p53 MUTATIONS IN SPORADIC ADRENOCORTICAL TUMORS
H. OHGAKI, P. KLEIHUES! and P.U. HEITZ
Department of Pathology, University Hospital, Zurich, Switzerland.
Non-familial human adrenocortical adenomas and carcino- mas were screened for mutations in exons 5-8 of the p53 tumor suppressor gene by single-strand-conformation-polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. Point mutations in codons 12, 13 and 61 in H-ras, K-ras and N-ras proto-oncogenes were similarly assessed by direct DNA sequencing. Three out of 15 primary adrenocortical carcinomas (20%) contained a mis-sense point mutation in the conserved regions (exons 5 and 8) of the p53 gene. Mutations were located in codon 157 (GTC → TTC; Val → Phe), codon 163 (TAC → AAC; Tyr → Asn), and codon 273 (CGT → TGT; Arg → Cys). The mutation in codon 157 was detected in the primary tumor as well as in brain and lymph-node metastases. Among 18 adrenocortical adenomas, there was only a single non-miscoding mutation in codon 295 (CCT → CCC; Pro → Pro). These data suggest that mutational inactivation of the p53 gene occurs in a minority (20%) of sporadic adrenocortical carcino- mas and that these mutations constitute a late event in the multi-step process of malignant transformation. No ras muta- tions were detected in any of these tumors, suggesting that these genes are not involved in the development of tumors originating from the adrenal cortex. @ 1993 Wiley-Liss, Inc.
Adrenocortical carcinomas constitute highly malignant tu- mors with a mortality rate of more than 50% (Weiss et al., 1989). They occur in all age groups, but are most common in the 5th to 7th decade (Barzilay and Pazianos, 1989). A minority of neoplasms affects children who may be genetically predisposed (Mcwhirter et al., 1989; Tsunematsu et al., 1991). Patients with the dominantly inherited Li-Fraumeni syndrome also develop adrenocortical carcinomas, although at an inci- dence of only 1% (Malkin et al., 1990; Srivastava et al., 1990). These patients carry germ-line mutations of the p53 tumor- suppressor gene, which are clustered in exon 7 (codons 245 to 258). Recently, Sameshima et al. (1992b) reported p53 germ- line mutations in exon 8 (codons 286 and 307) in 2 Li-Fraumeni- syndrome families identified by selection for the presence of childhood adrenocortical carcinomas.
The genetic alterations involved in the development of non-familial adrenocortical neoplasms are still poorly under- stood. Yano et al. (1989) reported a loss of heterozygosity of chromosomes 17p, 11p and 13q in 6 of 6 (100%), 4 of 6 (67%) and 3 of 6 (50%) adrenocortical carcinomas respectively. Since the p53 tumor-suppressor gene is located on chromosome 17p, we screened non-familial adrenocortical adenomas and carci- nomas for p53 point mutations. Since p53 and ras genes may cooperate in malignant transformation (Finlay et al., 1989), we also assessed the presence of point mutations in the ras genc family.
MATERIAL AND METHODS
Tumor samples
Eighteen adrenocortical adenomas, 15 adrenocortical carci- nomas and 3 metastases from adrenocortical carcinomas were collected during the years 1971 to 1991. The mean age of patients with adrenocortical adenomas was 45 years (range, 18 to 62 years; 5 males and 13 females). Patients with adrenocor- tical carcinomas had a mean age of 50 years (range; 20-84 years; 5 males, 10 females). The histopathological distinction between adenomas and carcinomas was based on the criteria defined by Weiss (1984).
Tissues were fixed by immersion in 4% formaldehyde and embedded in paraffin. DNA was extracted from paraffin
sections as described by Shibata et al. (1988), with slight modifications. Briefly, after comparison with serial sections stained with hematoxylin and eosin, tumors were scraped off the histological slide. Samples were de-paraffinized with xy- lene and washed with absolute ethanol. Dried samples were treated with 500 µg/ml of proteinase K (Boehringer, Mannheim, Germany) in 50 to 100 ul of digestion buffer (50 mM Tris, pH 8.5, 1 mM EDTA, and 0.5% Tween 20) at 55°℃ for 3 hr. After inactivation of proteinase K by heating at 95℃ for 10 min, samples were kept at -20℃ until PCR reaction.
