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

Abstracts
Methods. Detailed clinical and histopathologic review of a clinical case
and review of the literature using PUBMED.
Results. We report on a 70-year-old woman with FIGO Stage 1A Grade II
(pT1a pN0 L0 V0 R0) endometrial adenocarcinoma who presented 2 years
following total abdominal hysterectomy, bilateral salpingo-oophorectomy
and lymphonodectomy with left radius bone metastasis. A left forearm
amputation was performed. The tumor measured 7.1×6.9×6.5 cm.
The histological diagnosis was a poorly differentiated (G3), endometrioid
endometrial carcinoma. Both carcinomas were oestrogen and progesterone
receptor negative, vimentin, OSCAR and AE1/AE3 positive. Immunohistochemical
stains for S-100, desmin, a-smooth muscle actin and
CD 34 were negative.
Conclusions. This case highlights the rare and unusual presentation of
endometrial cancer. For this reason a review of the literature is also provided
for all cases with evidence of bone metastasis at presentation of the
disease and as a recurrence. Its diagnosis requires immunohistochemistry
and awareness of its possible existence.
Poster: Molekularpathologie I
SA-P-035
Identification of uncommon PIK3CA-mutations in lung cancer by
using pyrosequencing
V . Schildgen1 , J . Lüsebrink 1 , J .D . Appel1 , C . Wübben1 , W . Engel-Riedel1 ,
E . Stoelben1 , O . Schildgen1 , M . Brockmann1 1University Witten/Herdecke, Kliniken der Stadt Köln gGmbH, Köln
Aims. Phospatidylinositol-3-kinases (PI3K) play an important role in various
cell processes. Oncogenic mutations in the PIK3CA gene which codes
for the catalytic subunit have been identified in various malignancies
and activate the PI3K/AKT/mTOR pathway which is a critical driver of
tumorigenesis.
Methods. We tested 41 tumor samples with known KRAS, BRAF, and
EGFR mutation status for the occurrence of mutations in the PIK3CA
gene using a new commercial pyrosequencing kit.
Results. Pyrosequencing revealed 2 mutations (4.9%) in the PIK3CA
gene, one in exon 9 and one in exon 20. Both mutations have not yet been
identified in lung tumor tissue.
Conclusions. The screening of our small patient cohort by pyrosequencing
identified two mutations (4.8%) in PIK3CA, on in exon 9 (Q546H)
and on in exon 20 (M1043T). Both mutations have not yet been described
in lung tumours and seem to be rather uncommon mutations. Future
screening of large patient cohorts with pyrosequencing may contribute
to the detection of more mutations in lung cancer due to the low limit of
detections of this method and may contribute to a better understanding
of the interaction of mutations and tumorigenesis.
SA-P-036
Detection of EML4-ALK fusion oncogenes in NSCLC using a commercial
three-color FISH assay (ZytoLight SPEC TriCheck)
B . Schmitt 1 , H . Schultz1 , F . Stellmacher1 , E . Vollmer 1 , T . Goldmann1 1Borstel Research Center, Clinical & Experimental Pathology, Borstel
Aims. About 5% of non-small cell lung cancers (NSCLC) harbor an inversion
of the short arm of chromosome 2 that causes a rearrangement of
the ALK and EML4 genes. The resulting fusion oncoprotein comprises a
constitutively active tyrosine kinase. Those patients will benefit substantially
from treatment with crizotinib, a Met-/tyrosine kinase inhibitor,
highlighting the need for appropriate tests allowing one to reliably detect
such fusions in biopsy specimens. Existing techniques such as immunohistochemistry,
multiplex PCR, or 2-color FISH using break apart-probes
each possess specific strengths and weaknesses. By design, a novel
150 | Der Pathologe · Supplement 1 · 2012
commercial 3-color FISH assay offers several theoretical advantages.
However, its actual performance in research and diagnostic settings has
not been tested.
Methods. We analyzed formalin-fixed and paraffin embedded (FFPE)
tissue sections (pulmonary adenocarcinomas) and tissue microarrays
(TMA; assembled from adenocarcinomas, squamous and large cell carcinomas)
for EML4-ALK rearrangements by 3-color FISH assay using
ZytoLight SPEC TriCheck probes (Zytovision). Stained slides were evaluated
multiply by pathologists and molecular biologists according to
standardized criteria. Cancers exhibiting a characteristic fluorescence
pattern in at least 15% of tumor cells were taken as EML4-ALK fusion
positive.
Results. High quality signals were obtained in FFPE tissues, in individual
sections as well as in TMAs. Interobserver variability was negligible
for wild type identification. Discrimination between FISH signals from
wild type vs. EML4-ALK fusions in cancer tissue was clear and binary
owing to probe design, and without the problematic intermediate results
observed with break apart-probes in case of a small split. Evaluation was
more laborious and less reproducible, however, when more non-tumor
tissue was interspersed and tumor cells were harder to identify. Assay
hands-on time and total turnover time were less than for multiplex PCR.
Conclusions. Detecting EML4-ALK fusions based on a 3-color FISH assay
using the “ZytoLight SPEC TriCheck”-probes requires specialized
equipment, familiarization of the observer and reliable distinction of
tumor cells from non-tumour cells. The approach does not require assessment
by multiple observers, unlike assays using break-apart probes.
Overall, this approach appears particularly suited for clinical studies and
basic research in that it will also identify rare and putative novel fusion
variants, as well as deletions and amplifications.
SA-P-037
Systematic quantitative cross-validation and content analysis of
the 450k methylation array from Illumina
J . Rößler1 , O . Ammerpohl2 , J . Gutwein2 , U . Lehmann1 1 2 Medical School Hannover, Institute of Pathology, Hannover, University
Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel
Aims. The relationship between the beta-values provided by the newly
released 450k methylation array from Illumina for individual CpG dinucleotides
was compared with quantitative methylation levels obtained
by conventional pyrosequencing. In addition, the representation on the
Illumina array of two groups of genes, which are important in tumour
biology and display extensive aberrations in DNA methylation in cancer
(microRNAs and imprinted loci), was assessed in detail.
Methods. High molecular weight DNA was isolated from histologically
examined 18 fresh-frozen human breast cancer specimens, 4 normal
mammary epithelial fractions and 4 human breast cancer cell lines
(IPH-926, HCC1937, MDA-MB-134, PCM42) using standard procedures.
DNA was bisulfite treated and analyzed on 450k arrays following
the manufacturer’s protocol. For primary data processing and analysis
the software provided by Illumina was employed. These analyses were
complemented and extended by employing the Omics Explorer from
Qlucore and the R package IMA. The beta-values for individual loci were
cross-validated using conventional pyrosequencing of the same DNA
samples independently treated with sodium bisulfite.
Results. The newly released 450k array methylation array from Illumina
shows a high concordance with quantitative pyrosequencing if identical
CpG sites are analysed by the two different methods in cell lines (Spearman
r=0.88, p