Verhandlungen der Deutschen Gesellschaft fur Pathologie最新文献

Aims: NF-kappaB has been demonstrated to activate proliferative, inflammatory, and angiogenic processes in ovarian cancer cells in vitro. To add translational information on the situation in vivo, we determined the expression pattern of p65, an important subunit of the classic NF-kappaB pathway, in ovarian carcinoma tissue, and investigated in vivo and in vitro whether this pathway is implicated in the known overexpression of cyclooxygenase-2 (COX-2).

Methods: p65 siRNA, chemiluminescent NF-kappaB transcription factor assay, Taqman PCR, as well as immunoblotting were performed with OVCAR-3 ovarian cancer cells. 83 primary ovarian cancinomas as well as 17 cases of benign ovarian tissue were analyzed by p65 and COX-2 immunohistochemistry using a tissue microarray.

Results: DNA-binding avtivity as well as COX-2 mRNA and protein expression were strongly inducible by IL-1beta treatment in OVCAR-3 cells, while p65 siRNA inhibited IL-1beta-dependent p65 activity (p = 0.037) as well as COX-2 expression on the mRNA (p < 0.03) and on the protein level. In human tumor tissue, p65 protein expression was significantly associated with COX-2 expression (p = 0.002) as well as tumor grading (p = 0.005). Furthermore, p65 expression was a significant prognostic indicator of a reduced patient survival both in univariate (p = 0.038) and in multivariate analysis (p = 0.014).

Conclusion: Our study indicates a deregulation of the classical NF-kappaB pathway in ovarian cancer, which results in the overexpression of the NF-kappaB target gene COX-2. Components of this pathway might constitute novel attractive targets for a specific therapy of advanced ovarian cancer.

Based on the mechanism of their genetic instability human colorectal tumors can be subdived into two groups: (1) microsatellite stable (MSS) and (2) microsatellite instable (MSI-H) tumors. The reason for MSI-H is a defective mismatch repair (MMR) system. Besides the genetic mechanism MSI-H and MSS tumors differ also in other characteristics from each other. Mostly, MSI-H tumors are found in the proximal colon and display a moderate to weak differentiation. They display frequently a mucinous differentiation. MSI-H tumors harbour often tumor infiltrating lymphocytes (TIL) which are mostly of the CD8+ T-cell type. Moreover, MSI-H tumors display a higher frequency of apoptosis. But most importantly, patients with colorectal MSI-H tumors have a significant better prognosis compared to those with MSS tumors. Interestingly, cultured MSI-H colorectal cell lines are resistant to the adjuvant chemotherapeutical agent 5-fluorouracil (5FU) which is commonly used for the therapeutical treatment of colorectal cancers. The 5FU insensitivity is due to the defective MMR system which is responsible for the detection and repair of 5FU generated DNA helix-distorsions. Whereas 5FU has a strong impact on the survival of patients with MSS colorectal tumors, it is unclear if patients with MSI-H tumors benefit from adjuvant 5FU chemotherapy which is a conundrum as MSI-H cell lines are insensitive for this agent. Evidence is presented here, indicating that the results from studies demonstrating a benefit of MSI-H colorectal tumors from adjuvant 5FU chemotherapy are based on wrong statistical comparisons and that therefore patients with MSI-H colorectal tumors do not benefit from 5FU treatment.

Chromosomal abnormalities like translocations and their corresponding gene fusions were first suggested to be causal factors in the development of cancer by Boveri in 1914. At present more than 350 gene fusions involving 337 different genes have been identified and in particular in malignant hematological diseases and sarcomas an increasing number of chromosomal aberrations are being recognized as important diagnostic and prognostic parameters. By contrast this type of gene rearrangement has been until recently only rarely described in the common carcinomas. However, with new powerful techniques, that enable the detection of cytogenetically cryptic rearrangement, this number is likely to increase substantially and carcinomas characterized by fusions oncogenes indicate that the pathogenetic mechaisms involved in epithelial carcinogenesis may be similar to those known to operate in hematological and soft-tissue malignancies.

