Del'e!opmelltalapproaches ill cal1cer binlogy 3075 CORRELATION OF PCNA EXPRESSION WITH CUNICO-PATHOLOGIC FEATURES OF NEUROBLASTOMA Javier S. CASTRESANA 1.2, Lourdes GOMEZ2, Angel PESTANA " Purificaci6n GARCIA-MIGUEL 2, Antonio QUEIZAN3, Rocio MARTIN4,

INTRODUCTION AND AIM OF THE STUDY: Neuroblastoma, a tumor arising from the sympathetic nervous system, is one of the most common childhood tumors. Among the clinical factors with prognostic value, age at diagnosis and stage have been shown to be the most reliable. At the molecular level, a) MYCN oncogene amplification correlates with advanced disease stage, b) loss of heterozygosity studies show that chromosome arms 1p and 14q independently suffer losses of genetic material in neuroblastoma, and c) the expression of HRAS , SRC, TRK and LNGFR oncogenes correlate significantly with a better overall prognosis in neuroblastoma patients. Tumor suppressor genes and molecules involved in the control of the cell cycle are presently being tested to elucidate whether they are involved in neuroblastoma tumorigenesis. The p53 tumor suppressor gene is the most commonly mutated gene in human cancer. Proliferating cell nuclear antigen (PCNA) is a 36kD cell-eyeIe-related nuclear protein that is maximally elevated in late G1 and S phase of proliferating cells. The aim of this study was to determine the mutation pattern of the p53 tumor suppressor gene and PCNA expression in 29 neuroblastic tumors, in order to make correlations with known clinicopathologic features. MATERIAL AND METHODS: A series of 29 neuroblastic tumors from Hospital La Paz was studied. After surgical removal, the tumors were excised into two parts, one was fixed in 10% formalin for 24 h and embedded in paraffin for pathological study, and the other one, snap frozen in liquid nitrogen and stored at -70'C for molecular analysis. Tumors were classified according to their localisation, pathologic diagnosis, clinical stage, stroma content, and neuroblastic differentiation (Table 1). Detection of p53 mutations: After extraction of DNA from fresh-frozen tumors, we amplified exons 5 through 8 of the p53 gene by means of the polymerase chain reaction technique (PCR) and then we subjected those amplified radioactively labelled DNAs to single-strand conformation polymorphism (SSCP) analysis in 6% polyacrylamide (20:1 acrylamide:bis-acrylamide) and 4.5% polyacrylamide (49:1 acrylamide:bis-acrylamide) nondenaturing gels with or without 10% glycerol. Gels were run at 3-5 W for 18-24 h at room temperature (with glycerol) and at 4°C in a cold room without glycerol), dried at 80°C for 2 h, and exposed to X-ray films with intensifying screens at -70°C between one and 2 days. p53 and PCNA immunohistochemical expression: Sections were cut from the paraffin blocks, and routine immunohistochemical methods were used to detect p53 and PCNA proteins. Mouse monoclonal primary antibodies for p53 (Oncogene Research Products) and PCNA (Biomeda) were used at dilutions of 1:20 and 1:400 respectively. Sections were incubated with biotynilated goat antimouse IgG (Dako) as the secondary antibody, at a dilution of 1:400. Signal was visualized using the streptavidin-biotin-peroxidase method. The slides were counter stained with methyl green.