P53 Oncogene Mutations in Human Prostate Cancer Specimens

0022-534 7/94/1512-0492$03.00/0 Vol. 151, 492-495, February 1994

THE JOURNAL OF UROLOGY

Printed in U.S.A.

Copyright© 1994 by AMERICAN UROLOGICAL ASSOCIATION, INC.

p53 ONCOGENE MUTATIONS IN HUMAN PROSTATE CANCER SPECIMENS H. JAMES VOELLER, LAVENCIA Y. SUGARS, THOMAS PRETLOW AND EDWARD P. GELMANN* From the Division of Medical Oncology, Lombardi Cancer Research Center, Washington, D.C. and the Department of Pathology, Case Western Reserve University, Cleveland, Ohio

ABSTRACT

Eighty-five prostate cancer specimens from prostate resections were analyzed for the presence of p53 gene mutations by immunohistochemical staining for P53 protein. DNA from thirty-four of these samples was also analyzed for mutations in exons 5-8 by single-strand chain polymorphism (SSCP) analysis. One sample had P53 staining by immunohistochemistry, one sample was positive for a p53 mutation by SSCP, and one sample was positive by both techniques. Mutations in the two samples that were positive by SSCP were confirmed by nucleotide sequencing. In a separate study of ten lymph nodes that contained prostate cancer metastases, one had detectable P53 protein by immunohistochemical staining. Therefore, p53 mutations appear to be low frequency events m primary prostate cancer. KEY

WORDS: prostatic neoplasms; genes, p53; mutation

The p53 oncogene is mutated at a high incidence in a wide range of human cancers. p53 mutations occur at different "hot spots" of the protein-coding region that may cause mutant proteins to manifest different degrees of oncogenicity. 1 Moreover, specific nucleotide changes and recurrent alterations of specific codons in p53 have been found in some cancers and may be associated with specific carcinogenic insults. 2- 4 p53 mutations have been under active investigation in prostate cancer. Two large series analyzing tissue samples have been published. Visakorpi et al. performed immunohistochemical detection of P53 protein in 137 prostate cancer tissues and found 6% of samples with intense staining for P53. 5 A second report analyzing 150 specimens detected 12. 7% staining for P53 protein, with a strong predominance in patients with advanced stage cancers. 6 The notion that p53 mutations may be more common in aggressive prostate cancer is supported by the presence of p53 mutation in a segment of a primary prostate cancer specimen and in the entire lymph node metastasis from the same patient. 7 p53 mutations have also been found in three prostate cancer cell lines. 8 However, since we found that p53 mutations were present in some, but not all, strains of LNCaP, it appeared that at least some of the p53 mutations detected arose in vitro. 9 To study the prevalence of p53 gene mutations in prostate cancer, we investigated the structure of the p53 gene in surgical specimens from prostate cancer. We used both single-stranded chain polymorphism (SSCP) and nucleotide sequence analysis of tissue DNA to identify specific mutations in the p53 gene. These data were correlated with immunohistochemical staining of the tumor specimens for the P53 protein. MATERIALS AND METHODS

Tissue specimens. Fresh surgical prostate specimens were obtained at Georgetown University Hospital and the Washington Veterans Administration Hospital, Washington, D.C. Research use of human material was approved at both institutions by the respective Institutional Review Boards. Samples were transported to the laboratory on wet ice. Specimens were stored frozen or immediately processed for nucleic acid extraction. Accepted for publication August 25, 1993.

* Requests for reprints: Division of Medical Oncology, Lombardi Cancer Research Center, 3800 Reservoir Road N.W., Washington, D.C. 20057. This work was supported in part by National Cancer Institute Grants CA50355 and CA57178 to Dr. Gelmann and CA57179 to Dr. Pretlow. 492

