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Department of Pathology [A. R., W. H. W.], Department of Preventive Medicine [C. G.], and The Norris Comprehensive Cancer Center [M. F. P.], University of Southern California School of Medicine, Los Angeles, California 90033; Department of Obstetrics and Gynecology, Charité, Campus Virchow-Klinikum, Humboldt-University, Berlin, Germany [A. R., A. S., U. K., C. M., I. S., W. L.]; Department of Obstetrics and Gynecology, University of Ulm, Ulm, Germany [I. B. R., R. K.]; and Departments of Gynecologic Oncology [L. A. J.] and Pathology [A. E-N.], M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030; Department of Obstetrics and Gynecology, University of Graz, Graz, Austria, 112 [O. R.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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Experimental Design: One hundred seventy-eight ovarian carcinomas, snap frozen and stored at -80°C, were analyzed for mutations of the p53 gene (exons 2–11) by single-strand conformation polymorphism and DNA sequencing and for p53 overexpression by immunohistochemistry (monoclonal antibody DO7).
Results: p53 mutations were found in 56% (99 of 178) of the tumors, and 62% of these were located in evolutionary highly conserved domains of the gene. Time to progression and overall survival were significantly shortened in patients with p53 mutations compared with wild-type p53 (P = 0.029 and P = 0.014) and patients with mutations in highly conserved domains as opposed to nonconserved domains or wild-type p53 (P = 0.010 and P = 0.007). p53 protein overexpression (>10% positively stained nuclei) was found in 62% (110 of 178). Time to progression and overall survival were shorter in cases with p53 overexpression (cutpoint, 10%: P = 0.071 and P = 0.056) but only marginally significant. Resistance to adjuvant cisplatin or carboplatin chemotherapy was significantly more frequent in patients with p53 overexpression (P = 0.001) or p53 missense mutations (P = 0.008) than patients with normal p53.
Conclusions: p53 alterations correlate significantly with resistance to platinum-based chemotherapy, early relapse, and shortened overall survival in ovarian cancer patients in univariate analysis. In multivariable analysis though, p53 was not an independent prognostic factor.
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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14,500 deaths each year (1)
. Approximately 70% of women with ovarian cancer present with advanced stage disease with either regional or distant metastases at the time of diagnosis (1)
. Although relative 5-year survival data are 88 and 59% in FIGO3
stages I and II, respectively, the more advanced stages III and IV have a survival of approximately only 30 and 18%, respectively (2)
. The p53 tumor suppressor gene is the most frequently mutated gene in human cancers (3) and plays a critical role in the regulation of cell cycle and apoptosis. It has been found to be mutated in approximately 40–80% of epithelial ovarian cancers (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . In a previous study of 105 ovarian cancer patients, we found mutations in 57% of the cases (16) . It is thought that p53 protein alterations attributable to missense mutations, nonsense, or frameshift mutations provide a selective advantage for clonal expansion of neoplastic cells (17) .
Some studies have characterized p53 mutations in larger case numbers of ovarian cancer, but most of these have analyzed only a limited number of exons, mostly exons 5–8, which contain most of the mutations (4, 5, 6, 7 , 9 , 10 , 15 , 18, 19, 20, 21, 22, 23, 24) . Only a few studies have analyzed the entire open reading frame of the gene and have found mutations in 50–79% of the cases, with 5–20% of the mutations located outside exon 5–8 (5 , 8 , 12 , 14 , 16 , 25, 26, 27) . Therefore, a study that is limited to exons 5–8 will miss a substantial number of mutations.
Although p53 protein expression has been studied extensively by immunohistochemistry in ovarian cancer, the majority of these studies used archival formalin-fixed, paraffin-embedded tissue, which has a reduced immunoreactivity of many proteins after the process of fixation. In those studies that used frozen ovarian carcinoma specimens for immunohistochemical analysis of p53, the percentage of cases with p53 overexpression was 32–84% with an overall average of 50% (5 , 7 , 12 , 14, 15, 16 , 28, 29, 30, 31, 32, 33) .
The role of p53 alterations as a prognostic factor remains controversial. Several studies have identified p53 overexpression as a prognostic factor (30 , 32 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) , and a few studies have identified p53 overexpression as an independent prognostic factor in multivariate analysis (32 , 37 , 39 , 42) . But to date, only one study showed a marginally significant effect of p53 mutations on survival in ovarian cancer (16) .
