Molecular Alterations in the TP53 Gene of Peripheral Blood Cells of Patients W ith Chronic Myeloid Leukemia Shoshana Peller, 1 * Rivka Yona, 1 Yulia Kopilova, 1 Miron Prokocimer, 2 N aomi Goldfinger, 4 Akin Uysal, 3 Halil G. Karabulut, 3 Ajlan T ukun, 3 Isik Bokesoy, 3 Gurol Tuncman, 3 and Varda Rotter 4 1 D epartment of Hematology Assaf-Harofe Medical Center, Z erifin, Israel 2 Beilinson H ospital, Petach Tikvah, Israel 3 H ematology-O ncology and Medical Biology D epartments, Ankara U niversity Faculty of Medicine, Turkey 4 D epartment of Molecular Cell Biology, The W eizmann Institute of Science, Rehovot, Israel The TP5 3 gene has been extensively studied in patients with chronic myeloid leukemia (C ML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. W e report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic D N A, we sequenced PC R products of the coding exons and most introns of the TP5 3 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP5 3 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP5 3 mutations, we believe that wild type TP5 3 accumulates in blood cells of CML patients. O ur results, therefore, indicate that molecular changes in coding regions of the TP5 3 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP5 3 in C ML cells may indicate an additional mechanism involving this gene in the pathogenesis of this disease. Genes Chromosomes Cancer 2 1 :2 – 7 , 1 9 9 8 .  1998 W iley-Liss, Inc. INTRODUCTION Mutations in the TP53 gene have been observed in a wide spectrum of human tumors. Most muta- tions cluster to the four highly conserved ‘‘hot spots’’ of the gene (Hollstein et al., 1991; Caron de Fromentel et al., 1992). Mutations in some tumor types have been correlated with advanced stages (Nigro et al., 1989; Rodriguez et al., 1990; Lu et al., 1994). In other cases, mutations in TP53 were found to occur early in tumor development, although these same mutations were maintained in metasta- ses (Davidoff et al., 1991; Sameshima et al., 1992; Reichel et al., 1994). Chronic myeloid leukemia (CML) is a malig- nancy of the hematopoietic system characterized by a translocation between chromosomes 9 and 22 (Kurzrock et al., 1988). The disease has a triphasic course: the initial chronic phase, an accelerated phase, and a terminal blast crisis. The extensive studies of the TP53 gene in CML were recently reviewed by Prokocimer et al. (1994) and Nakai et al. (1995). There is a high variability in the reported incidence of mutations in CML, but most studies agree that the progression of the disease to blast crisis is accompanied by a rising incidence in TP53 mutations (Ahuja et al., 1989, 1991; Kelman et al., 1989; Feinstein et al., 1992; Nakai et al., 1992). The various aberrations of the TP53 gene in these patients have included mis- sense mutations, nonsense mutations, frame shifts, and a case with a mutation at the splice acceptor site of intron 4, resulting in abnormal splicing (Ahuja et al., 1989, 1991; Mori et al., 1992; Nakai et al., 1992). We have analyzed, in 29 blood samples of CML patients, the coding regions and most introns of the gene by direct sequencing. We also report here for the first time the overexpression of wild-type TP53 protein in peripheral blood cells of CML patients determined by immunocytochemistry, which may indicate an additional mechanism involving this gene in the pathogenesis of this disease. MATERIALS AND METHODS Patients Blood samples from 26 patients with CML were studied (Table 1), in 9 women and 17 men. Conven- tional criteria were used for diagnosis; this included Supported by: Hirsch and Genia Wasserman Medical Research Fund, Tel-Aviv University, Israel; Israel Cancer Association (the Huma Monka Averbuch Fund); Contract Grant number 940014-B. Correspondence to: Dr. S. Peller, Department of Hematology, Assaf-Harofe Medical Center, Zerifin, 70300, Israel. Received 22 January 1997; Accepted 11 June 1997 GENES, CHROM OSOM ES & CANCER 21:2–7 (1998) 3 RESEARCH ARTICLES 3  1998 W iley-Liss, Inc.