 DNA were analyzed by a LightCycler assay or amplified by full-COLD-PCR using different protocols and afterwards sequenced by pyrosequencing to determine the detection-limit of these methods. Results. For the LightCycler-assay, the best results are ren<strong>der</strong>ed with a combination of 10 cycles conventional PCR followed by 45 cycles COLD- PCR using 84°C as denaturation temperature. A dilution of down to 0.125% mt-DNA/total-DNA is still detectable. With COLD-PCR amplified DNA, a dilution of 0.125% mt-DNA/total-DNA is still detectable by pyrosequencing in reproducible results. Conclusions. Our results show the exceeding potential of COLD-PCR in enrichment of DNA of un<strong>der</strong>represented clones in clinical samples. Because of this, problems like dilution of potentially mutated tumor cells (showing for example EGFR-resistance mutation T790M) with non-resistant tumor cells or benign cells debt to macrodissection or tumor heterogeneity resulting in failing to detect clinically relevant minor clones could be overcome. FR-033 Fast and reliable detection of mutations in exon 9 of the KIT gene by high resolution melting analysis H . Künstlinger1 , M . Kleine1 , J . Fassunke1 , E . Wardelmann1 , R . Büttner1 , S . Merkelbach-Bruse1 , H .-U . Schildhaus1 1University Hospital Cologne, Institute of Pathology, Köln Aims. Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract. They harbour activating mutations in the KIT or platelet-<strong>der</strong>ived growth factor (PDGF) receptor. Imatinib mesylate is a potent inhibitor of KIT signalling and is therefore widely used as targeted therapy for GISTs. The mutational status of KIT exon 9 is of special importance for the therapy with Imatinib, because cases with exon 9 mutations need a higher dose of Imatinib. Therefore, in metastasized or high-risk GISTs as well as in primary notoperable tumours it is necessary to obtain the mutational result as fast as possible after diagnosis. At present, mutational analysis of KIT and PDGFR is routinely carried out by Sanger sequencing. This method has certainly its lasting relevance for DNA sections with high variability of mutations (e.g. KIT exon 11), but is relatively expensive and time consuming. Therefore alternative methods need to be established for other exons (like KIT exon 9) or other certain mutational types to enable a fast and cost efficient detection. Methods. High Resolution Melting (HRM) is a post-PCR mutation scanning tool that detects the change in fluorescence caused by the progressive release of a saturating intercalating dye from DNA duplexes while they are denatured by slight increases in temperature. HRM assays were developed using specifically designed primers and genomic DNA isolated from formalin-fixed paraffin-embedded GIST samples. Melting curve analyses were performed on the LightCycler 480 platform (Roche) and mutation analyses were additionally confirmed by Sanger Sequencing. Results. Conditions for High Resolution Melting analysis of KIT exon 9 could be established using more than 60 GIST samples with known mutational status of the KIT gene. Sensitivity was determined as a minimal proportion of 12.5% mutated alleles. A prospective screening of more than 100 additional GIST samples showed a complete concordance between HRM assay and traditional Sanger sequencing. Conclusions. The established high resolution melting assay represents a highly reliable method for the detection of mutations in exon 9 of the KIT gene. It allows a faster and more cost-effective mutational analysis of KIT exon 9 in the future, which is especially important for dose finding of Imatinib in GIST therapy. The determined sensitivity is comparable to the sensitivity of currently performed Sanger sequencing. FR-034 Morphological and clinical characterization of a novel mouse model for mutation-activated JAK1 S . Wagner1 , E . Janas2 , B . Lorenz-Depiereux3 , J . Calzada-Wack 2 , A . Benet-Pagès3 , S . Eck3 , J .A . Aguilar Pimentel4 , B . Rathkolb4 , V . Gailus-Durner4 , H . Fuchs4 , H . Höfler2 , M . Hrabé de Angelis1 , T . Strom3 , F . Neff2 1Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg, 2Helmholtz Zentrum München, Institute of Pathology, Neuherberg, 3Helmholtz Zentrum München, Institute of Human Genetics, Neuherberg, 4Helmholtz Zentrum München, German Mouse Clinic, Neuherberg Aims. The members of the Janus kinase family (JAK1, JAK2, JAK3) play important roles in signalling downstream of cytokine receptor activation and are implicated in various physiological processes including the hematopoietic, immune and neuronal systems. However, the lack of successful mouse models for mutation-activated JAK1-induced diseases hampers the un<strong>der</strong>standing of disease pathology. Here, we have produced a novel mutant mouse line leading to an amino acid substitution in the pseudokinase domain of JAK1 using N-ethyl-N-nitrosourea (ENU) mutagenesis. This mutation corresponds to a JAK1-activating mutation (Ser646Phe) described in humans and associated with acute lymphoblastic leukemia (Mullighan et al. 2009). Methods. The ENU mutagenesis was generated in C3HeB/FeJ genetic background. Mutation screening was performed after linkage analysis using single nucleotide polymorphisms (SNP) and chromosome sorting by next generation sequencing. A total of 64 mice at the age of 18 to 31 weeks were analyzed for clinical and immunological parameters as well as histology. Thirty organs were examined by H&E staining and immunohistochemistry. Results. All mutant mice showed a loss of ear cartilage starting with 4 months of age without signs of inflammation, a significant loss in body weight due to an alternation in body composition. In serum, a significant increase of auto-antibodies was observed. Most strikingly, histopathological analysis revealed a nodular regenerative hyperplasia of the liver with a remarkable increased neovascularisation. This vascularisation was also observed in the skin of ears indicating a systemic vasculitis. Conclusions. A significantly increase in auto-antibodies together with the pathological changes observed implicates that the introduced mutation in the pseudokinase domain of JAK1 induces a systemic autoimmune disease. Der Pathologe · Supplement 1 · 2012 | 57
Abstracts FR-035 MAP3K7 deletions have prognostic relevance in prostate cancer M . Kluth 1 , A . Krohn 1 , J . Hesse 1 , R . Simon 1 , T . Schlomm 2 , H . Huland 2 , G . Sauter 1 , S . Minner 1 1 University Medical Center Hamburg-Eppendorf, Hamburg, 2 Martini-Clinic, Hamburg Aims. MAP3K7 (mitogen-activated protein kinase kinase kinase 7) gene is a component of the MAPK-pathway and plays a central role in cell growth, differentiation and apoptosis. The MAP3K7 gene is located at 6q15, a region, which is commonly deleted in prostate cancer. The aim of this study was to examine the potential relevance of MAP3K7 (6q15) deletions in prostate cancer with respect to tumor phenotype and clinical outcome. Methods. A prostate tissue microarray (TMA) with clinical follow-up data consisting of over 4500 prostate cancer samples was analyzed for MAP3K7 deletions by fluorescence in situ hybridization (FISH). Results were correlated with tumor phenotype, clinical outcome and ERG expression. In addition, 15 tumors with known MAP3K7 deletion and 14 tumors without MAP3K7 deletions were examined for the presence of MAP3K7 mutations (Exon 1–17). Results. Heterozygous MAP3K7 deletions were found in 18.5% (423/2289) of all analyzable prostate cancers. MAP3K7 deletions were associated with advanced tumor stage (p
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Inhalt Der Pathologe · Supplement
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Inhalt Der Pathologe · Supplement
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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 56 and 57: (i.e. investment in equipment and e
- 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
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- 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
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prognostic impact was found in rect
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provided information on survival ti
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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
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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