Platinum-based combination chemotherapy with either cisplatin or carboplatin and paclitaxel is the standard treatment for ovarian cancer. However, resistance to chemotherapy remains a complex problem. Although 50% of patients are already resistant to chemotherapy, a substantial number of those, who were originally responsive, develop resistance to platinum-based chemotherapy during the course of their treatment (45)
. In cell culture experiments, there is evidence that the efficacy of various chemotherapeutic agents including cisplatin requires a functional p53 protein for efficient induction of apoptosis and that loss of p53 function enhances resistance to cytotoxic agents used in cancer therapy (46, 47, 48, 49)
.
In this investigation, we characterized p53 mutations and p53 expression in a series of 178 ovarian carcinoma patients. We have tried to avoid potential methodological problems encountered with both DNA sequence analysis and with immunohistochemistry by screening the entire coding region of the gene (exons 2–11) by PCR/SSCP, followed by DNA sequence analysis and by using frozen tissue for immunohistochemistry. The aim of the study was to further clarify the role of p53 alterations as a predictor of responsiveness to chemotherapy, and as a prognostic marker of disease outcome in epithelial ovarian cancers.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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. Patient ages ranged from 23 to 84 years, with a median age of 57 years. The racial-ethnic groups represented were: white, 92%; African-American, 5%; Hispanic, 3%; and Asian, 1%.
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. Adjuvant chemotherapy was given to 115 (76%) of the patients with tumor stages Ib–IV. Among these, 54 (47%) of the patients were treated with cisplatin-cyclophosphamide, 20 patients (17%) with carboplatin-cyclophosphamide, and 41 (36%) of the patients with other chemotherapy regimens. Thirty-six patients (24%) were not treated with adjuvant chemotherapy because of early stage disease, old age, or because they refused treatment. In 27 patients, information about chemotherapy treatment was insufficient. The response to chemotherapy was defined as: (a) platinum refractory, if there was no change or progressive disease during therapy; (b) platinum resistant, if the patient responded initially but relapsed or progressed within 6 months after the last cycle of chemotherapy; and (c) as platinum sensitive, if no relapse or progression was noted within 6 months after the last cycle of chemotherapy.
Clinical follow-up and overall survival information was available for all 178 patients, and information about progression of the disease was available for 138 (78%) patients. Follow-up ranged from 1 month to 12 years, with a median follow-up of 31 months in the total cohort and 52 months for the survivors. During the time of follow-up, 117 (66%) deaths occurred. One hundred and four patients died from ovarian cancer, 6 patients by intercurrent disease or accident, and 1 patient as a complication of surgery. In 7 cases, no information about the cause of death was available. Sixty (34%) patients were alive. At the time of last contact, 48 patients (27%) showed no evidence of disease. 76 patients (43%) had been documented to have recurrent disease, 3 patients had known residual disease with steady state, 11 patients (6%) had primarily progressive disease, and in 29 (16%) patients, who died from the tumor, the date of relapse was not available from the patient charts. In 11 patients, the disease status was not sufficiently documented.
Histological Classification, Grading, and DNA Ploidy Analysis.
All tumors were classified and graded according to the criteria defined by the WHO. The 178 epithelial ovarian carcinomas studied for p53 alterations included 111 (62%) serous and 30 (17%) endometrioid, 8 (5%) mucinous, 6 (3%) clear cell, 1 (0.6%) malignant Brenner tumor, 10 (6%) undifferentiated, 10 (6%) mixed epithelial, and 2 (1%) unclassified epithelial tumor. The evaluation of grading was based on the degree of histological differentiation with the formation of papillary, tubular, glandular, or cystic structures versus solid structures. One hundred and two cases included in this study had been characterized previously for DNA ploidy and S-phase fraction with a Cell Analysis System Image Analyser (CAS 200; Becton Dickinson; Table 1
) (51)
.
Immunohistochemistry for p53 Analysis.
Frozen tissue samples were embedded in OCT compound, sectioned at 4 µm in a TissueTek II cryostat, thaw-mounted on glass slides, and fixed immediately in acetone for 10 min. The initial tissue section, stained with H&E, was used to confirm that ovarian tumor of the appropriate histopathological type was present in the frozen specimen. Subsequent tissue sections were immunostained for p53 or used as a negative control section. p53 immunostaining was performed by successive incubation in a primary mouse monoclonal p53 antibody [DO-7, 0.95 µg/ml; 1:100 dilution; DAKO Corp.], a bridging secondary rabbit antimouse IgG antibody and a tertiary mouse peroxidase antiperoxidase antibody as described elsewhere (16)
.
Frozen cell pellets of human breast cancer cell lines known to have mutant p53 (SK-BR-3 and T47D; American Type Culture Collection) were used as positive control specimens for the immunohistochemistry. A negative control was prepared for each unknown tissue by substituting normal mouse IgG (2.5 µg/ml; 1:1000 dilution; Zymed Laboratories, Inc.) for the primary p53 antibody.
