DETERMINAZIONE MOLECOLARE DEL RISCHIO DI METASTASI EPATICHE NEI PAZIENTI CON CARCINOMA COLORETTALE. PRIN 2005; metastasi epatiche da cancro del colon-retto: fattori di rischio, prognosi e trattamento su basi molecolari (responsabile nazionale: BIASCO G.)

Prognostic assessment and therapy of patients with CRC mainly relies on accurate staging of the disease. However, the clinical outcome of patients with stage II and III tumors (approximately 70% of cases) is largely unpredictable. Although recent advances in our knowledge of the molecular biology of CRC have led to the identification of potential prognostic and predictive factors and new active compounds are now available to treat this tumor, to improve patients' management there is clearly the need to identify molecular markers that could reliably predict the clinical course of the disease and the response to different therapeutic regimens. Objective of this project is the identification of molecular markers predicting: a) risk of liver metastases and clinical outcome among patients with stage II and III colorectal cancer; b) responsiveness to chemotherapy with 5-FU or other cytotoxic drugs. In particular, genetic and epigenetic changes will be investigated. In addition, the prognostic value of a pathologic parameter indicative of effective local cytotoxic anti-tumor immune response will be evaluated. The relationship between genetic alterations (MSI status, grade of chromosomal instability and allelic losses of several chromosomal arms) and metastasis-free survival and overall survival will be studied in a large series of patients (more than 400) with stage II or III CRC, surgically resected between January 1999 and December 2002, who received adjuvant chemotherapy. These patients have been followed at the Division of Clinical Oncology of the S. Anna Hospital (Ferrara, Italy). Data obtained from the study of this series of patients will be compared with those previously obtained from the analysis of a large number of stage II and III patients who did not received adjuvant chemotherapy. In this way interactions between genetic changes, adjuvant chemotherapy and clinical outcome will be better investigated. Furthermore, among patients who have developed hepatic metastases the relationship between genetic characteristics of the tumors and response to chemotherapy will be evaluated. MSI and allelic losses will be determined by microsatellite analysis using DNA extracted from fresh-frozen or formalin-fixed paraffin-embedded tissue specimens and a fluorescence-based PCR method. Products of PCR amplification will be evaluated by an automated DNA sequencer. Allelic loss will be assessed at chromosomes 18q, 17p, 8p, 5q and other chromosomal arms. In the majority of tumors, immunohistochemical analysis of MLH1 and MSH2 expression will be performed. In this series of tumors, the density of intraepithelial cytotoxic T lymphocytes (CD8+ TILs) will also be determined, using immunocytochemistry and a digital image analysis system. CD8+ TIL density will be related to clinical outcome and patients with MSI-H and non-MSI-H tumors will be analyzed separately. Epigenetic changes occur at high frequency in CRCs and are thought to play a relevant role in the development of this tumor type. In addition, the existence of a hypermethylator phenotype termed CIMP characterized by the contemporary promoter methylation of several genes has been assumed. However, the prognostic and predictive value of epigenetic changes in CRC is poorly understood. The study of epigenetic alterations will be conducted in two phases. In the first phase, a consecutive series of 100 CRCs will be investigated. These tumors have been previously characterized for MSI status, DNA ploidy, MLH1/MSH2 and p53 expression, and multiple allelic losses (18q, 17p, 8p and 5q). Methylation status of the promoter of several genes, such as MLH1, MGMT, CHFR, HLTF, RASFF1, p16, p14 and APC, and of CpG islands (MINT1, MINT2, MINT12 and MINT31) will be investigated by methylation specific PCR. The "grade of epigenetic instability" will be assessed as the fraction of genes/loci hypermethylated by the total number of genes/loci examined. As the concept of CIMP phenotype is controversial, a special effort will be focused to relate grade of epigenetic instability to clinico-pathologic features, MSI status and other genetic changes. Furthermore, the relationship between methylation status of the different genes and of grade of epigenetic instability and patients' long-term clinical outcome will be evaluated. In the second phase, methylation status of selected genes/loci will be assessed in the large series of stage II and stage III tumors, to evaluate more precisely their prognostic value and to determine their relation with benefit from chemotherapy. Preliminary results from our laboratory seem to demonstrate that loss of MGMT protein expression might represent a more specific indicator of MGMT gene inactivation than promoter hypermethylation. For this reason, in our project immunohistochemical analysis of the expression of selected genes will be performed in conjuction with promoter methylation status evaluation. To this end, tissue microarrays will be prepared and utilized. As inactivation of the DNA repair gene MGMT seems to play a major role in the development of a subgroup of CRCs and seems also to be involved in the clinical response to chemotherapy with different drugs, immunohistochemical analysis of MGMT protein expression will be done in the whole series of stage II and III carcinomas. At the end of the study, a comprehensive statistical analysis will be performed to select molecular parameters more closely related to the development of liver metastases, to disease-free and overall survival, and also to benefit from chemotherapy.