1.2 Detection and validation of new biomarkers in colorectal cancer

Cooperation: Cluster for Translational Oncology
Institute for Surgical Oncology
Institute for Stem-Cell Transplantation
Department of Pathology Donauspital SMZ Ost

Based on a well defined and selected group of patients the overall objective of the project is to investigate certain molecules as potential biomarkers. We intend to evaluate if these molecules will help to determine the malignant phenotype and metastatic potential of colorectal cancer and aid in the assessment of the prognosis of operated patients with colorectal cancer.

Tumor specimens of 300 patients with colorectal carcinoma and suitable UICC stages and established clinical classification as relapses or disease-free survivors had been identified and are used for identification of expression of certain genes using immunohistochemistry. After identification of the appropriate candidate gene, genetic and epigenetic analyses are planed.

Patient selection is completed and was done as described below. Tissue samples of 300 patients who were operated for colorectal cancer at the department of surgery Donauspital SMZ Ost between 1992 and 2002 will be included in the study.
Inclusion criteria:
1.) Surgery for primary colorectal cancer and minimum follow up period 60 months (n: 300).
2.) No history of additional cancer.
3.) Patients age between 18 and 80 years.
4.) Tumor stage UICC II (n: 150) and UICC III (n: 150).
5.) Seventy fife patients of each stage group experienced a relapse of disease within 5 years postoperative and 75 did not.
Exclusion criteria
1.) Apparent polyposis syndrome or a hereditary cancer syndrome.
2. An additional inflammatory condition of the colon (i.e. Diverticulitis, Inflammatory bowel disease).
3. Emergency operations for i.e. an obstructive situation of the colon or rectum.
4.) Neo-adjuvant therapy for rectal cancer.

After expression analysis by immunohistochemistry it is planed to perform genetic and epigenetic analysis of the respective genes as well as prospective confirmatory studies on fresh tumor tissue of benign and malign neoplastic lesions. Ethical approval is grantaed.
Several grant applications for financing are awaiting their respective decision.

1.3. Repeated bone marrow assessment in colorectal cancer for detection of disseminated tumor cells

Cooperation: Cluster for Translational Oncology
Institute for Surgical Oncology
Institute for Stem-Cell Transplantation
Department of Pathology Donauspital SMZ Ost

Five year follow up will be completed by the end of 2009.
A preliminary publication reporting the clinical significance of repeated bone marrow assessment for DTCs is accepted for publication in “Colorectal Disease”. The official Journal of the European Association of Coloproctology; The Association of Coloproctology of Great Britain and Ireland; The Spanisch Society of Coloproctology”.


LBI of Clinical Oncology and Photodynamic Therapy

One of the several experimental projects was performed as part of an international cooperation, the other projects within different institutions of the LBG cluster, combining different techniques and expertise. The projects aimed at evaluation of preclinical toxicity and cytotoxicity of new drug compounds and possible drugs sources in order to find new agents for the targeting of repopulating tumor cells, that are responsible for tumor relapses.

(1) In vitro testing of a therapeutic drug in a long-term hepatocyte system (HEPAC)
The company Primacyt (Cell Culture Technology GmbH, Schwerin, Germany) established an in vitro cytotoxicity test system (HEPAC) using human hepatocytes, that allows for a 28-day continous cell culture evaluation of potential drug candidates in order to replace toxicity tests in experimental animal models and to provide relevant preclinical human data. In our institution, validation of the HEPAC system was done using the antitumor compound cis-hydroxyproline (CHP) for which animal toxicity test results and phase I clinical data were available. Comparison of the results obtained using the HEPAC system, rat toxicity tests and laboratory clinical parameters obtained in patients revealed a better correlation for the HEPAC results and the clinical data than for the animal toxicity testing.
Abstract: cis-hydroxyproline is being clinically evaluated as an anticancer drug. Since this compound targets the production of L-proline-rich proteins and critical L-proline residues, its impact on long-term cultures of human hepatocytes and toxicity in rats was studied to investigate possible effects on hepatic function, previously reported in rat hepatocytes. In the HEPAC2 human hepatocyte culture system, concentrations of CHP below 3.2 mg/ml had no significant effects on the release of lactate dehydrogenase (LDH), albumin, and urea. In rats, continuous administration of three different doses of CHP were tested for 28 days and resulted in signs of liver damage, as indicated by elevations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at a dose of 903 mg/kg, corresponding to a plasma concentration of approximately 200 μg/ml. Data from a clinical study of CHP in bladder and prostate cancer patients showed no adverse effects of administration of 8 g CHP/day, 4 days/week for 3 weeks in liver parameters ALAT, ASAT, γ-glutamyltransferase (γ-GT) and alkaline phosphatase (AP). In conclusion, the HEPAC2 human hepatocyte culture system correlates well with clinical results of a Phase II study of CHP, whereas a previous rat hepatocyte culture system predicted the compound would have toxic effects. The HEPAC2 system therefore constitutes a valuable tool for the preclinical screening of the hepatotoxicity of chemotherapeutic and other drugs, thereby reducing the need for experimental animals.
According to these results the in vitro HEPAC system constitutes a promising tool for the replacement of animal toxicity tests, providing data more relevant for human clinical studies than xenogenic systems. This work was rewarded with the 27th Research Award for the Development of Methods sparing Animal Experiments, granted by the German Ministry for Food, Agriculture and Consumer Protection, Berlin, Germany.

