p53 Mutations in Adrenal Tumors: Caucasian Patients Do Not Show the Exon 4 “Hot Spot” Found in Taiwan*
MARTIN REINCKE, CHRISTOPH WACHENFELD, PATRICIA MORA, ANDREA THUMSER, CORNELIA JAURSCH-HANCKE, SALEH ABDELHAMID, GEORGE P. CHROUSOS, AND BRUNO ALLOLIO
Schwerpunkt Endokrinologie (M.R., C.W., P.M., A.T., B.A.), Medizinische Universitätsklinik Würzburg, Deutsche Klinik für Diagnostik (C.J-H., S.A.), Wiesbaden, Germany; Developmental Endocrinology Branch (G.P.C.), NICHD, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Mutations in the p53 tumor suppressor gene are frequently present in human cancers but have rarely been described in benign tumors. We previously reported mutations in the “hot spots” between exons 5-8 of the p53 gene in adrenocortical carcinomas but not in adenomas. Recently, a previously unknown hot spot in exon 4 of the p53 gene was described in adrenal adenomas and pheochromocytomas of Taiwan- ese patients. We, therefore, investigated whether these mutations are also present in Caucasian patients from the U.S. and Europe. We analyzed tumor tissue of 12 aldosterone-producing adenomas, 7 cor- tisol-producing adenomas, and 6 pheochromocytomas. Overexpres- sion of the p53 protein was investigated by immunohistochemistry. Point mutations within exon 4 were identified by polymerase chain
reaction (PCR) amplification and direct sequencing of the PCR prod- uct. The pYNZ22 microsatellite located on chromosome 17p, close to the p53 gene, was used to screen for allelic loss (LOH) of the p53 gene. Overexpression of p53 was not identified in any of the adenomas and pheochromocytomas. Point mutations within exon 4 were found in 0/25 tumors. LOH was present in 1/13 informative adenomas and 0/2 informative pheochromocytomas. We conclude that p53 mutations do not play a major role in the tumorigenesis of adrenal adenomas and pheochromocytomas of Caucasian patients. Thus, ethnic and envi- ronmental factors may be responsible for the mutational spectrum found in Taiwanese patients. (J Clin Endocrinol Metab 81: 3636- 3638, 1996)
M UTATIONS in the p53 tumor suppressor gene are the most common genetic alterations in human tumors identified to date (1). The p53 gene functions as a tumor suppressor gene and more specifically as a cell-cycle regu- lator. The gene is located on chromosome 17p, encodes a protein of 53-kilodalton, and consists of 11 exons (2). Inac- tivation of p53 by point mutations and/ or deletions results in loss of tumor suppressor function. p53 mutations are rarely observed in benign tumors, are generally a late event in tumorigenesis, and are associated with a more malignant phenotype (3-5).
More than 90% of p53 mutations found in human tumors are located in 4 “hot spots” within exons 5 to 8 (1, 6). These exons contain four highly conserved domains essential for a normal function of the protein. Mutations outside these areas are scattered throughout the remaining exons, and their pathophysiological significance has been the matter of some debate (7). However, recently, Lin et al. (8) reported a new mutational “hot spot” within exon 4 in benign adrenal tu- mors, with 9 of 15 (60%) adrenocortical adenomas and 3 of 6 (50%) pheochromocytomas having evidence for p53 mu- tations. Sequencing of this exon confirmed the presence of point mutations at codons 100, 102, or 104, respectively, in the majority of tumors.
Received February 27, 1996. Revision received May 31, 1996. Ac- cepted June 11, 1996.
* Supported by a grant of the Wilhelm Sander-Stiftung, München (M.R.)
This finding is in contrast to our own experience, using immunohistochemistry, that p53 mutations occur in adre- nocortical carcinomas but not in benign adenomas (9). How- ever, the number of tissues was small, limiting more general conclusions drawn from our studies. We, therefore, investi- gated the frequency of p53 mutations in a large series of Caucasian patients with benign adrenal tumors.
Patients and Methods
Patients and DNA isolation
We studied 25 Caucasian patients with a variety of adrenal diseases (clinical data, see Table 1). The mean age of the patients was 50 yr. Neoplastic tissue was obtained at adrenalectomy. Diagnosis was made by clinical and histological data following standardized criteria (10). Blood was simultaneously collected for leukocyte DNA extraction in 20 patients. DNA was isolated from frozen tissue and leucocytes using a commercial kit (Quiagen, Hilden, Germany). Tissue from 2 additional patients with adrenocortical carcinomas harbouring p53 mutations served as controls, data of which were published in part previously (9).
Immunohistochemistry
Immunohistochemistry was performed on frozen tumor sections us- ing the monoclonal immunoglobulin G (IgG)mouse antibody PAb 1801 (Ab 2, Oncogene Science, Manhassed, NY), which reacts specifically with an epitope between amino acids 32 and 79, detecting both wild-type and mutant p53 protein (9). Control sections were incubated with se- rially diluted primary antibody and with a negative control monoclonal antibody.