Nuclear immunostaining patterns were evaluated by three observers (A. R., W. W., M. P.). The immunoreactivity for p53 antibody was scored as the percentage of positively stained nuclei by counting 100–200 (minimum 100) tumor cells. Tumors with 10% or more nuclei showing immunostaining were considered to have p53 overexpression.
SSCP for p53 Analysis.
Frozen tissue sections stained with H&E were used to confirm that the majority of the tissues selected for analysis was composed of tumor cells. DNA was extracted from 10–20 serial frozen tissue sections (10 µm thick) of the tumor collected in Eppendorf tubes. The extraction solution consisted of 300 µl of 10 mM Tris-HCl, 25 mM EDTA, 100 mM NaCl, 0.5% SDS, and proteinase K (0.1 mg/ml) incubated overnight at 50°C. After complete digestion, DNA was purified by centrifugation after deproteination with phenol:chloroform:isoamyl alcohol (50:49:1) treatment and precipitation with ethanol and sodium acetate overnight at -20°C. The DNA yield was determined by spectrophotometry and analyzed by SSCP and DNA sequencing as described below.
The PCR was used to amplify each of the exons contributing to the open reading frame of the p53 gene. One pair of primers was used for each exon, except exon 4, where in part of the cases, two pairs of oligonucleotide primers had been used as published previously (16) . Each of the oligonucleotide primer pairs were designed to span not only the exon of interest but also sufficient flanking intron sequence so that splice junction mutations would be included for analysis. The 3' oligonucleotide primer of exon 11 was not outside of the splice junction but was 196 nucleotides downstream of the translation termination codon. The sequence for each primer pair was as follows: exon 2, 5'-CAGGGTTGGAAGCGTCTCAT-3' and 5'-CTTCCCACAGGTCTCTGCTA-3'; exon 3, 5'-TAGCAGAGACCTGTGGGAAGC-3' and 5'-AGAGCAGTCAGAGGACCAGGT-3'; exon 4, 5'-CGTTCTGGTAAGGACAAGGGT-3' and 5'-AAGAAATGCAGGGGGATACGG-3'; exon 5, 5'-CTGTTCACTTGTGCCCTGAC-3' and 5'-AACCAGCCCTGTCGTCTCTC-3'; exon 6, 5'-GCTGGAGAGACGACAGGGCT-3' and 5'-CAACCACCCTTAACCCCTCC-3'; exon 7, 5'-CTTGCCACAGGTCTCCCCAA-3' and 5'-AGGGGTCAGCGGCAAGCAGA-3'; exon 8, 5'-TTCCTTACTGCCTCTTGCTT-3' and 5'-AGGCATAACTGCACCCTTGG-3'; exon 9, 5'-AGCAAGCAGGACAAGAAGCG-3' and 5'-GCAAATGCCCCAATTGCAGG-3'; exon 10, 5'-CGATGTTGCTTTTGATCCGTCA-3' and 5'-ATCCTATGGCTTTCCAACCTAG-3'; and exon 11, 5'-TCCCGTTGTCCCAGCCTTAG-3' and 5'-TGGTATGTCCTACTCCCCATC-3'. The primer sequence for exon 7 and exon 8 was used as described by Kohler et al. (19) . Each exon of p53 was amplified by the PCR technique through 35 reaction cycles in a thermal cycler using 100 ng of genomic DNA, 4 µM deoxynucleotide triphosphates, 6 µCi of [33P]dATP, 6 µCi [33P]dCTP, 1 pmol of the appropriate oligonucleotide primer pair, 0.6–1.6 mM MgCl, and 1 unit of Taq polymerase in a 25-µl reaction mix. Conformational differences in the PCR products were resolved on a nondenaturing, MDE (mutation detection enhancement) polyacrylamide gel with the addition of 5% glycerol at room temperature (52) . Specimens containing known mutations were processed as positive control samples for exons 5–8, and normal ovarian tissue DNA was used as a negative control. All samples identified by SSCP as having altered mobility were further characterized by DNA sequencing for the exon putatively identified as mutated.
DNA Sequence Analysis for p53.
DNA segments identified as having altered mobility by SSCP were evaluated by conventional DNA sequence analysis methods (53
, 54) . Ovarian carcinoma template DNA was reamplified with the appropriate PCR primer pair, and amplified PCR product was purified (PCR Purification kit; Qiagen, Inc.) and pretreated with 0.4 units/µl shrimp alkaline phosphatase, and exonuclease 2 units/µl PCR product. Both the sense and antisense strands were analyzed by the dideoxynucleotide chain termination technique with PCR sense and antisense primers using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham) according to the manufacturer’s instructions. Forty-two ovarian carcinoma cases, which had no mobility shift identified by SSCP screening, had been subjected to complete DNA sequence analysis of exons 2–11 by automated DNA sequence analysis, as described previously, to identify those mutations that SSCP failed to identify (16
, 55)
.
