Principal Investigator: Prof. Zoran Krivokapic, Faculty of Medicine, University of Belgrade

This project has been designed to consist of three parts or sub-projects dealing with the investigation of various aspects of malignant disease from fundamental research to determination of prognostic factors and treatment modalities, both in the early and in the advanced phases of the disease. Research within the proposed project would include testing of mechanisms that would lead to cell cycle aberrations in patients with colon and rectal cancer. Also, the role of stress proteins Hsp90/70 in colorectal carcinogenesis is to be examined as well as the importance of disregulation of proteins that regulate the adhesion process and the antitumor immune response. One of the objectives of the proposed project is the determination of proteolytic activity and expression of matrix metalloproteinase (MMP-2 and MMP-9) in serum and in the tissues of patients with rectal cancer and their correlation with tumor stage. Activity of MMP-2 and MMP-9 in tissues would be determined by using the immunohistochemical method. The second purpose of this study is to determine the concentration of microelements zinc and copper and their correlation. The concentration of trace elements would be determined by using flame atomic absorption spectrometry. The superoxide dismutase enzyme would be determined by using standard methodology. The research is also based on the genetic phenomenon of microsatellite instability (MSI) in colorectal cancer (CRC). The analysis is performed using Pentaplex PCR with five marked primers, followed by the analysis of the fragments to obtain the result (fragment analysis). In addition to the MSI status, the mutation status of K-ras and B-raf genes using the RFLP method is to be assessed and subsequently confirmed by direct sequencing. The significance of MSI whould also be explored, as well as that of B-raf and K-ras mutations, for sporadic CRC among our patients. The analysis would be performed on DNA isolated from malignant and non-malignant tissue of patients with colorectal cancer and patients with benign colon disease. In all patients the response to a variety of therapeutic treatments in a specific period of time will be monitored. DNA analysis would be performed by using methods based on PCR technique, and entail laser detection of fluorophore marked DNA. Testing based on digital images and excitation-emission spectra has not yet been included in this scientific area, i.e. in the diagnosis and treatment of colorectal carcinoma. Testing based on digital images relies on knowledge that malignant tissue is organized into specific structures that can be analyzed and detected by using specific software. The excitation-emission spectrum method involves the usage of opto-magnetic spectroscopy (OMC). The sample is exposed to two rays of light directed from two angles (perpendicular to the substrate and at the Brewster's angle). When the sample is illuminated perpendicularly, the reflected light contains information about the electromagnetic properties of the sample. When the ray comes at the Brewster's angle, linear polarization of light occurs and the light reflected has only the electrical component. The measured difference of the two reflections reveals the magnetic properties of the surface of the sample - the opto-magnetic fingerprint. The aim of this study is to increase the sensitivity and specificity of the test in occult bleeding, introduce a noninvasive way to determine preoperatively, the degree of differentiation of rectal tumors, as well as the quality of healthy and diseased tissue before and after preoperative chemo/radio therapy. In determination of the local disease stage, high-resolution magnetic resonance imaging of the pelvis minor with will be used. In all patients, this assessment will be done before surgery and then after any preoperative radio/chemotherapy. The aim of this study, inter alia, would be to determine valid indications for intersphincteric resection of the rectum. Preoperative testing of the function of the pelvic floor muscle by anal manometry would be performed. Endoanal ultrasonography would be used to estimate the thickness of the anal sphincter. The proposed project includes verification of the optimal radiation protocol depending on the stage of the disease (T3, T4, N1-2 in mesorectum in long course or short course protocols and the impact on the local recurrence rate and five-year survival). The implementation of the project on advanced illness treatment modalities is expected to identify predictive markers of response to chemotherapy and thus enable the optimization of chemotherapy. We also expect to determine the prognostic significance of response to chemotherapy in the neoadjuvant approach as well as in metastatic cases, and to form a prediction model for the given disease stage that would include other parameters too. The place and role of biological therapy in combination with cytotoxic chemotherapy and radiation treatment in all disease stages would be determined. Quantification of patient's quality of life during and after combined chemo/radiotherapy, and surgical treatment would also be achieved. In the treatment of peritoneal dissemination of disease survival length and time to relapse would be determined in relation to intraoperative PCI score, completeness of cytoreduction, residual status and the chemotherapy protocol used.