Morphogenesis of malignant tumors is characterized by proliferation, invasive growth and metastasis. Although these properties are determined mainly by genetic derangements, their biological behavior within tissues is strictly regulated by tumor microenvironment, which consists of extracellular matrix, proteinases, soluble factors (cytokines/growth factors/chemokines), stromal cells and blood vessels. Thus, modulation of the microenvironment is implicated in morphogenesis in tumorigenesis. ADAMs (a disintegrin and metalloproteinases) are a new gene family of membrane-anchored and secreted proteins that have proteolytic and/or adhesive properties. They are involved in biological events including cell adhesion, cell fusion, membrane protein shedding and proteolysis. We examined the expression of the proteinase-type ADAMs in the invasive breast and lung carcinoma tissues, and found that membrane-anchored ADAM28m and secreted ADAM28s are selectively overexpressed in activated forms by carcinoma cells. The mRNA expression levels directly correlated with the proliferative activity of the carcinoma cells in both carcinomas, and with lymph node metastasis in the lung carcinomas. Our experimental studies showed that ADAM28 plays a key role in cancer cell proliferation through enhancing bioavailability of insulin-like growth factor-I (IGF-I) released from the IGF-I/IGF-binding protein 3 (IGFBP-3) complex by selective cleavage of IGFBP-3. We also identified P-selectin glycoprotein ligand-1 (PSGL-1) as a binding protein to ADAM28 by yeast two-hybrid system, and demonstrated that ADAM28s promotes PSGL-1/P-selectin-mediated HL-60 cell rolling adhesion to endothelial cells and subsequent transendothelial migration into tissue spaces. Altogether, our data suggest the possibility that ADAM28 expressed by cancer cells is involved in cancer cell proliferation and metastases in human cancers through modulation of tumor microenvironment and cell adhesion.

Aims: Fetal skeletogenesis has been extensively investigated in the vertebrates and both, cellular phenotypes and regulatory mechanisms are well characterized. This knowledge has been used in the past years to elucidate the biology of mesenchymal neoplasias of the skeleton, in particular of the cartilage forming tumors.

Results: The hallmark of chondrogenic tumors is the presence of cells, which can show the full differentiation potential of physiologic chondrocytes depending on the tumor entity investigated. The phenotypic plasticity of chondrocytes explains the striking heterogeneity of cells and extracellular matrix found not only in between different, but also within chondrogenic tumor types. Hereditary exostosis is one example for which genetic analysis as well as the knowledge of regulatory pathways have contributed to our understanding of tumor development: the deficiency in functional EXT gene products explains the pathogenesis of these neoplasms.

Conclusions: In principle, chondrogenic neoplasias follow differentiation rules delineated during fetal skeletogenesis. Tumor classifications, so far based only on histomorphological criteria can be extended by molecular markers, which have the potential to contribute to a biology-orientated classification of skeletal neoplasms

Aims: DNA methylation has been shown to play an important role in breast cancer pathogenesis, but up until now it is not clear how the tissue components contribute to the overall methylation of the sample, because microdissection does not provide sufficient material for most standard methylation assays.

Methods: We developed a technology to analyse several methylation markers in a limited number of cells dissected from tissue sections. To evaluate the technology, we analysed, among others, the methylation markers PITX2, RASSF1A and TFF1 in 79 samples of three PITX2 methylation positive invasive ductal carcinomas. The microdissected samples were from the invasive part, the intraductal part, the stroma, tumor infiltrating lymphocytes, chest wall muscle, adipose tissue and healthy ducts.

Results: The multiplexed methylation analysis allows for the quantitative analysis of methylation patterns in microdissected samples with as few as 100 genome copies. In all analysed patients PITX2 and RASSF1A were highly methylated in invasive and intraductal carcinoma cells compared to other tissue components. TFF1 behaved inversely. PITX2 showed some methylation in normal adjacent breast tissue. The methylation of the individual markers varied little within one tissue type and between blocks.

Conclusions: This technology is a powerful tool to analyse the methylation of multiple markers in different microdissected tissue components. Methylation patterns may differ significantly between different markers and tissue components. This technology may help to analyse different transitions states of breast and other cancers.

To analyze the consequence of ,gain of function' and ,loss of function' of transcription factor AP-2gamma in mice Using pronuclear injection, mice harbouring a mammary gland specific transgene were established and analyzed. Constitutive and conditional knockout mice complement the analysis, showing the consequences of loss of function for specific tissues. Gain of AP-2 expression on it's own in mammary gland results in enhanced proliferation which is compensated by accelerated apoptosis. Only after mating with a second transgenic mouseline, the MMTV-ErbB2 oncomouse model, an effect on tumor progression was observed. Hence, AP-2 might influence progression, but not initiation of mammary tumors. Knockout mouse models reveal that AP-2gamma is essential for the extraembryonic cells indicating a role for trophoblast cell lineage These and other studies indicate that AP-2gamma might be a molecule which enables proliferation of undifferentiated precursors or transient amplifying cells. Reexpression in tumors correlates with dedifferentiation and progression.