Statistical Methods.
Statistical analyses were implemented using the SAS software packages. Overall survival was defined as the time from initial surgery following the diagnosis of ovarian cancer until death or the date of last follow-up, if the patient was still alive. All causes of death were counted as failures. Time to progression was defined as the time from initial surgery until documentation of disease progression. Patients who had not yet progressed were censored at the date of last follow-up in which the disease status was assessed; no patients died prior to progressing, unless they died from intercurrent disease. For 29 (16%) of the patients who died of their ovarian cancer, the date of progression was not documented. To estimate the date of progression, the median time from progression to death was calculated using the Kaplan-Meier estimator (56)
for those patients (n = 83) who had progressed and whose date of progression was known. This value, 273 days, was subtracted from the survival time of the patients whose date of recurrence was undocumented. In 5 patients, this value was greater than the time from surgery to death, and in this situation, the time to progression was taken as one-half of the interval between surgery and death. In 11 cases, no follow-up regarding disease status was available; these patients were not included in the analysis of time to progression but were included in the summary of baseline characteristics. Medians, quartiles, and percentages were used to summarize the patient characteristics and to illustrate associations. Pearson’s 
2 test for association and the Mantel-Haenszel test for trend (57)
were used to evaluate the strengths of the observed associations. Kaplan-Meier plots, relative risks, and Ps derived from the partial likelihood ratio test based on Cox’s proportional hazards model (58)
were used to summarize the relationships.
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RESULTS |
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TopABSTRACTIntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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. Ninety-two of these were mutations in exons 4–9. Eight were splice site mutations in introns 4–8. Forty-five of the sequence alterations were polymorphisms or intron alterations of undetermined significance. Thirteen of these were exon polymorphisms, and 32 were intron alterations, some of which are known as polymorphisms. Nine sequence alterations, which are known as a Codon 72 Arg
Pro polymorphism, were found in exon 4.4
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). One ovarian cancer patient had a mutation in exons 7 and 8, both with alteration of the amino acid sequence. The majority of mutations (86%) was found in exon 5 (22 mutations), exon 6 (10 mutations), exon 7 (25 mutations), and exon 8 (29 mutations; Fig. 2
; Table 2
). However, 14% of the mutations were found outside codons 126–306 in exon 4 (3 mutations), exon 9 (3 mutations), intron 4 (3 splice site mutations), intron 5 (1 splice site mutation), intron 6 (1 splice site mutation), intron 7 (2 splice site mutations), and intron 8 (1 splice site mutation).
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. Four "silent" DNA sequence alterations, located in exon 4 (codon 36), exon 6 (codon 213 and 224), and exon 7 (codon 231), which did not result in an amino acid alteration, were considered polymorphisms.
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. Region II, which spans part of exons 4 and 5, contained 10 mutations including 3 splice site mutations in intron 4 (10%), region III contained 8 mutations (8%), region IV 23 mutations (23%), and region V a total of 21 mutations (21%). Thirty-eight % of the mutations were found in nonconserved regions of the gene. The codons most frequently mutated were codons 175, 179, 234, 242, 248, 273, 275, and 281, which were each mutated in three or more different carcinomas (Fig. 2)
.
Fourty-two mutations (42%) were classified as transition mutations, 46 (45%) as transversions, and 13% were deletions or insertions. We found 29 G:C to A:T transitions and 13 A:T to G:C transitions, and 16 of 29 G:C to A:T transitions (55%) were located in CpG sites that are known to be potential sites of DNA methylation. Of 46 transversions, we found 20 G:C to T:A mutations, 12 G:C to C:G, 8 T:A to A:T, and 6 A:T to G:C mutations (Table 1)
.
p53 Protein Overexpression.
p53 protein overexpression, using a cutpoint of 
10% positively stained nuclei, was found in 62% (110 of 178) of the cases. The cutoff for p53 overexpression was chosen as 10% as in our previous study (16)
. Twenty-two (12%) cases had immunostaining <10%, and 46 (26%) cases had no immunostaining of tumor cell nuclei, and together were considered to have normal p53 expression. None of the ovarian carcinomas had specific cytoplasmic staining. p53 immunostaining was not observed in the benign tissue of ovarian carcinomas including fibrous connective tissue, blood vessels, and inflammatory cells.
