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

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 rendered 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 underrepresented 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-derived 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 understanding 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.
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