Ref. Dickens H, Ullrich A, Runge D, Mueller B, Olszewski U, Hamilton G (2008) Anticancer drug cis-4-hydroxy-L-proline: Correlation of preclinical toxicology with clinical parameters of liver function. Molecular Medicine Reports 1: 459-464

(2) Preclinical characterization of the platinum(IV) drug Oxoplatin.                         Oxoplatin (cis,trans,cis-diammine-dihydroxo-dichloro-platinum(IV)), presently under investigation as an oral antitumor agent, was screened in vitro using MTT proliferation assays in a panel of 38 diverse human tumor cell lines. With exception of renal cell and ovarian cancer, cell lines sensitive to oxoplatin were detected for all other tumor entities. Using flow cytometry, the drug was shown to induce cell cycle arrest (propidium iodide staining), reactive oxygen species (ROS; dihydroethidium fluorescence) and cell death (annexinV / propidium iodide staining). Generally, the mean IC50 value for oxoplatin (sensitive cell lines: 7.6 ± 5.8 µg/ml; n = 27) was ~ 2.5-fold higher than for cisplatin. However, the small cell lung cancer (SCLC) cell line H526 showed similar sensitivity to oxoplatin and cisplatin and was used for genome wide expression profiling (Applied Biosystems Human Genome Survey Microarray V2.0) following application of the two compounds. Only 35 % of the downregulated and 10 % of the upregulated genes were identical in response to cisplatin or oxoplatin, respectively. Diametrically regulated genes included ribosomal proteins, DEAD box polypeptide 1, tubulins, pyruvate kinase, stathmin and high mobility group nucleosomal BD2 (HMGN2), indicating that oxoplatin is not a simple prodrug of cisplatin, and that these two platinum compounds comprise distinct mechanisms of action. Since untreated H526 cells exhibited marked acidification of the medium to pH = 6.5 rapidly, effects of acids on oxoplatin were investigated. While addition of 50 – 800 µg/ml lactate had no effect on the cytotoxic activity of oxoplatin against COLO 205 cells, pretreatment of the compound with 5 mM ascorbic acid yielded enhanced activity (~ 2.5-fold), and thiols activated oxoplatin to a minor extent (< 10 %). Following a 15 min exposure of oxoplatin to 0.1 M HCl representing gastric acid, the resulting platinum species cis-diammine-tetrachloroplatinum(IV) revealed a more than twofold increase in cytotoxicity against various cell lines. In conclusion, oxoplatin constitutes an effective oral anticancer agent that can be converted into more active platinum intermediates different from cisplatin by acidic conditions depending on its pharmaceutical formulation. Platinum(IV) compounds are expected to have a higher probability to reach the tumor tissue intact and to be activated eventually at the intended target site than platinum(II) drugs.
Ref. Olszewski U, Ach F, Zeillinger R, Ulsperger E, Baumgartner G, Hamilton G (2008) Expression profiling reveals different targeted genes of the oral platinum(IV) prodrug oxoplatin and its putative activation product cisplatin. P. 120; EACR 20 Meeting, Lyon, France, July 4th – 8th, 2008