PCR-SSCP analysis
For pre-screening of the samples for mutations in the p53 gene, PCR-single-strand-conformation-polymorphism (SSCP) analysis was performed according to a slight modification of the method of Orita et al. (1989). Briefly, PCR was performed with 2 ul of DNA solution, 2.5 pmol of each primer, 50 p.M of dNTPs, 1 µCi of [@-32P]-dCTP (Amersham, Aylesbury, UK, specific activity 3000 Ci/mmol), 10 mM Tris (pH 8.8), 50 mM KCI, and 1 mM MgCl2, 0.5 U Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) in a final volume of 10 pl. After adding 10 ul of mineral oil (Sigma, St. Louis, MO), 40 cycles of denaturation (95℃) for 50 sec, annealing (63℃ for exons 5, 6, 7; 58℃ for exon 8) for 50 sec and extension (72℃) for 70 sec were done in an automated DNA Thermal Cycler (Perkin-Elmer Cetus). Primer sequences were as follows: 5’-TTCCTCTTCCTGCAGTACTC and 5’-ACCCTGGG- CAACCAGCCCTGT for exon 5, 5’-ACAGGGCTGGTTGC- CCAGGGT and 5’-AGTTGCAAACCAGACCTCAG for exon 6, 5’-GTGTTGTCTCCTAGGTTGGC and 5’-GTCAGAG- GCAAGCAGAGGCT for exon 7, and 5’-TATCCTGTAG- TAGTGTAATC and 5’-AAGTGAATCTGAGGCATAAC for exon 8. After PCR, the reaction mixture (1.5 ul) was mixed with 2 ul 0.2 M NaOH and 9 ul of sequencing stop solution (USB, Cleveland, OH). Samples were heated at 95℃ for 10 min and immediately loaded onto a 6% polyacrylamide non- denaturating gel containing 7.5% glycerol. Gels were run at 7 W for 13 to 15 hr at room temperature and dried at 80°C. Autoradiography was performed with an intensifying screen for 5 to 48 hr and the patterns of single-strand DNA were checked for differences from those of normal DNA.
Direct DNA sequencing of PCR products
For the samples which scored positive with SSCP analysis, PCR was performed with 10 ul of DNA solution, 10 pmol of cach primer, 200 pM dNTPs, 10 mM Tris (pH 8.8), 50 mM KCI, 1 mM MgCl2, and 2.5 U Taq polymerase in a total volume of 60 ul. After amplification, 50 ul of the PCR reaction was electrophoresed on a 6% polyacrylamide gel. The amplified bands were cut out, eluted in 0.5 M ammonium acetate and 1 mM EDTA at 37℃ overnight and precipitated with ethanol. Dried DNA was re-suspended in 15 ul of TE buffer.
Sanger dideoxynucleotide sequencing was performed using [@-32P]-dATP and primers for amplification. The template-
1To whom correspondence and reprint request should be sent, at Institute of Neuropathology, Department of Pathology, University Hospital, CH-8091 Zurich, Switzerland. Fax: 01-255-4402.
Received: December 24, 1992 and in revised form February 13, 1993.
primer mixture in 10 ul reaction buffer (10% dimethylsulfox- ide, 20 mM Tris-HCI, pH 7.5, 10 mM MgCl2, and 25 mM NaCl) was heated at 95℃ for 5 min and immediately placed in liquid nitrogen. After adding 0.1 M dithiothreitol, 0.5 pCi [@-32P]- dATP and 2U Sequenase version 2.0 DNA polymerase (USB), samples were divided into 4 wells each containing termination mixtures and incubated at 37℃ for 10 min. Samples were mixed with 4 ul stop solution, heated at 80℃ for 2 min and immediately loaded onto a 6% polyacrylamide/7 M urea gel. Gels were dried and autoradiographed for 1 to 5 days.
Exons 1 and 2 of H-, K- and N-ras genes were amplified and directly sequenced as described above. Primers for amplifica- tions were 5’-CTGAGGAGCGATGACGGAAT and 5’-AGTGGGGTCGTATTCGTCCA for exon 1 of H-ras, 5’- CTACCGGAAGCAGGTGGTCA and 5’-CGCATGTACT- GGTCCCGCAT for exon 2 of H-ras, 5’-GACTGAATATAAA- CTTGTGG and 5’-CTATTGTTGGATCATATTCG for exon 1 of K-ras, 5’-TTCCTACAGGAAGCAAGTAG and 5’- CACAAAGAAAGCCCTCCCCA for exon 2 of K-ras, 5’-TGACTGAGTACAAACTGGT and 5’-GGGCCT- CACCTCTATGGTGG for exon 1 of N-ras and 5’-TCTTA- CAGAAAACAAGTGGT and 5’-ATACACAGAGGAA- GCCTTCG for exon 2 of N-ras genes. The primers for sequencing were 5’-TCCACAAAATGGTTCT for exon 1 of H-ras, 5’-AGACGTGCCTGTTGGACATC for exon 2 of H-ras, 5’-TCTGAATTAGCTGTATCGTC for exon 1 of K-ras, 5’- GTAATTGATGGAGAAACCTG for exon 2 of K-ras, 5’- GATTAGCTGTATTGTCAGTG for exon 1 of N-ras, and 5’-GGTGAAACCTGTTTGTTGGA for exon 2 of N-ras genes.
RESULTS
SSCP analysis showed 6 samples with altered migration, indicating the presence of a mutation. Sequencing analysis confirmed miscoding p53 point mutations in 3 of 15 (20%) primary adrenocortical carcinomas (Table I). One CGT → TGT transition mutation was found in the carcinoma of a 26-year-old female patient and affected codon 273 (Arg → Cys). Another mutation was identified in the adrenocortical carci- noma of a 59-year-old male patient. This TAC → AAC trans- version occurred at codon 163 and led to amino-acid substitu- tion Tyr → Asn. In addition, a GTC → TTC transversion (Val → Phe) in codon 157 was present in a primary adrenocor- tical carcinoma in a 37-year-old female patient. The same mutations were also found in 2 metastatic lesions (brain and lymph nodes) from this adrenocortical carcinoma (Table I) but not in the normal part of adrenal cortex from the same patient. The typical DNA sequencing autoradiographs are shown in Figure 1. One adenoma contained a non-miscoding mutation at codon 295 (CCT → CCC; Pro → Pro). All other adenomas scored negative. No mutation was found in exons 1 and 2 of H-ras, K-ras and N-ras genes in any of the adrenocortical adenomas and carcinomas.