Aims: Receptor tyrosine kinases have been shown to be a challenging target for the treatment of hematologic diseases and solid tumors. One very effective tyrosine kinase inhibitor is imatinib mesylate inhibiting the KIT receptor tyrosine kinase in gastrointestinal stromal tumors (GISTs) which often carry activating mutations in the KIT gene. Our own observations show that the location of the underlying mutations influence the response to treatment with imatinib.

Methods: In Bonn, nearly 1000 GISTs have been characterized molecularily before and/or under treatment with tyrosine kinase inhibitors. Tumor-DNA was extracted from paraffine-embedded material, amplified in all known hot spots of KIT (exons 9, 11, 13, 14, 17) and PDGFRcx (exons 12, 14, 18) and sequenced directly.

Results: The best response to treatment with imatinib is achieved in GISTs with an underlying KIT mutation in exon 11 encoding the juxtamembranous domain. Exon 9 mutated GISTs respond in only half of the cases. GISTs with mutations in the tyrosine kinase domain 1 or 2 are very rare (less than 1%) and are thought to be resistant. PDGFRalpha-mutated GISTs are resistant when carrying the most common point mutation D842V (exon 18) and may respond when deletions occur in exon 18. Low response rates are achieved in tumors without detectable mutation. Under treatment, secondary KIT mutations may occur leading to resistance to treatment.

Conclusions: The molecular status of GISTs plays a central role for treatment response. Its evaluation will be mandatory in the future at least in tumors with intermediate or high risk criteria.

Aims: This study addresses the question whether microRNA genes are affected by aberrant hypermethylation and subsequent transcriptional repression in human breast cancer.

Methods: All known human microRNA genes were screened for the association with CpG islands using different algorithms. Methylation status of candidate genes was assessed in a panel of breast cancer cell lines, various normal tissue samples and primary breast cancer specimen using COBRA, bisulfite sequencing and pyrosequencing. Transcriptional silencing was measured by real-time RT-PCR. The effect of demethylation on microRNA gene expression was analysed in breast cancer cell lines after treatment with the DNMT inhibitor 5'-deoxy-2-azacytidine.

Results: Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in preinvasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8).

Conclusions: In addition to deletion and mutation, microRNA genes are also affected by aberrant hypermethylation in human breast cancer. This epigenetic inactivation is frequent and occurs early during breast cancer progression.

Aim: Overexpression of Aurora-A/STK15 kinase (hereafter AUKRA) is seen in a variety of epithelial cancers, such as gastrointestinal and gynaecological carcinomas. Its role as prognostic and/or predictive marker for adjuvant therapy of patients with advanced ovarian cancer is however still unclear. Therefore, the present study aimed at determining (1) the clinical value of AURKA expression (mRNA and protein) in 115 patients with ovarian carcinomas and (2) the basis of AURKA overexpression at the DNA level.

Methods: Formalin-fixed and Paraffin-embedded tissue samples (ovarian carcinoma: n=115; non-neoplastic ovaries: n=28) were processed for microdissection and quantitative RT-PCR as well as for semi-quantitative immunohistochemistry (IHC) of tissue microarrays according to standardised protocols. Fluorescence in Situ Hybridisation (FISH) was performed in a sub-set of cases (n=37) to analyse AURKA DNA copy numbers.

Results: The results demonstrate significantly elevated AURKA expression at the mRNA and protein level in ovarian carcinomas as compared to non-neoplastic ovaries (p < 0.0001). AURKA protein overexpression was observed in 68/107 (63.5%) of cases. For patients with stage III ovarian carcinoma having been optimally debulked and receiving adjuvant Taxane-based chemotherapy, AURKA overexpression was significantly linked to prolonged overall survival (p = 0.02). Finally AURKA overexpression was associated with increased AURKA DNA copy numbers (p = 0.01).

Conclusion: In summary, AURKA overexpression, which is regulated at the DNA level, is a novel predictive marker for a subgroup of patients with stage III ovarian carcinomas.