Comparison of p53 Overexpression with p53 Mutations.
p53 overexpression detected by immunohistochemical staining was significantly correlated with p53 mutation (P < 0.001) detected by DNA sequence analysis. The percentage of cases with overexpression was 81% in ovarian carcinomas with p53 mutation and 38% in cases with wild-type p53 (Table 3)
. The percentage of cases with p53 immunostaining varied according to the type of mutation. The correlation between protein overexpression and mutations was mainly attributable to the high proportion of missense mutations that had p53 immunostaining. Sixty-seven of 71 (94%) of the cases with missense mutations showed p53 overexpression, whereas immunostaining >10% was only seen in 46% (13 of 28) of the ovarian carcinomas with nonmissense mutations. This is not significantly different from the frequency of overexpression in 30 of 79 tumors with wild-type p53 sequence (38%; P = 0.43; Table 3
). Nineteen % (19 of 99) of the cases with p53 mutations failed to show immunostaining, which was largely related to nonmissense mutations.
Correlation of p53 Alterations with Histopathological and Clinical Data.
p53 mutations were significantly more frequent in ovarian carcinomas with advanced FIGO stages III and IV (P < 0.001), poor differentiation (P < 0.001), residual tumor burden after debulking surgery (P < 0.04), DNA aneuploidy (P = 0.003), and high S-phase fraction as measured by image cytometry (P < 0.001). Patients with p53 mutations also had a higher median age than patients with wild-type p53 (P = 0.041). Overexpression of the p53 protein was correlated only with poor differentiation (P < 0.001) and high S-phase fraction (P < 0.001; Table 1
).
Impact of p53 Alterations on Chemotherapy Response.
Patients with p53-overexpressing tumors were significantly more often resistant or refractory to a platinum-based chemotherapy (61%) than patients with nonoverexpressing tumors (22%; P = 0.001; Table 4
). Among patients with p53 mutations, 56% as opposed to only 35% of the patients with wild-type p53 were resistant or refractory to platinum-based chemotherapy, but the result did not achieve formal statistical significance (P = 0.071). However, the response to chemotherapy was correlated with the type of mutation. If the subgroup patients with missense mutations were evaluated, 19 of 29 (66%) were platinum resistant as compared with only 15 of 44 (34%) patients with nonmissense mutations or p53 wild-type (P = 0.008; Table 4
). Overall, time to progression in patients who had received adjuvant platinum-based chemotherapy was significantly shorter for those with p53 alterations than those with normal p53 (P = 0.037; Fig. 3
A).
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B; Table 5
). In 61 of 99 (62%) tumors, the p53 mutation was in an evolutionary highly conserved domain. The clinical outcome of these as compared with patients with either wild-type p53 or a mutation in a nonconserved domain showed a significant difference in time to progression (P = 0.010) and overall survival (P = 0.007; Fig. 3
C). Furthermore, time to progression and overall survival were better in patients with normal p53 expression as opposed to p53 overexpression, but the results reached only marginally statistical significance (P = 0.071, P = 0.056 log-rank; Table 5
). Overall, the most favorable prognosis in terms of overall survival was seen in patients who had wild-type p53 sequence and normal p53 expression as opposed to those who had either one or both alterations of the p53 gene and protein expression (P = 0.007; Fig. 3
D).
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Univariate analysis of overall survival identified residual disease, FIGO stage, age, grade, lymph node status, and S-phase fraction as prognostic factors besides p53 mutation and p53 alteration (Table 5)
. In multivariable analysis though, only residual disease (P < 0.001), FIGO stage (P = 0.001), grade (P = 0.009), and patient age (P = 0.01) were shown to be independent predictors of survival (Table 5)
.
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DISCUSSION |
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TopABSTRACTIntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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(a) As in our previous study (16) , we analyzed frozen tissue to avoid problems related to loss of antigenicity that is observed with immunohistochemistry in paraffin-embedded tissue.
(b) We assessed the frequency of mutations throughout the entire coding region of the gene by analyzing exons 2–11 by SSCP and subsequent DNA sequencing.
(c) Information on adjuvant treatment was available, and the results of the p53 sequence analysis and immunohistochemistry were compared with responsiveness to platinum-based chemotherapy.
(d) Finally, clinical follow-up information concerning outcome was available in all of the cases, and information about relapse or progression of the disease was available in 78% of the patients, which allowed evaluation of the prognostic relevance of p53 alterations in a sufficient number of cases.