(3) Antitumor activity of a Zanthoxylum fruit extract.
The majority of new antitumor drugs were isolated from plants and organisms or used natural compounds as drug leads. In search of new anticancer compounds we investigated fruits of Xanthoxylum sp. plants, that are in use as spice and folk medicine in Asia as antiseptic, adstringent, carminative, stomachic, and anthelmintic drug. The essential oils of the fruits are known to contain linalool, E-carveol, limonene and methyl cinnamate as main constituents. Dried fruits were extracted with ethanol. Proliferation assays were performed for four days and viable cells assessed using MTT assays. Cell cycle analysis on ethanol-fixed and propidium iodide/RNAse A-treated cells was carried out by flow cytometry. RNA was extracted from treated and control cells and gene expression analysis performed using the human genome survey microarray V2.0 (32K, Applied Biosystems, Foster City, CA, USA). Cytotoxic activities of dilutions of the extract were evaluated in a panel of leukemia, small cell lung cancer (SCLC), pancreatic and colon cancer cell lines. IC50 values ranged from 0.6 mg/ml for HL-60 leukemia cells to 1.8 mg/ml for drug-resistant NCI-H526 SCLC cells. COLO 205 colon cancer cells revealed S-phase arrest in response to Zanthoxylum extract, whereas the more chemosensitive HL-60 cells accumulated in G1/0 phase. Sensitive cells exhibited signs of mixed apoptotic/necrotic cell death as demonstrated by annexinV/ propidium iodide as well as toluidine blue staining of semi-thin cell-culture sections. Genome-wide expression analysis of extract-treated versus control COLO 205 colon cancer cells revealed autophagin, TRAIL receptor, protocadherin, cyclin C, prostaglandin receptor, metallothioneines, and S100 protein as the most markedly induced, and myeloperoxidase, RAS-GEF, ceramide kinase, translation/elongation factors and metallothioneine proteinases as the most downregulated genes. In conclusion, the extract of fructus Z. armati induces cell cycle arrest and cell death in diverse cancer cell lines, involving increased expression of autophagin and TRAIL receptor, compatible with induction of an oxidative cellular stress response and autophagic/apoptotic cell death.
Ref. Olszewski U, Zeillinger R, Hamilton G (2008) Antitumor Activity of Fructus Zanthoxyli. P32; PSE Conference Natural Products in Cancer Therapy, Naples, Italy, September 23rd – 26th, 2008

(4) M65 cytokeratin fragment as colon cancer tumor and relapse marker.
Cell death in epithelial tumor cells results in release of soluble cytokeratin fragments, namely M65 and the caspase-cleaved fragment M30, specific for apoptotic cell death. The serum concentrations of both fragments were measured in colon cancer patients prio to and one week after tumor surgery. M65 can be used to discriminate between noncancer and low-grade colon cancer patients and furthermore patients that showed persistent high M65 values despite surgery were subject ot early ttumor relapses.
Ref. Ausch CA, Olszewski U, Buxhofer-Ausch V, Ogris E, Hinterberger W, Schiessel R, Hamilton G (2008) Evaluation of perioperative measurements of cytokeratin 18 (M65) and caspase-cleaved cytokeratin 18 fragment (M30) in colon cancer. Abstract No. 7; 2008 ASCO-NCI-EORTC Annual Meeting on Molecular Markers in Cancer, Hollywood, FL, USA, October 30th – November 1st, 2008

(5) Ongoing projects.
In cooperation with two groups at German universities (Greifswald and Rostock) 5 different cell lines each exhibiting drug resistance against different platinum complexes were obtained and subjected to genome-wide expression analysis in order to investigate the different gene products involved in distinct drug resistance profiles (manuscript submitted).
Small cell lung cancer (SCLC) constitutes a subgroup of lung tumors associated with a dismal prognosis and an extremely poor survival rate. Initial response to chemotherapy is followed by drug-resistant relapses within approximately 12 months. We obtained a panel of drug-sensitive and highly resistant SCLC cell lines and initiated genome-wide exression analysis in parallel with the testing of drug sensitivity profiles. Two cell lines have been established within the cluster project and testing has included new chemotherapeutic drugs, like epothilones, pemetrexed, bortezomib and small-molecule signal transduction inhibitors among others. Where available, clinical data for the newer antitumor drugs have been recorded by the participating hospitals.