Pergamon EurqmiJomalofCanVol. 31A,No.5,pp.73~738,1995 Copyright 0 1995Ekvier Science Ltd Printed in Great Britain. AUrights reserved 09594049195 $9.50+0.00 Detection of 2753 Gene Mutations in Human Sarcomas J.S. Castresana, M.-P. Rubio, L. Ghmez, A. Kreicbergs, A. Zetterberg and C. Barrios To determine the frequency and type of TP53 mutations in human sarcomas, we examined exons 5-8 of the TP53 gene in 48 sarcomas using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. Nine tumours had mobility-shifts on SSCP analysis, and sequencing of six of these tumours revealed 10 mutations: one insertion, two deletions and seven point mutations (four transitions and three transversions). Four of these mutations resulted in frame-shifts, one in a truncated protein, four cases in mono-allelic point mutations and one case in an altered splice site. These data show that approximately 20% of sarcomas harbour TP53gene alterations and illustrate a variety of TP53 gene mutation types. Key words: tumour suppressor gene, TP53, point mutation, sarcomas, PCR-SSCP, diiect sequencing EurJ Cancer, Vol. 31A, No. 5, pp. 735-738,199s zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE INTRODUCTION THE INACTIVATION of tumour suppressor genes appears to be integral to the development of several types of human tumours [ 11, including human sarcomas [2, 31. Evidence has implicated the TP53 gene as a tumour suppressor gene [4] and alterations of the gene are frequently observed in many human tumours [S], including neoplasms of the colon [6], lung [7], breast [8] and brain [9, lo]. Studies of human sarcomas have also shown TP53 alterations, but have produced varied results. For instance, a variety of mechanisms of gene inactivation have been observed, including point mutations, rearrangements and deletions. The frequencies of TP53 mutations have varied, from approximately one-sixth [Ill to one-third1 [12] of all sarcomas, with most studies showing the majority of mutations in osteosarcomas. Furthermore, germline mutations of the TP53 gene have been associated with soft tissue a.nd bone sarcomas, both in the Li- Fraumeni syndrome [ 131 and in sporadic sarcoma patients [ 121. To determine the frequency and type of TP53 mutations in human sarcomas, we examined exons 5-8 of the TP53 gene in 48 sarcomas using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. Correspondence zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA to J.S. Castresana at the Xnstituto de Investigaciones Biomedicas,CSIC, Arturo Duperier 4, E-28029Madrid, Spain. J.S. Castresana, M.-P. Rubio and L. amez areat the Molecular Neuro- Oncology Laboratory and the Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, 149-13th Street, Charles- town, Massachusetts 02129, U.S.A.; J. S. Castresana and A. Zetterberg are at the Department of Tumour Pathology, Karolinska Hospital and Institute, S-10401 Stockholm, Sweden; A. Kreicbergs and C. Barrios are at the Department of Orthopaedics, Karolinska Hospital and Insti- tute, S-10401 Stockholm, Sweden; C. Barrios is also at the Orthopaedics and Trauma Institute, Deparrment of Surgery, Valencia University Medical School, Blasco Ibanez 17, E-46010 Valencia, Spain. Revised 20 Jul. 1994; accepted .22 Jul. 1994. MATERIALS AND METHODS zyxwvutsrqponmlk Tissue specimens and histopathology Tumour samples were obtained from 48 patients with muscu- loskeletal sarcomas surgically resected at the Department of Orthopaedics, Karolinska Hospital, Stockholm, Sweden. No patient had been treated by chemotherapy or radiotherapy before surgery. There were 13 bone tumours (sevenosteosarcomas, four chondrosarcomas and two Ewing’s sarcomas) and 35 soft tissue sarcomas (12 malignant fibrous histiocytoma (MFH), nine neurofibrosarcomas, four liposarcomas, two fibrosarcomas, two angiosarcomas, two hemangiopericytomas, one malignant mesenchymoma, one leiomyosarcoma, one lymphangiosarcoma and one rhabdomyosarcoma). Purification of the DNA Immediately after surgical removal, the tumour samples were frozen in liquid nitrogen and stored at -70°C until DNA extraction. High molecular weight DNA was isolated by lysis of small pieces of tumour tissue in a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 7.6, 5 mM EDTA (ethylenediaminetetraacetic acid), 0.5% SDS (sodium dodecyl sulfate) and 250 p&ml proteinase K at 56°C overnight. The DNA was purified by repeated extractions with phenol/chloro- form/isoamyl alcohol (25:24:1), precipitated in 100% ethanol and 0.3 M sodium acetate, and dissolved in TE (10 mM Tris pH 7.4,0.1 mM EDTA) buffer. PCR-SSCP analysis of the TP53 gene SSCP analysis was performed according to the procedure of Orita and associates [14], with minor mod&cations. Exons 5,6, 7 and 8 of the TP53 gene were amplified using five sets of primers previously described [12]; portions of the Seth exon were amplified with two different sets of primers. The reaction mixtures contained 50-100 ng DNA, 200 pM dATP, 200 pM dTTP, 200 p,M GTP, 20 pM dCTP, 1 &i of alpha-32P-dCTP (Amersham, Aylesbury U.K.; specific activity, 3000 Ci/mmol), 735