Data on patient age, race and final pathologic diagnosis were obtained for correlation with genetic analysis. All slides were reviewed by one of us (E.P. G.) and confirmation of cancer grade was done by Janice Lage, M.D., Director of Surgical Pathology, Georgetown University School of Medicine. Lymph nodes were formalin -fixed, paraffin embedded archival specimens and were reviewed by one of us (T.P.) to confirm the presence of metastases. Nucleic acid extraction. Frozen surgical specimens were thawed on ice, and sections were removed with a scalpel, minced into small pieces and collected in 15 ml. polypropylene tubes. High molecular weight DNA was extracted and purified using standard procedures. Single-stranded polymorphism analysis. The PCR-SSCP method described by Orita et al. 10 was modified and used to detect the presence of p53 gene mutations. In brief, 100 ng. of genomic DNA was amplified in a reaction containing 10 mM. Tris-Cl, pH 8.3, 50 mM. KCl, 1.5 mM. MgCl2 , 25 µM. each dNTP, 10 pmol. each primer, 0.1 µl. 32 P dCTP (3000 Ci/mmol., Amersham, Arlington Heights, Illinois) and 0.25 units of Taq polymerase (Perkin Elmer, Norwalk, Connecticut). Thirty cycles at 94C for 30 seconds, 55C for 30 seconds and 72C for 60 seconds were employed. One µl. of the PCR reaction was mixed with 9 µl. of loading buffer (90% formamide, 20 mM. EDTA, 0.05% xylene cyanol and bromophenol blue), denatured at 95C for 2 to 3 minutes, chilled briefly on ice and loaded, 2 µl. per lane, onto a polyacrylamide gel. Six percent polyacrylamide gels containing 10% glycerol were electrophoresed in 0.5 X TBE at 40 watts for 4 to 6 hours at 4C; 0.5 X MDE gels (AT Biochem, Malvern, Pennsylvania) containing 10% glycerol were electrophoresed at 8 to 10 watts for 16 to 30 hours at room temperature with cooling by a fan. The gels were transferred to Whatman 3MM paper, dried, and autoradiographed for 12 to 24 hours, and DNA was amplified and radiolabelled for each of exons 5-8 by polymerase chain reaction (PCR). The PCR Primers for p53 Exons 5-8 were: Exon 5: TTC-CTC-TTCCTG-CAG-T AC-T; AGC-TGC-TCA-CCA-TCG-CTA-T. Exon 6: TGG-TTG-CCC-AGG-GTC-CCC-AG; GGA-GGG-CCACTG-ACA-ACC-A. Exon 7: TGT-TGT-CTC-CTA-GGTTGG-CT, CAA-GTG-GCT-CCT-GAC-CTG-GA. Exon 8: CCT-ATC-CTG-AGT-AGT-GGT-AA, TCC-TGC-TTGCTT-ACC-TCG-CT. Cloning. Single-strand chain polymorphism bands with normal and altered mobility were separately cut out of a dried gel with a scalpel and removed to a microcentrifuge tube. The DNA

