Current funding :

Current projects :

On the way to a prognostic chip for colorectal carcinomas

Colorectal cancer is one of the leading causes of death in developed countries. Although prognosis for colorectal carcinomas has improved during the last twenty years, nearly one-half of patients succumb as a result of formation of metastasis. In an initial study expression profiles of early colonic carcinomas have been studied by whole human genome oligonucleotide microarrays. GeneChip analysis led to the identification of a number of candidate genes showing a strongly differential expression between epithelial cells of healthy and tumor patients, but also between non- and metastasizing carcinomas. Moreover, several genes showing a strong over- or underexpression in carcinoma cells of patients with poor survival were identified. The next step was the development of a low-density microarray with the aim to provide a cheap and easy to use chip for the clinical routine. Oligonucleotides (70-mers) for 300 candidate genes identified in the initial genome wide study were spotted onto glass slides. The prognosis chip was completed by the addition of probes for 90 stably expressed genes, so-called housekeepers, and a number of common prognostic molecules and tissue markers. Colorectal carcinoma samples and normal mucosa were available from the tumorbank of the RRK hospital. Cryosections were prepared of these samples, and after pathological survey and sample processing of sections containing at least 60 % of carcinoma tissue, single color labelled cDNA was hybridized to the chip. Meanwhile, in a retrospective study 70 colorectal carcinoma samples with a long-term follow-up of more than 5 years have been examined. Bioinformatic analysis proved the robustness of the profiling. Using unsupervised random-forest clustering a number of genes which show an expression correlating well with disease-free survival was identified. Our results show that by using the prognosis chip a better prediction of patient survival can be achieved than by using histopathological parameters such as invasiveness (T) and grading (G) alone. In a ongoing prospective study we are now evaluating the prognostic value of the microarray for patients who have been treated by surgery more recently.

Clinical relevance of glycan structures – GlycoProfiler-Chip

At least 1 % of the transcribed human genome is devoted to the production of proteins involved in synthesis, degradation and function of glycoproteins. Many of these glycoproteins are components of the cell membrane; therefore, it is assumed that glycans function as mediators of the interaction between cells or between cells and signalling molecules. Of clinical relevance are alterations in the glycosylation profile which accompany onset and progression of carcinomas and are of crucial relevance for tumor metastasis. The Thomsen-Friedenreich glycotope, but also the glycanstructures Sialyl-LeX and Sialyl-LeA, ligands of the selectins, are in use as markers for tumour prognosis and tumour recurrence. Due to their complexity and due to the limited amount of material, the quantitative analysis of single glycan structures in clinical samples is rather difficult. However, in many cases the structures of the cell surface glycan chains can be inferred, at least partially, from the protein- or even the mRNA-expression pattern of the anabolic enzymes, the glycosyltransferases. Moreover, we and others have found that tumor-associated glycosyltransferase mRNA-expression by itself could be of prognostic value for predicting patient survival. Therefore, monitoring of glycosyltransferase mRNA-expression using highly parallel methods such as microarrays is of strong interest. In principle, DNA-microarrays are well suited to determine which molecules of the glycosylation machinery are actively transcribed within a cell line or tissue. The advantage of these microarrays is the comprehensiveness of the analyis, which encompasses all or most of the molecules of the glycosylation machinery, its speed and its modesty in terms of the need of sample material. The aim of the project is to develop a low-density microarray for the analysis and monitoring of the glycosylation of cells used for the production of glycoproteins. This array might be suitable also for the diagnosis and prognosis of carcinomatous diseases. Meanwhile, the "GlycoProfiler" contains probes for about 220 genes of the glycosylation machinery. Several studies using this microarray have shown its reliability in comparison to quantitative RT-PCR. Furthermore, our results demonstrated that microarray-analyses are helpful for the understanding of cell glycosylation and even the functionality of a specific glycosylation pattern.

Endogenous Protoporphyrin IX Accumulation in Gastrointestinal Cancers

Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism. Previously, we demonstrated accumulation of the heme precursor protoporphyrin-IX (PpIX) in gastrointestinal tumor tissues. In order to elucidate the mechanisms of PpIX accumulation, by quantitative RT-PCR we studied expression of the relevant enzymes of the heme-synthetic pathway. Meanwhile, we were able to describe a significant down regulation of ferrochelatase (FECH) mRNA expression in gastric, colonic, and rectal carcinomas. Accordingly,in an in vitro model of several carcinoma cell lines, ferrochelatase downregulation and loss of enzymatic activity corresponded with an enhanced PpIX-dependent fluorescence. Direct detection of PpIX in minute amounts was achieved by a specifically developed pulsed solid-state laser dual delay fluorimetry setup. Silencing of FECH using siRNA-technology led to an up to 50 fold increased PpIX-accumulation, imageable by a specifically adapted two photon microscopy unit. Our results show that in malignant tissue a transcriptional downregulation of FECH occurs, which causes endogenous PpIX accumulation. Furthermore, accumulation of intracellular PpIX due to FECH siRNA-silencing provides a small molecule based approach to molecular imaging and molecular therapy.
Possible applications of this method are e.g. : i) FECH-silencing might be suitable for therapeutic approaches, e.g. it might be possible to achieve via light treatment a selective destruction of siRNA-transfected cells. With regard to conventional cytotoxic constructs used in gene therapy approaches, the cytotoxic effect in this case requires and relies upon a local component, light activation. Therefore, if a gene therapeutic construct has affected cells in light-protected areas of the body, a restriction of specific light activation to the target areas may provide cytotoxicity with limited side effects. Local application of siRNA will improve the specificity of photodynamic therapy with regard to the treated area and even the specific cell type which is attacked. This might lead to a new kind of local cancer therapy which is essentially non-invasive and well tolerated by patients. ii) Detection of fluorescently labelled of cells in the body by specific delivery of FECH-siRNA to specific cell types or tissues ii) Induction of endogenous fluorescence by FECH-silencing as a general read-out for determining the efficiency of siRNA-delivery in various transfections systems.
Patent: EPO 06018993.3-1212

