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

Abstracts
mary antibody which is intended to be used for IHC-P. Subsequently,
antibody specificity can be tested on multiple cell lines using a FFPE cell
line microarray (CMA). Correlating cmA IHC-P profiles with mRNA
gene expression profiling data may be helpful. If the target is ubiquitously
expressed, evaluation of antibody specificity may require more sophisticated
technologies like siRNA knock down of target in cancer cell lines.
The siRNA knock down or a transfection of cell lines with wild type or
mutated target generates gene expression modified cell lines which may
be assembled to a gene expression modified cmA. Thereby IHC-P can be
linked to functional assays.
Results. In-depth antibody validation discloses that primary antibodies
may be not selective in IHC-P, although the antibody datasheet or published
data suggest the IHC-P application. This observation applies not
only to polyclonal antibodies but also to monoclonals. Using antibodies
that demonstrate a specific staining, IHC-P may correlate remarkably
with alternative protein detection methods like Luminex’s multiplex bead-based
immunoassay that is limited to fresh samples.
Conclusions. The data underline the need for in-house validation of primary
antibodies prior to their use as analytic tool in IHC-P. It is likely
that the unsatisfactory validation status of published antibodies represents
one of the causes for contradictory IHC-P data in the literature.
Concerted activities should be considered to improve the current situation.
Poster: Molekularpathologie II
SA-P-054
The homeobox gene HOPX is methylated in human lung cancer,
and the methylation status of HOPX is a diagnostic marker for
patients with lung adenocarcinoma
Y . Chen1 , T . Cui1 , L . Yang 1 , N . Posorski 2 , I . Petersen 1
1 2 University Hospital Jena, Institute of Pathology, Jena, University Hospital
Jena, Institute of Human genetics, Jena
Aims. The homeobox gene HOPX has been considered as a tumor suppressor
in various cancers. Our previous studies showed that HOPX is
downregulated in lung cancer cell lines and primary lung tumors, and
forced expression of HOPX by stable transfection could inhibit tumor
cell growth. However, the regulation of HOPX in lung cancer has not yet
been well elucidated. Therefore the aim of the study is to investigate the
epigenetic regulation, analyse the potential target genes of HOPX, and
evaluate the clinical relevance of HOPX in lung cancer.
Methods. HOPX protein expression was analysed by western blotting.
Demethylation test by 5-aza-2-deoxycytidine (DAC), bisulfite sequencing
(BS), and methylation-specific-PCR (MSP) were carried out to analyse
the methylation status of HOPX in lung cancer cells and primary
lung tumor samples. cDNA microarray analysis was performed to identify
target genes of HOPX.
Results. In line with decreased expression of HOPX mRNA in lung
cancer cell lines, western blotting showed that HOPX protein was downregulated
compared to normal HBEC cells. Ten out of 12 lung cancer
cell lines restored HOPX expression after treatment with DAC , and the
methylation status of HOPX in the promoter region and exon 1 was confirmed
by bisulfite sequencing. MSP showed that HOPX was methylated
in 64 out of 88 (72.7%) primary lung tumor samples, and methylation of
HOPX was significantly associated with lung adenocarcinoma (p200 patients.
Methods. We performed quantitative real-time PCR to analyse the DNA
methylation of the SHOX2 gene compared with beta-actin using the Epi-
ProLung Test (Epigenomics) in >200 patient undergoing routine diagnostics
for lung cancer at the Charité in 2011. A dichotomized methylation
status was determined by using a ddCT cut-off value of 9.5.
Results. From the 239 cases, 173 were SHOX2-, 44 SHOX2+ and 17 undetermined.
The cytological analysis yielded 200 cases unsuspicious of
cancer (or with only mild atypia) and 34 cases moderately to highly suspicious
of cancer. This comparison between SHOX2 and cytology displays
a sensitivity of 73% and a specificity of 78.4% (AUC 75.1%, SHOX2
testing benchmarked with cytology).
Conclusions. The results demonstrate the utility of SHOX2 methylation
testing for routine diagnostics of lung cancer in direct comparison with
cytology in lavage samples. Clinical, radiological and histological data
will be included upon availability to determine the absolute performance
of SHOX2 testing.
SA-P-056
Detection of a heterogeneous BRAFV600E mutation status in a
patient with metastasized malignant melanoma
R . Marienfeld1 , J . Heinrich2 , S . Jung2 , P . Möller1 , K . Scharffetter-Kochanek3 ,
M . Huber3 , T . Menzel4 , L .-A . Schneider3 , T . Barth2 1 2 University of Ulm, Institute of Pathology, Ulm, University of Ulm, Institute
of Pathology, 3University of Ulm Medical Center, Department of Dermatology
and Allergology, 4Dermatophathology Friedrichshafen
Aims. Targeted tumor therapy is expected to lead to a breakthrough in
the treatment of advanced stage melanoma. By end of this year Vermurafenib®
(Roche PLX4032) is expected to be licensed as the first BRAF
pathway inhibiting substance for metastasised melanoma carrying a
V600E BRAF mutation. Up to 50% of all melanoma patients are estimated
to carry a mutation in the codon 600 of the BRAF gene. It is therefore
in the focus of scientists and clinicians since targeted therapy is supposedly
less harmful and more effective than conventional chemotherapy.
Mutational analysis of the codon 600 of the BRAF gene is strictly required
to determine the eligibility of the patient for a targeted therapy
with Vermurafenib®. The goal our study is to determine the reliability of a
BRAF mutational analysis on the basis of a single tissue specimen. Here,
we present the BRAF mutational analysis of four different specimens of a
patient with metastasized melanoma.
Methods. Microdissection of FFPE tumor tissue. DNA isolation and PCR
amplification of the BRAF gene segment. Pyrosequencing of codon 600.
Results. The patient is a 38-year-old woman with a pTxpN3pM1 metastasized
acrolentigeneous melanoma. We analysed the BRAF mutation
status in the primary tumor as well as in three different biopsies from a
lung axillary lymph node and brain obtained with a time difference of
six years. BRAF mutation has been shown to be an early event in melanoma
and thus should be present in the first biopsy. However, whereas
the analysis of the first biopsy from lymph node revealed a wild type