We found p53 mutations in 56% of the ovarian carcinomas (99 of 178), which is a slightly higher percentage than that observed in most other studies. Sixty-four of these alterations have been described previously in a study of 105 ovarian carcinoma patients (16) . The frequency of p53 mutations in studies using frozen tissues including our previous study is 50% and ranges from 20 to 73% (4, 5, 6, 7 , 12 , 14, 15, 16 , 20 , 23 , 50) . In studies using paraffin-embedded tissues, the frequency of mutations was 47%, ranging from 26 to 79% (8, 9, 10, 11 , 18 , 21) . Several studies have analyzed part of the p53 coding region, usually exons 5–8 or exons 4–9, and found between 20 and 52% mutations (6 , 9 10 11 , 18 , 20 , 21 , 23 , 59) with the exception of Kohler et al. (7) , who found 73%, and Jacobs et al. (60) , who found 66% mutations. The percentage of mutations in studies analyzing the entire coding region of p53 is 59% (207 of 349) overall and varies from 52 to 79%, which is consistent with our own results (5 , 8 , 12 , 14 , 16) .
The mutations were located predominantly (92%) in exons 5–8 (codons 126–307) and their associated splice junctions, whereas 8% of the mutations were outside of these exons in exon 4, exon 9, and their splice junctions. These results demonstrated that 8% of the mutations could only be identified by evaluation of codons 1–125 and 308–393 (211 codons). The results are consistent with our previous study and those of other authors who analyzed the entire coding sequence of the gene. The frequency of mutations in exons 2–4 and 9–11 was reported to be between 0 and 20% (24 of 258) with an overall average of 11% (5
, 8
, 12
, 14
, 16
, 25)
. This means that studies analyzing only exons 5–8 of the p53 gene will miss 11% of the mutations.
Type of Mutations.
In our study, 72% of identified mutations were missense mutations. This is consistent with the results of most other authors, who found between 62 and 85% missense mutations (6
, 8
, 12, 13, 14, 15
, 21)
. Deletions are usually described in 7–13% of cases (6
, 8
, 13
, 15
, 16)
, which is similar to our finding of 10%. But some authors find deletions in a percentage as high as 22% by SSCP and sequencing of exons 2–11 (14)
and 27% of all mutations by sequencing exon 2–11 (12)
. Insertions have been found less often and have been described as only 2.5–6% of the p53 mutations in ovarian cancers (6
, 12
, 14)
. We identified three insertions (3%), one of which was combined with a deletion. Splice site mutations are considered rare mutations, and thus far only 12 splice site mutations have been described in the ovarian cancer literature including our previous study, with a percentage of 1–5% (8
, 12, 13, 14, 15
, 21)
. Including three previously published splice site mutations (16)
, we found eight splice site mutations accounting for 8% of all mutations identified, which is higher than in any of the other studies.
p53 Mutations in Evolutionary Highly Conserved Domains.
The majority of mutations in this study (62%) were located within highly conserved domains of p53 (domains II, III, IV, and V). These domains are almost equivalent to the loop-sheet-helix (domains II and V), loop 2 (domain III), and loop 3 (domain IV) regions of p53 (61
, 62)
. The loop-sheet-helix is responsible for direct DNA interactions with the major groove; loop 3 is responsible for direct interactions with the minor groove of DNA, and loops 2 and 3 together are responsible for maintaining the needed three-dimensional conformation (61
, 62)
. Tumors with mutations in these conserved domains were, in our study cohort, associated with more aggressive clinical behavior compared with those with mutations in nonconserved regions or wild-type p53 sequence (P = 0.007).
p53 Protein Overexpression.
Several investigators have used frozen ovarian carcinoma specimens for immunohistochemical analysis of p53 and found p53 overexpression in 32–84% with an average percentage of 51% (5
, 7
, 12
, 14 , 15
, 16
, 28, 29, 30, 31, 32, 33)
. We found p53 overexpression in 62% of the cases. A few authors have performed both immunohistochemical and mutation analysis (5
, 12
, 14
, 16
, 22
, 23
, 28)
. Only three of the latter studies involved >50 carcinomas (12
, 14 , 16)
. In studies that used formalin-fixed, paraffin-embedded material and analyzed >50 cases of FIGO I-IV ovarian carcinomas, a frequency of 49% p53 overexpression with a range of 26% to 70% was found (21
, 22
, 34, 35, 36, 37
, 40, 41, 42, 43, 44
, 51 , 63, 64, 65)
. With the exception of four studies (8
, 12
, 18 , 23)
, three of which were of a small number of cases, the frequency of p53 overexpression is consistently higher than the frequency of p53 mutations. Our observations of a 62% immunostaining rate and a 56% mutation rate for p53 were consistent with these generalizations.
Correlation between p53 Mutations and Overexpression.