p53 ONCOGENE MUTATIONS IN PROSTATE CANCER was eluted in 100 µl. H 20 at 37C for 1 hour. Ten µl. of eluted DNA was amplified by PCR in a 100 µl. reaction containing 10 mM. Tris-Cl, pH 8.3, 50 mM. KCl, 1.5 mM. MgCb, 200 µM. each dNTP, 100 pmol. each primer and 2.5 units Taq polymerase. Cycle parameters were the same as for PCR-SSCP, with a final 10 minute extension at 72C. The "ragged ends" of the PCR product were mended in a 100 µl. reaction using 25 to 50 µl. of the PCR reaction in 50 mM. Tris-Cl, pH 7.6, 10 mM. MgCb, 10 mM. 2-mercaptoethanol, 50 µg./ml. BSA, 50 µM. dNTP, 1 mM. ATP, 15 units T4 polynucleotide kinase (US Biochemical, Indianapolis, Indiana), and 13.5 units Klenow (Promega, Madison, Wisconsin), at 37C for 1 hour. After the addition of 1.0 µl. 0.5 M. EDTA, pH 8.0, the samples were ethanol precipitated and electrophoresed through a 1.5% agarose gel. The PCR product was cut out of the gel and purified using the Gene Clean kit or the Mermaid Kit (Bio 101, LaJolla, California). The PCR products were then subcloned into pGEM7Zf (Promega) vector that had been treated with Smal and calf intestinal alkaline phosphatase. Clones containing the correct insert were used for sequencing. Sequencing. Plasmid DNA was denatured and sequenced with the Sequenase Version 2.0 kit (US Biochemical, Indianapolis, Indiana) using 35 8 dATP (Amersham) and T7 SP6 promoter primers (Promega). Six clones from each mutant PCR-SSCP product and 4 clones from the normal PCR-SSCP product were sequenced for each sample. Immunohistochemical staining for P53 protein. Murine antiP53 monoclonal Ab 1801 (Ab-2) was purchased from Oncogene Science, Inc. (Mineola, New York). The secondary antibody was affinity-purified biotinylated horse anti-mouse lgG (H&L) and was purchased from Vector Laboratories, Inc. (Burlingame, California). Pathology specimens of primary prostate tissue were methanol fixed and paraffin-embedded. Five micron sections were cut and placed on 3-amino-propyl triethyl silane (TES)-treated slides, and staining was performed with monoclonal antibody PAb 1801. The primary antibody was applied at 1:600 in blocking serum (horse serum 1:66.67 in phosphatebuffered saline [PBS]) overnight at 4C. The secondary antibody was used at 1:200 in blocking serum for 60 minutes at room temperature. Avidin-biotin complex (Biotin Super ABC Kit, Biomeda Corp., Foster City, California) was applied at 1:50 for avidin and 1:100 for biotin in blocking serum. DAB(3,3' -diaminobenzidine tetrahydrochloride solution, Sigma Chemical Co., St. Louis, Missouri) was used at 1:100 and 30% H202 at 1:2873.6 in PBS for 15 minutes with stirring. Sections were counterstained with methyl green. RESULTS

Patient characteristics. Primary prostate tissue samples were taken during transurethral resection or radical prostatectomy. All of the 85 samples included in this analysis were reviewed to confirm the diagnosis of prostate cancer. Also, 70 of these specimens contained regions of benign prostatic hyperplasia (BPH). We analyzed P53 staining in both areas of prostate cancer and of BPH. Information about the patients is shown in table 1. The median age was 67, and the range was 56 to 90 years old. Thirty-five specimens were from black men and 50 from white men. The distribution of Gleason's pattern scores is shown in table 1. Fifteen of the 85 specimens had Gleason's pattern scores of 8, 9, or 10. TABLE

493

Single-strand chain polymorphism analysis. Initially we performed SSCP analysis on 32 samples using primers for exons 5, 6, 7 and 8 since the mutational "hot spots" found in other cancers are located within these four exons. 1 Single-strand chain polymorphism is used to identify p53 gene exons that contain mutations and thereby migrate aberrantly on a nondenaturing gel. In addition to the 32 cancer specimens, 7 samples of BPH were analyzed. Each exon was analyzed on a separate gel and coelectrophoresed with normal tissue DNAs and positive control DNAs from cell lines with known p53 mutations. For some samples, exons 5 and 6 were analyzed together using PCR primers that spanned both exons and the intervening sequences. Aberrantly migrating bands were seen in 3 of the 32 cancer samples, one in exon 5 and two in the combined exon 5/6 analysis. The seven BPH specimens were all negative. The positive lanes from the SSCP analysis are shown in figure 1. Sample 51 was found to have a nucleotide sequence polymorphism at codon 213. One sample that contained a mutation, 76, was from a 66-year-old white man with pathologic stage B2-C, Gleason's score 8, prostate cancer. A second mutated sample, 129, was from a 73-year-old black man with moderately to poorly differentiated adenocarcinoma of the prostate (Gleason's score 3 + 3), stage unknown. After we screened additional samples by immunohistochemical staining for P53 protein (see below), the one additional sample that had positive staining was not found to have exon 5-8 abnormalities by SSCP. Nucleotide sequence analysis. We performed nucleotide sequence analysis of the aberrant p53 exons. The relative intensities of the anomalous and native bands in the SSCP gels indicated that the putative p53 gene mutations in samples 51, 76 and 129 were present in a minority of the cells from which DNA was extracted. Because this low fraction of mutant DNA would be difficult to analyze by direct sequencing from PCR reactions of the total tissue DNA, we enriched the mutant DNAs by excising the aberrant bands from the SSCP gel, cloning the fragments and sequencing the cloned p53 fragments containing the putative mutations. Segments from sequencing gels are shown for the three patient samples (fig. 2). Sample 129 mutation is A- T transversion in codon 132, lys -+ met. The sequence for sample 129 has been confirmed by sequencing several DNA clones from separate PCR reactions performed on normal and mutant DNAs. The p53 gene codon 132 lys - met mutation present in sample 129 has been reported in other cancers. 1 Sequence analysis of sample 76 is also shown in figure 2. Sequencing clones derived from two separate PCR reactions show a C - T transition in codon 152, pro - leu. Lastly, sequence analysis of sample 51 shows that this sample contained a polymorphism at codon 213 that has previously been reported in approxi1