DISCUSSION
There is increasing evidence that the multistage develop- ment of malignant tumors is associated with the cumulative acquisition of genetic alterations which comprise both the activation of proto-oncogenes and the inactivation of tumor suppressor genes (Weinberg, 1989). In human neoplasms, the most frequently involved proto-oncogenes are of the ras gene family, while the p53 gene is the most frequent tumor- suppressor gene (Nigro et al., 1989; Hollstein et al., 1991; Levine et al., 1991). The p53 gene encodes a nuclear phospho- protein and is considered to play an essential role in the regulation of cell proliferation (Boyd and Barrett, 1990). While the wild-type p53 acts as a tumor-suppressor gene, p53 mutations occurring within highly conserved domains not only
| Age | Gender | Diagnosis | Exon | Codon | Mutation | Amino-acid substitution |
|---|---|---|---|---|---|---|
| 35 | F | Adenoma | 8 | 295 | CCT - CCC | Pro -> Pro |
| 26 | F | Carcinoma | 8 | 273 | CGT -> TGT | Arg -> Cys |
| 37 | F | Carcinoma | 5 | 157 | GTC -> TTC | Val -> Phe |
| Brain metas- tasis | 5 | 157 | GTC -> TTC | Val -> Phe | ||
| Lymph-node metastasis | 5 | 157 | GTC-> TTC | Val -> Phe | ||
| 59 | F | Carcinoma | 5 | 163 | TAC -> AAC | Tyr -> Asn |
ACGT
ACGT
ACGT
En
우
TVA
G/T
codon 163
codon 273
codon 157
cause loss of tumor-suppressor function but may even activate p53 to an oncogene in a dominant negative fashion (Finlay et al., 1989; Eiyahu et al., 1989). Various human tumors have been shown to contain either loss of both alleles of the p53 gene, the loss of one p53 allele with an associated point mutation, insertion or deletion of the remaining allele, or inactivation of the p53 gene in one allele but a normal (wild-type) sequence in the other.
The present study shows that 3 of 15 (20%) primary adrenocortical carcinomas contained miscoding point muta- tions in the conserved region of the p53 tumor-suppressor gene, suggesting that loss of its function may play an important role in the development of some non-familial adrenocortical carcinomas. The low frequency of p53 mutations observed in this study and the high frequency (100%) of 17p loss described by Yano et al. (1989) suggest the existence of an additional tumor-suppressor gene on the short arm of chromosome 17. This has also been suggested on the basis of molecular genetic analyses of human brain and breast neoplasms. Frankel et al. (1992) reported that 36% of gliomas with loss of heterozygosity in 17p did not contain p53 mutations. Similarly, 8 out of 8 medulloblastomas (Saylors et al., 1991) and 7 out of 7 pediatric primitive neuro-ectodermal tumors (Biegel et al., 1992) with allelic loss of 17p lacked p53 mutations. The results of the deletion mapping on chromosome 17p by Sato et al. (1990) implied the loss of an as-yet unidentified tumor-suppressor gene distal to the p53 locus in primary human breast cancer.
The emergence of adrenocortical carcinomas from adreno- cortical hyperplasia has occasionally been observed (Hamwi et al., 1957; Bauman and Bauman, 1982), but a clear-cut adenoma-
to-carcinoma sequence has not been established. On the basis of histopathological criteria alone, the distinction between adenomas and carcinomas may be difficult, particularly in well-differentiated tumors (Weiss et al., 1989; Weiss, 1984; Von Slooten et al., 1985). Occasionally, proof of the malignant nature relies on the presence of metastases. In the present study, only one adenoma (6%) contained a p53 mutation and this was not miscoding (Pro → Pro). The increased frequency in carcinomas suggests that, in this tumor type, p53 mutations constitute a late event in the evolution of malignant transforma- tion. The observation of identical p53 mutations in one primary adrenocortical carcinoma and its lymph-node and brain metastases indicates that metastatic spread to other tissues is not associated with selection against p53 inactivation.
A similar correlation was reported for primary and metastatic lung tumors (Sameshima et al., 1992a).
In contrast to many tumors at other organ sites where ras mutations are frequent, notably pancreas, lung and colon (Bos, 1989), the present study clearly shows that activation of ras genes by point mutations is not involved in the evolution of adrenocortical tumors.
ACKNOWLEDGEMENTS
We thank Dr. E.W. Newcomb, Department of Pathology, New York University, for critical review of the manuscript. The excellent technical assistance of Ms. S. Graf and Ms. P. Saremaslani is gratefully acknowledged. This work was sup- ported by the Cancer League of the Canton of Zurich and by the Swiss National Science Foundation.
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