A strong correlation was observed between p53 immunostaining and p53 mutations (P < 0.001), but this was mainly an effect of the high rate of immunostaining in ovarian carcinomas with missense mutations (94%). Only 46% of nonmissense mutations were identified by immunostaining, and this percentage was not significantly different from the percentage of staining in wild-type p53 cases (38%; P = 0.43). Therefore, it can be concluded that immunohistochemistry is not suitable for the detection of nonmissense mutations. The low rate of immunostaining in cases with nonmissense mutations is assumed to be caused by alteration and truncation of the protein attributable to introduction of a stop codon in nonsense mutations, frameshifts caused by deletions or insertions, and alterations of transcribed RNA in splice site mutations. But p53 protein may not only be qualitatively altered but also quantitatively reduced, possibly because of destruction of the mRNA as a result of sequence changes.
A high percentage of cases with wild-type p53 sequence (38%) showed overexpression of the p53 protein. Because this cannot be explained by sequence alterations, the stabilization and accumulation of a presumably normal p53 protein may be caused by other genes interacting with p53. Alterations of the mdm2 gene, which down-regulates p53 in an autoregulatory feedback and promotes nuclear export and degradation of the p53 protein (66 , 67) , may play an important role in these cases.
p53 and Response to Chemotherapy.
Cell culture experiments have shown that the sensitivity of tumor cells to various chemotherapeutic agents depends on the efficient induction of apoptosis mediated by a functional p53 protein and that loss of p53 can enhance resistance to chemotherapy (46, 47, 48)
, but the results concerning p53 status and chemotherapy sensitivity remain controversial in cell culture experiments.
The wild-type p53-expressing A2780 human ovarian cancer cell line acquired cross-resistance to cisplatin and doxorubicin by transfection with a dominant-negative mutant p53 gene, whereas it retained (47) or even increased (68) sensitivity to Taxol. In another study (48) , cisplatin caused strong induction of p53, WAF1, and Bax in the cisplatin-sensitive A2780 cell line, whereas there was no such effect in a cisplatin-resistant cell line A2780-DX3, which furthermore showed a significant proportion of potentially inactive p53 protein located in the cytoplasm instead of the nucleus.
However, some of the studies using cell culture experiments report contradictory results. In a panel of cell lines, cisplatin was more efficient against mutant/null p53 cell lines than wild-type cell lines, whereas the novel platinum analogue diaminocyclohexane-trans-diacetato-dichloroplatinum was considerably more toxic in wild-type cell lines (49) . Similarly, in isogenic A2780 human ovarian cancer cell lines that differ only in p53 function by transfection of HPV-E6, the p53-deficient cell line was more sensitive to cisplatin and the novel platinum agent ZD0473 (69 , 70) .
Recently, genetic suppressor elements have been identified that correspond to various regions within the p53 gene and can act as dominant-negative peptides or antisense RNA molecules (71) . A synthetic peptide, representing the predicted amino acid sequence of this genetic suppressor element, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence-specific DNA binding activity of p53 protein in vitro. This indicates that inactivation of p53 function confers cisplatin resistance in these ovarian tumor cells.
The hypothesis that ovarian cancer cells with functional p53 are more sensitive to cisplatin is further supported by the findings of gene therapy studies. Introduction of wild-type p53 protein via adenovirus gene transfer into A2780/CP cisplatin-resistant cells significantly sensitized these cells to platinum cytotoxicity, indicating that p53 was involved in resistance to cisplatin (72) .
Further strong evidence for the importance of a functional p53 protein for the efficacy of cisplatin and carboplatin is given by a database of the National Cancer Institute on drug activity in cell lines (73) . This database compares activity patterns of chemotherapeutic agents and possible targets or modulators of activity in the cells, such as oncogenes, tumor suppressor genes, drug resistance-mediating transporters, and others for >60,000 compounds against a panel of 60 human cancer cell lines. The results show a strong correlation between p53 wild-type sequence or p53 function and efficacy of cisplatin and carboplatin (73) . As opposed to this, antimitotic tubulin-active agents such as Taxol show a strong negative correlation between p53 wild-type as well as p53 function and activity of the drug (73) .