Molecular imaging of breast cancer in a transgene mouse model

The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma and is increasingly diagnosed since the introduction of high-quality mammography screening. As a model serves a set of transgenic mice (WAP-T and WAP-T-NP lines) presenting with various morphological forms of DCIS-like lesions. In these mice the SV40 large tumor antigen is specifically induced in epithelial cells of the terminal duct lobular units. As a consequence of continuous expression of the oncogene, the animals develop multifocal DCIS and consequently invasive carcinoma within strain specific periods of latency. DCIS lesions in transgenic mice exhibit distinct architectural and cytological features which closely resemble those commonly present in humans. Purpose of the study is to detect early breast cancer and its precursors by in vivo optical molecular imaging in an animal model. One molecular target for imaging are molecules associated with tumour angiogenesis.

Early diagnosis and monitoring of breast cancer

Breast cancer is in most cases a chronic disease, with diagnosis and treatment followed by extended periods of both low grade disease and apparent disease free intervals. Early diagnosis is prerequisite to better therapy and a higher chance of being cured. Once it is treated, recurrence of the disease requires further treatment where possible. Diagnosis is currently performed by imaging and biopsy.  This application will explore a SmartHEALTH system for home monitoring, or which might be used in the presence of the patient’s GP. A panel of approximately 8-12 marker proteins – specific to breast cancer – will be detected. The overall objectives of the SmartHEALTH project is to develop innovative Point of Care test systems for biomarker determination for the future patient management at all levels of disease management, i.e. risk assessment, screening/early diagnosis, classification of disease and selection of therapy, and monitoring of disease progress. The SmartHealth system shall be used within several disease areas, however in the project the use of biomarker determination at different stages in the clinical evolution of cancer are used to exemplify the different applications of the SmartHealth diagnostic system. The clinical applications selected in the SmartHEALTH project address the different sample materials and biomarkers (body fluids, cells, tissue biopsies, proteins, DNA and RNA) and cover the different test locales for biomarker analyses. Therefore the chosen clinical applications can be used to demonstrate all functions intended to be included in the SmartHEALTH diagnostic system.

Current publications

  Rohwer N, Lobitz S, Daskalow K, Jöns T, Vieth M, Kemmner W, Wiedenmann B, Cramer T, Höcker M.
HIF-1a determines the metastatic potential of gastric cancer cells. Br J Cancer, 2009, in press

  Anders M, Vieth M, Röcken C, Ebert M, Pross M, Gretschel S, Schlag PM, Wiedenmann B, Kemmner W, Höcker M.
Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression.
results of a prospective randomized trial. Br J Cancer, 2009, in press

  Schulze T, Kemmner W, Weitz J, Wernecke KD, Schirrmacher V, Schlag PM.
Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in
colorectal cancer patients following resection of liver metastases:
results of a prospective randomized trial. Cancer Immunol Immunother 2009 58:61-9

  Astrosini C, Roeefzaad C, Dai YY, Dieckgraefe BK, Jöns T, Kemmner W.
REG1A expression is a prognostic marker in colorectal cancer
and associated with peritoneal carcinomatosis. Int J Cancer 2008,123:409-413

  Kemmner W, Wan K, Rüttinger S, Ebert B, Macdonald R, Klamm U, Moesta T.
Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation
in colon cancer - a new tool for molecular imaging. FASEB J 2008,22:500-509

  Gretschel S, Astrosini C, Vieth M, Jöns T, Tomov T, Höcker M, Schlag PM, Kemmner W.
Markers of tumour angiogenesis and tumour cells in bone marrow in gastric cancer patients.
Eur J Surg Oncol 2007, in press

  Bembenek A, Li J, Loddenkemper C, Kemmner W, Stein H, Wernecke KD, Schlag PM.
Presence of mature DC-Lamp(+) dendritic cells in sentinel and non-sentinel lymph nodes
of breast cancer patients. Eur J Surg Oncol 2007, in press

  André S, Sanchez-Ruderisch H, Nakagawa H, Buchholz M, Kopitz J, Forberich P,
Kemmner W, Böck C, Deguchi K, Detjen KM, Wiedenmann B, von Knebel Doeberitz M,
Gress TM, Nishimura SI, Rosewicz S, and Gabius HJ. Tumor Suppressor p16INK4a:
Modulator of Glycomic Profile and Galectin-1 Expression to Increase Susceptibility
to Carbohydrate-Dependent Induction of Anoikis in Pancreatic Carcinoma Cells.
FEBS J 2007;274(13):3233-56

  Nazarenko I, Kristiansen G, Fonfara S, Guenther R, Gieseler C, Kemmner W,
Schafer R, Petersen I, and Sers C.  H-REV107-1 stimulates growth in non-small cell
lung carcinomas via the activation of mitogenic signalling.
Am J Pathol, 2006;169:1427-1439