Loss of heterozygosity (LOH)
LOH indicating allelic loss was analyzed with the pYNZ22 micro- satellite, a variable number of tandem repeat (VNTR) markers at 17p near the p53 gene. The rate of heterozygosity in examined populations
| Diagnosis | n | Range of age in yr | Mean age in yr | Tumor size in cm | Sex (F/M) |
|---|---|---|---|---|---|
| Aldosterone-producing adenoma | 12 | 40-66 | 53.2 | 1.0-4.0 | 9/3 |
| Cortisol-producing adenoma | 7 | 0.5-65 | 46 | 2.5-7.0 | 6/1 |
| Pheochromocytoma | 6 | 26-71 | 49.5 | 2.0-9.0 | 4/2 |
| Adrenocortical carcinoma | 2 | 17-79 | 48 | 7.0-9.0 | 2/0 |
| Diagnosis | n | Detected mutations in exon 4 | Detected heterozygosity at codon 72 | Detected over-expression | Detected LOH |
|---|---|---|---|---|---|
| Aldosterone-producing adenoma | 12 | 0/12 | 5/12 | 0% | 1/7 |
| Cortisol-producing adenoma | 7 | 0/7 | 2/7 | 0% | 0/6 |
| Pheochromocytoma | 6 | 0/6 | 4/6 | 0% | 0/2 |
| Adrenocortical carcinoma | 2 | 0/2 | 0/2 | 50% | 2/2 |
is approximately 80% (11). Aliquots of 0.1-1.0 µg DNA from frozen tissue or leukocyte DNA were amplified by PCR as described elsewhere (12).
PCR amplification and direct sequencing of exon 4 of the p53 gene
PCR (13) was used to amplify a DNA fragment including exon 4, which contains the “hot spot” found in Taiwanese patients. We used the following primers: 5’-CAG TCA GAT CCT AGC GTC GAG C-3<, 5’-ACA GCT GCA CAG GGC AGG TCT T-3’ (0.2 mmol/L each). Using 1 pL of this double-stranded PCR product as template and a 100-fold excess of a nested sense or antisense primer (5’-GAC CTA TGG AAA CTG TGA GTG-3’, 5’-CAT TAA GTC TCA TGA AGC CA-3’), we am- plified single-stranded DNA in an asymmetric PCR (14). Single stranded DNA was purificated with a Quiagen PCR Purification Kit (Quiagen, Hilden, Germany) and directly sequenced using the dideoxy-chain- termination method of Sanger (15) (Sequenase 2.0, United States Bio- chemicals, Cleveland, OH). All samples were sequenced in up- and downstream direction, using the following primers: 5’-TGG TCC TCT GAC TGC TCT-3’, 5’-GCA ACT GAC CGT GCA AGT CAC AG-3’.
Results
Immunohistochemistry
p53 overexpression was not detected in any of the adrenal adenomas and pheochromocytomas, in contrast to adreno- cortical carcinoma tissue that was run in parallel in all ex- periments as a positive control tissue (Table 2).
Loss of heterozygosity of 17p
LOH was assessed using the pYNZ22 microsatelite marker in 18 cases. Fifteen (83.3%) were heterozygous for this locus. LOH of 17p was found in 1 aldosteronoma but in none of the remaining 14 tumors (Table 2).
Sequencing of exon 4 of the p53 gene
The sequence of exon 4 surrounding the “hot spots” at codon 100 to 104 identified in Taiwanese patients was suc- cessfully amplified and visualized by sequencing in all tumor samples. No homozygous or heterozygous mutation at the “hot spot” or at any other codon of exon 4 was detected in any of these tumors. However, 11 of 25 (44%) patients were heterozygous for the polymorphism at codon 72 (16) (Table 2).
Discussion
Several lines of evidence suggest that mutant p53 is in- volved in adrenal tumorigenesis. First, allelic loss of the chromosomal locus of the p53 gene, 17p, has been found in adrenocortical carcinomas (17). Second, the Li-Fraumeni syn- drome, a hereditary tumor syndrome associated with devel- opment of breast carcinoma, adrenal carcinoma, and other tumors early in life, is caused by mutations at one of the evolutionary highly conserved domains within exon 5-8 of the p53 gene (18). Third, we and others recently identified p53 mutations in adrenocortical carcinomas and carcinoma cell lines but not in benign adenomas of the adrenal cortex (5, 9, 19).
We now show that previously identified “hot spot” mu- tations within exon 4, found in hyperfunctioning adenomas and pheochromocytomas of Taiwanese patients, are not present in a Caucasian population from the U.S. and Europe. We neither identified p53 overexpression by means of im- munohistochemistry nor did we find point mutations by direct sequencing of the entire sequence of exon 4. Loss of heterozygosity of 17p was present in only 1 tumor of 15 informative patients.
The discrepancies between our results and the data of Lin et al. (8) can be explained in several ways. Ethnic and envi- romental factors could contribute to the genetic instability of the p53 gene in Taiwanese patients. For example, aflatoxin B1 is a potent liver carcinogen with widespread human expo- sure in high-risk areas like southeastern Asia. Mutational “hot spots” in the p53 gene with G to T mutations at codon 249 have been identified in patients with hepatocellular car- cinoma from Quidong (People’s Republic of China) and Southern Africa. In mutagenesis experiments with aflatoxin B1, induction of base substitutions, principally G to T trans- versions, have been demonstrated in vivo and in vitro (20). These data show that similar, but as yet unidentified envi- ronmental mutagens may cause p53 mutations and could be responsible for the mutational spectrum observed in adrenal tumors of Taiwanese patients.
Another explanation would be based on technical consid- erations. Mutational pseudo “hot spots” identified by PCR- based techniques in tumor samples may be caused inadvert-
edly by cross-contamination of mutated genes, such as from PCR-amplified DNA, in the reaction buffers, primers, or genomic DNA preparations, even if proper precautions like the ones in the study of Lin et al. are used. This could explain why the majority of the mutations found in their tumors apparently were heterozygous mutations, an uncommen finding in other human tumors. In addition, p53 mutations within exon 4 have rarely been found in other malignant and benign human tumors calling the pathophysiologic impor- tance of mutations in this area into question. The significance of the findings of Lin et al. is further reduced by an apparent lack of a more aggressive behaviour or a larger tumor mass at surgery in their series compared with tumors without p53 mutations or tumors from other geographic areas. This shows that the malignant potential of their tumors with exon 4 mutations seems to be essentially similar to that of tumors without these mutations.
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