The overall objective of the project is an integrated development of a molecular diagnostic test system that not only supports targeted, individualized tumor therapy, but also allows a deeper insight into the molecular mechanisms of carcinogenesis by revealing new scientific evidence. Based on previous research on novel tumor suppressors and tumor marker genes, which partners published in more than fourty high impact research papers, they expect to discover and characterize new therapeutic drug target molecule(s) and to obtain information facilitating the early diagnosis and potential prevention of certain malignancies. Partnership plans to focus its research efforts on common cancers of public health significance, with particular focus on colon and lung cancers. It is one of the main objectives to establish a multidisciplinary consortium which is intended to act as a special mediator between basic research and clinical practice.Specific objectives can be the following: 1. Identification of novel tumor markers by characterizing molecular mechanisms in generation of underlying mutations the main facilitators of carcinogenesisWe have published more than forty publications on the characterization of the main genetic elements affecting mutagenesis (e.g. translesion DNA polymerases, HLTF, SHPRH and Rad6-Rad18) in high-impact international journals (Nature, Nature Genetics, Molecular Cell, PNAS). In the course of the planned project we wish to determine the role of these molecules in the formation of point mutations in the molecular marker genes (e.g. EGFR, K-RAS, BRAF, PIK3CA) utilized in individualized tumor therapy, an explosively developing field. 2. Development of a patentable molecular diagnostic test system serving early tumor diagnosis and targeted molecular tumor therapy3. Linking the population heterogeneity of the stress response and membrane structure in primary tumor cell cultures Achievements: For the detection of mutations occuring in codon 12.-13. of the K-RAS protooncogene BRC developed a new method: the so-called PNA (Peptide Nucleic Acid) mediated allelediscrimination. SZTE collected and characterized more than 100 FFPE samples from colorectal cancer and lung adenocarcinoma. Partner from NoviSad started preparation of tumor cell suspensions and their characterization using ICH analyses. Two primary cell cultures were established from tumor cell suspensions, which later gave two stromal fibroblast-like cell lines. BRC continued the sensitivity assays started by SZTE. BRC compared their sensitivity to cisplatin,doxorubicin, 5-fluorouracil, hidroxyurea and MMS. SZTE examined the microsatellite instability of 72 samples. Among these tumors 10 was microsatellite instable (7 MSI-L and 3 MSI-H) and 14 was EMAST positive. SZTE also determined the mutational status of BRAF of these 10 microsatelliteinstable samples by allele specific PCR. NoviSad Partner continued with preparation of tumor cell suspensions and their characterization using immunocytochemical (ICH) analyses. Immunohistochemical (IHH) identification of specific markers was performed on both primary cell suspensions and tumor tissue. SZTE developed the molecular analysis of a new tumor marker, the HLTF gene. We planned to determine the HLTF promoter methylation status of 72 colon cancer samples. SZTE used bisulfite treatment of DNA to determine the pattern of methylation. BRC continued to test FFPE samples for K-Ras mutation with our KRAS Diagnostic Kit using HCT116 (K-RAS mutant) as positive control. For the detection of mutation V600E of B-RAF gene BRC developed another method besides theallele-specific PCR reaction: the HRM (High Resolution Melting). BRC also started to develop diagnostic method for the detection of frequent mutations in gene PIK3CA. BRC continued the sensitivity assays that were started in the previous period. BRC used HeLa cell lines silented byRAD18 siRNA, HLTF siRNA and control siRNA and cell lines transfected with RAD18 and HLTF overexpressing plasmids. We compared their sensitivity to 5-fluorouracil, doxorubicin,methotrexate, MMS, etopozid and avastin. welve primary tumor cell (PTC) suspensions were prepared and their characterization was performed by NoviSad Partner.