Because a dysfunctional p53 cannot mediate the apoptotic process, tumors with p53 mutations or altered p53 protein may become resistant to platinum-based chemotherapy (74) . In our study in a subgroup of 72 patients who received cisplatin or carboplatin-cyclophosphamide combination therapy, we found a significant correlation between p53 protein overexpression and resistance to platinum-based chemotherapy (P = 0.001). If all p53 mutations were taken into account, there was only a trend but not a significant association with treatment response (P = 0.071). Interestingly though, we found that missense mutations, when compared with wild-type p53 or other mutations, were correlated with a high percentage of chemotherapy resistance in this cohort (P = 0.008). This is consistent with the results of three other studies that have demonstrated a correlation between p53 alterations and resistance to platinum-based chemotherapy in ovarian cancer (13 , 31 , 65) . One study analyzed p53 overexpression and mutations in 32 cases of ovarian cancer FIGO stages III and IV and found a higher frequency of chemotherapy resistance both in p53-overexpressing tumors and in tumors with missense mutations (13) . Another study, analyzing 33 cases of advanced ovarian cancer FIGO stages III and IV found a significant association between chemotherapy resistance and p53 immunostaining, as well as SSCP alterations (31) . These results are further supported by two immunohistochemical studies. p53 overexpression was found to be associated with poor response to either paclitaxel/platinum or cyclophosphamide/platinum chemotherapy in 54 stages III and IV patients with results approaching statistical significance (75) . A higher frequency of early tumor progression was found in 28 of 59 patients who had received either cisplatin and treosulfan or treosulfan alone (76) . In a more recent study, Ferrandina et al. (65) analyzed 168 primary stage III–IV ovarian carcinomas and observed a significant correlation of p53 overexpression with resistance to platinum-based chemotherapy in those patients who underwent pathological assessment of response. However, three other studies using immunohistochemistry (36 , 39 , 40) and one study using temperature gradient gel electrophoresis and immunohistochemistry (42) did not find a difference in treatment response between patients with or without p53 alterations.
p53 Alterations and Survival.
To date, several studies have found a correlation between p53 overexpression and shortened survival (30
, 32
, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
, but only few studies have identified p53 as an independent prognostic factor in multivariate analysis (32
, 37
, 39
, 42
, 44)
. However, p53 protein expression was not a significant predictor of poor outcome in several other studies (8
, 16
, 19
, 21
, 22
, 28
, 33
, 63
, 65)
. Only five studies described a correlation between p53 overexpression and time to recurrence (36
, 38
, 42, 43, 44)
. None of seven studies that analyzed p53 sequence alterations and clinical follow-up data found a significant correlation between p53 mutations and shortened relapse-free or overall survival (4
, 7
, 8
, 21
, 22 , 24
, 31)
. In our previous study, p53 mutations were associated with shortened overall survival, but the results reached only marginal statistical significance (P = 0.049; Ref. 16
). Most of the p53 studies examined either p53 expression or p53 mutations but not both in study populations of limited size.
In our study, p53 overexpression with a cutpoint of 10% reached only marginal statistical significance as a predictor of poor clinical outcome (P = 0.056). p53 mutations, and especially those that were located in highly conserved domains, were clearly correlated with poor overall survival (P = 0.014 and P = 0.007). An association between p53 mutations and overexpression was observed (P < 0.001), and the most favorable prognosis with a significantly longer time to progression and overall survival was seen in patients with neither p53 mutation nor overexpression (P = 0.035, P = 0.007).
Summarizing these results, our investigation analyzed p53 protein expression and p53 sequence alterations in the entire coding region of the gene using frozen ovarian cancer tissue with complete clinical follow-up information. p53 mutations and especially those in evolutionary conserved domains correlated with shortened time to progression and shortened overall survival. Expression of p53 protein was not significantly associated with poor clinical outcome. In multivariable Cox regression analysis, p53 alterations were not an independent prognostic factor. Most importantly, evaluation of adjuvant treatment showed that p53 overexpression as well as p53 missense mutations were correlated with resistance to platinum-based chemotherapy. This may provide further clinical evidence that the sensitivity of ovarian cancer cells for cisplatin and carboplatin depends on the efficient induction of apoptosis mediated by a functional p53 protein.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants CA48780 and CA50589 from the National Cancer Institute, Grants IN-21-29/003 and PDT-411 from the American Cancer Society, Grant NIH NCRR GCRC MO1 RR-43 from the Molecular Core Laboratory Facilities of the GCRC, Grant DFG RU 476/2-1 from the Deutsche Forschungsgemeinschaft, and a grant from the University of Southern California Ovarian Cancer Research Fund. A. R. was supported by an Alexander von Humboldt Fellowship. 
2 To whom requests for reprints should be addressed, at Norris Comprehensive Cancer Center, Norris Topping Tower, Mailslot # 73, University of Southern California Los Angeles, 1441 Eastlake Avenue, Los Angeles, CA 90033. Phone: (323) 865-0563; Fax: (323) 865-0122; E-mail: villalob{at}hsc.usc.edu
3 The abbreviations used are: FIGO, Fédération Internationale des Gynaecologistes et Obstetristes; SSCP, single-strand conformation polymorphism.
4 A. Reles, I. Schmidt, C. Gee, C. Minguillon, W. Lichtenegger, and M. F. Press, unpublished data.
Received 9/25/00; revised 7/16/01; accepted 7/18/01.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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STOP mutations of the TP53 gene in human ovarian cancer in the southwestern part of the Netherlands. Int. J. Cancer, 76: 299-303, 1998.[CrossRef][Medline]