2

3

4 S 6 7 8

1. Patient information

Gleason score

>7 4-7 <4 Age, median No. specimens from black patients stained by IHC No. specimens from white patients stained by IHC

15 52 5

67.4 years 35

50

Fm. 1. Single-strand chain polymorphism analysis of prostate cancer tissues. Lanes 1-3 contain PCR-amplified p53 exon 5. 1) Sample 129, 2) placental DNA, 3) SK-Br-3 human breast cancer cells known to have exon 5 mutation. 14 Lanes 4-8 contain PCR-amplified DNA of exons 5 and 6. 4) Sample 76, 5) Sample 51, 6) placental DNA, 7) human breast cancer cells MDA-MB-436 known to have exon 6 mutation,14 8) normal human thymus DNA.

494

p53 ONCOGENE MUTATIONS IN PROSTATE CANCER Sample 129 mutant 134

wild type T T T G T

T T ............... T

133

134/.

133

A G

A A

132

132

C 131

A A

130

C T C

131

130

Sample 76 mutant

B

C G

154

G

C C C G T•

153

152

C

C C C A

151

150

C

A

Sample 51 polymorphism

C A

A C A

210

C T

211

212

\

j.

213

214

\

215

Fm. 2. Nucleotide sequence analysis fragments of p53 DNA cloned from prostate cancer tissues. Top panel shows segments from sequencing gels of normal and aberrant bands isolated and cloned from SSCP gel or sample 129. Middle panel shows a segment from sequencing gel of mutant allele from sample 76. Lower panel shows sequence of polymorphic allele of sample 51.

FIG. 3. P53 immunostaining of prostate cancer. Photographs were taken at 400X magnification with Nomarski differential optics. A, sample 76 immunostaining showing neoplastic cells with nuclear p53 staining. B, sample 190 immunostaining for p53. C, positive lymph node showing immunostaining for p53.

TABLE 2.

mately 10% of samples in another study. This is an A/G polymorphism in the third nucleotide of codon 213. Immunohistochemical staining. The tissue from prostates that were subjected to SSCP analysis and 51 additional paraffin -embedded samples were sectioned. They were examined histologically after staining with hematoxylin and eosin (H&E) to confirm the presence of malignancy in the laboratory sample and stained immunohistochemically for the presence of P53. Two samples, 76 and 190, were positive by immunohistochemical staining of malignant epithelium (fig. 3, A and B). Sample 76 had been shown to contain a p53 mutation by SSCP and nucleotide sequencing. Sample 190 was normal by SSCP. Sample 129, which was shown to have a p53 mutation, did not show detectable nuclear staining of P53 protein. A summary of the data on abnormal p53 genes is presented in table 2. There was no staining of BPH in any of the 70 samples that contained hyperplastic ducts. Because there have been suggestions that P53-positive pros11

Samples positive for p53 mutations by immunohistochemical (IHC) and PCR-SSCP analysis

Sample#

Age/Race

IHC

PCR-SSCP

Gleason score

76

64, W

+

+, exon 6, codon

8

152, C---->T +, exon 5, codon 132, A---->T

6

W

129

73,B

190

69,W

+

7

= white patient; B = black patient.

