p53 Gene Mutation, Tumor p53 Protein Overexpression, and Serum p53 Autoantibody Generation in Patients with Breast Cancer KATERINA ANGELOPOULOU, 1 HE YU, 3 BHUPINDER BHARAJ, 1 MAURIZIA GIAI, 4 and ELEFTHERIOS P. DIAMANDIS 1,2 1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario; 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 3 Department of Medicine, LSUMC School of Medicine, Shreveport, LA; and 4 Department of Gynecologic Oncology, Institute of Obstetrics and Gynecology, University of Turin, Turin, Italy Objectives: Autoantibodies against the p53 tumor suppressor protein have been detected in the serum of a proportion of patients with various cancers. The generation of such antibodies has been proposed to be due to either tumor p53 protein accumulation or to the type of p53 gene mutation. These hypotheses are examined in the present study. Design and methods: Using immunofluorometric assays, we stud- ied 195 patients with primary breast cancer for the presence of p53 antibodies in serum and p53 protein accumulation in the corre- sponding tumor. Seventeen patients (9%) were p53 antibody- positive and 77 (40%) overexpressed p53. Ten of the 17 p53 antibody-positive patients had tumor p53 accumulation and 7 were negative for p53. Statistical analysis revealed a weak association between the presence of p53 antibodies and p53 protein accumu- lation (p = 0.05). Direct DNA sequencing of exons 1–11 of the p53 gene was performed for 16 p53 antibody-positive and 16 p53 antibody-negative patients. Results: Five of the seropositive and eight of the seronegative patients had a p53 gene mutation. Four of the five mutations in the p53 antibody-positive patients affected a Tyr residue, whereas none of the gene abnormalities in the seronegative patients had such an effect. Conclusions: We conclude that p53 antibodies tend to develop in patients with tumor p53 accumulation, but p53 accumulation is neither sufficient nor necessary for the generation of the immune response. Further, p53 antibody-positive patients do not have higher frequency of p53 gene mutations than p53 antibody-negative patients, but the former patient group is associated with a Tyr substitution in the protein product. Copyright © 2000 The Canadian Society of Clinical Chemists KEY WORDS: p53 tumor suppressor; breast cancer; tumor markers; autoantibodies; gene mutation. Introduction T he p53 gene is localized on the short arm of chromosome 17 and it encodes a 393-amino acid phosphoprotein, which is present at very low levels in normal cells. This molecule appears to play a major role in the maintenance of genomic integrity (1). Following DNA damage, p53 can either arrest the cells at the G1 phase of the cell cycle, thus providing time for the damage to be repaired (2– 4), or induce apoptosis (5). Both pathways prevent replication of damaged DNA and further accumula- tion of mutations. Cells containing biologically inac- tive p53 protein are devoid of such protective mech- anisms and they are genetically unstable. Genetic mutation is the most common pathway for p53 inactivation. Being mutated in approximately 50% of all tumors, p53 is currently considered the most frequently altered gene in human tumorigenesis. Since the discovery of this gene, more than 4000 abnormalities have been reported, the majority of them being missense point mutations that result in single amino acid substitutions. These mutations cluster between exons 5–9, which correspond to highly conserved domains of the protein (6). Many mutants have a different conformation and a longer lifetime compared to the wild-type protein. In- creased lifetime causes mutant p53 protein accumu- lation in the tumor cells, which is detectable by conventional immunohistochemical or other immu- nologic methods. Crawford et al. (7) reported for the first time the presence of autoantibodies against p53 in serum of patients with breast cancer. During the past few years, analysis of a large number of sera from patients with various malignancies revealed that the most immunogenic tumors are those of the lung (8 –13), ovary (9,10,14 –16), colon (9,10,17), breast Correspondence: E. P. Diamandis, M.D., Ph.D., FRCPC, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5. E-mail: ediamandis@ mtsinai.on.ca Manuscript received May 20, 1999; revised September 30, 1999; accepted September 30, 1999. Clinical Biochemistry, Vol. 33, No. 1, 53– 62, 2000 Copyright © 2000 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/00/$–see front matter PII S0009-9120(99)00084-3 CLINICAL BIOCHEMISTRY, VOLUME 33, FEBRUARY 2000 53