tate cancers may be associated with higher stage 6 or favor metastatic spread, 7 we stained ten positive lymph nodes obtained at radical prostatectomy for P53. These lymph node samples were not represented among the 85 prostate tissues described above. One of the ten lymph node specimens was positive for P53 staining. Unlike the prostate specimens that contained rare isolated ducts that were positive for P53, the lymph node contained p53-staining cells throughout (fig. 3, C).

p53 ONCOGENE MUTATIONS IN PROSTATE CANCER DISCUSSION

We screened 85 surgical prostate cancer specimens for p53 mutations and found mutations that altered the protein coding sequence in two specimens and evidence of elevated P53 expression in a third specimen. The level of p53 mutation in prostate cancer is lower than in other carcinomas such as breast and colon cancer, where substantially higher percentages of samples . . have p53 mutations. Other studies have found the occurrence of p53 mutat10n m prostate cancer to range from 6% to 13%. 5- 7 The significance of p53 mutation in prostate cancer is as yet unclear. Several groups have found that P53 staining detects only a small fraction of malignant cells in positive specimens. Often, the P53 staining is concentrated in isolated sections of the cancer. This suggests that p53 mutations may be late events that arise in derivative clones of a preexisting malignancy. Our overall rate of p53 mutations was 3.5% among prostate samples and 10% of metastases. These rates are somewhat lower than the reports of Visakorpi 5 and Bookstein. 6 Differences may have been due to immunohistochemical techniques. Bookstein used two monoclonal antibodies, CM-1 and P Ab1801 although he did not state whether or not each sample was stai~ing with both antibodies. Our rate of p53 mutation detection did not differ greatly between SSCP and immunohistochemistry. We only detected one sample with each technique that was not detected by the other. Bookstein performed nucleic acid analysis on 14 positively staining samples and found that he could detect mutations in 9. In prostate cancer, where only a subset of the malignant cells appears to carry p53 mutations, it is important to use complementary analytical techniques to maximize the detection of mutations. Including the cases in this report, nucleotide sequences of thirteen p53 mutations in prostate cancer specimens have now been published. One report found five G:C ---'> A:T transitions in nine cases. However, among the other four sequences, only one of ours sample 76 is a G:C ---'> A:T transition. Three other ' sequence changes were' an T:A -> C:G transition at co don 172, 7 A:T - C:G transversion at codon 1978 and A:T - T:A transversion at codon 132, described here. It is difficult to conclude that there are similarities in codon frequency or nucleotide changes that have characterized prostate cancer thus far. Therefore, analysis of p53 mutations in prostate cancer thus far has not suggested a common etiology for a subset of patients. p53 mutations could have implications for prognosis in prostate cancer. The incidence of p53 mutations in primary prostate cancer appears to be approximately 10%. However if future studies confirm that p53 is an independent variable associated with a poor prognosis, then identification of cases that have this mutation may have an implication for treatment of a subset of patients who will do poorly with standard therapies. One study has suggested that the presence of P53 protein overexpression is associated with tumor cell aneuploidy. 5 Tumor cell aneuploidy is a known poor prognostic factor in prostate cancer and occurs in about 15% of cases at presentations. 12 · 13 P53 protein overexpression was only found in 8% of 137 samples tested by Visakorpi et al. 5 Aneuploidy and p53 mut~tions represented two overlapping subsets of prostate cancer patients. Because of the small numbers of patients with either p53 mutations or aneuploidy or both, larger scale studies will be required to define the impact on prostate cancer prognosis of these two markers singly or in combination. We also note that in our study nucleic acid analysis identified two samples with p53 gene mutations. Had we used immunohistochemistry alone, we would have missed the mutation in sample 129. In the three prostate cancer specimens in our analysis that contained p53 mutations, the mutations were present in a small fraction of the cells subjected to analysis. The immunohistochemical staining of sample 76 indicated that a very small percentage of malignant glands in the sample

495

analyzed had a p53 mutation. Therefore, in of tissues that appear to be heterogeneous for p53 mutations, it may be important to use at least two detection methods to analyze samples. We speculate that specimen heterogeneity was the reason we were unable to detect a sequence alteration in sample 190 and that others found no mutations in 5 of 14 positively staining samples. We also note that our SSCP analysis focussed on exons 5-8. In other cancers, but not in prostate cancer, p53 mutations have been found infrequently in exons 4 and 9. We can also speculate that there are other molecules that may stabilize P53 protein, thereby facilitating antibody staining and contributing to oncogenicity. It is possible that studies of metastatic prostate cancer lesions will identify a higher percentage of tissues with p53 mutations than is found in primary specimens. In one case a p53 mutation was found to occur heterogeneously in a cancerous prostate specimen, but was also detected in a pelvic lymph node metastasis. 7 Analysis of ten lymph nodes reported here found one positive for P53 staining. More extensive analysis of p53 mutations in prostate cancer metastases is necessary to confirm the suggestion that mutations are more common in advanced disease. 6 Acknowledgements. Keith Orford helped with nucleic acid extractions, Janice Lage with histology and Soon Paik with photomicroscopy. Greg Winstead, Blonka Winkfield and Clint Vickers performed tissue collection and storage. We are grateful to our surgical collaborators John Lynch and Timothy Tehan. Owen Blair and the Lombardi Cancer Research Center Flow Cytometry Facility performed the F ACS analysis. REFERENCES 1. Hollstein, M., Sidransky, D., Vogelstein, B. and Harris, C. C.: p53 mutations in human cancers. Science, 253: 49, 1991. 2. Bressac, B., Kew, M., Wands, J. and Ozturk, M.: Selective G to T mutations ofp53 gene in hepatocellular carcinoma from southern Africa. Nature, 350: 429, 1991. 3. Hsu, LC., Metcalf, RA., Sun, T., Welsh, J. A., Wang, N. J. and Harris, C. C.: Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350: 427, 1991. 4. Puisieux, A., Lim, S., Groopman, J. and Ozturk, M.: Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res., 51: 6185, 1991. 5. Visakorpi, T., Kallionieni, 0.-P., Heikkinen, A., Koivula, T. and Isola, J .: Small subgroup of aggressive, highly proliferative prostatic carcinomas defined by p53 accumulation. J. Natl. Cancer Inst., 84: 883, 1992. 6. Bookstein, R., MacGrogan, D., Hilsenbeck, S. G., Sharkey, F. and Allred, D. C.: p53 is mutated in a subset of advanced-stage prostate cancers. Cancer Res., 53: 3369, 1993. 7. Effort, P., Neubauer, A., Walther, P. J. and Liu, E.T.: Alterations of the p53 gene are associated with the progression of a human prostate carcinoma. J. Urol, 147: 789, 1992. 8. Isaacs W. B., Carter, B. S. and Ewing, C. M.: Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res., 51: 4716, 1991. 9. Carroll, A. G., Voeller, H. J., Sugars, L. and Gelmann, E. P.: p53 mutations in human prostate cancer cell lines. Prostate, in press, 1993. 10. Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K.: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5: 874, 1989. 11. Carbone, D., Chiba, I. and Mitsudomi, T.: Polymorphism at codon 213 within the p53 gene. Oncogene, 6: 1691, 1991. 12. Montgomery, B. T., Nativ, 0., Blute, M. L., Farrow, G. M., Myers, R. P., Zincke, H., Therneau, T. M. and Lieber, M. M.: Stage B prostate adenocarcinoma flow cytometric nuclear DNA ploidy analysis. Arch. Surg., 125: 327, 1990. 13. Forsslund, G., Esposti, P.-1., Nilsson, B. and Zetterberg, A.: The prognostic significance of nuclear DNA content in prostatic carcinoma. Cancer, 69: 1432, 1992. 14. Nigro, J.M., Baker, S. J., Preisinger, A. C., Jessup, J.M., Hostetter, R., Cleary, K., Bigner, S. H. and Davidson, N.: Mutations in the p53 gene occur in diverse human tumour types. Nature, 342: 705, 1989.