The P53 Mutational Spectrum Associated with BRCA1 Mutant Ovarian Cancer1

Vol. 7, 831–838, April 2001 Clinical Cancer Research 831

The p53 Mutational Spectrum Associated with BRCA1 Mutant Ovarian Cancer1

Richard E. Buller,2 Thomas A. Lallas, Conclusions: Ovarian cancers containing somatic or Mark S. Shahin, Anil K. Sood, germ-line BRCA1 mutations are uniformly accompanied by Melanie Hatterman-Zogg, Barrie Anderson, p53 dysfunction. This finding offers additional support to observations regarding the importance of p53/BRCA1 inter- Joel I. Sorosky, and Patricia A. Kirby actions in ovarian carcinogenesis. Division of Gynecologic Oncology, Departments of Obstetrics and Gynecology [R. E. B., T. A. L., M. S. S., A. K. S., M. H-Z., B. A., J. I. S.] and Pathology [P. A. K.], The University of Iowa Hospitals INTRODUCTION and Clinics, Iowa City, Iowa 52242-1009 Mutation of the p53 tumor suppressor gene is one of the most frequent molecular genetic events in cancer (1–3). Char- acteristic p53 mutational spectra have been described for Bur- ABSTRACT kitt’s lymphoma (4), aflatoxin-induced hepatocellular carci- Purpose: Cancer-specific p53 mutational spectra have noma (5), benzo(a)pyrene-induced lung cancers (6), and skin been identified. Data from murine models and human cancers where UV light is causal (2, 7). Several reports suggest BRCA1-related hereditary breast cancers suggest that both that novel p53 mutations and an overall high frequency of the unique and specific BRCA1-associated p53 mutations may p53 mutations associate with familial (8) and hereditary (9–11) be found in BRCA1-related ovarian cancers. breast cancers. There are few reports of p53 mutations in Experimental Design: The p53 mutational spectrum BRCA1-related ovarian cancers (12–15). The total number of from ovarian cancers containing either somatic or germ-line BRCA1 mutant cancers analyzed is 46. Each report has limita- BRCA1 mutations was compared with that of sporadic ovar- tions, such as evaluating a single ethnic group, Ashkenazi Jews, ian cancers defined as those diagnosed with a negative fam- without a control population (12), evaluation only of primary ily history for breast/ovarian cancer in a three-generation peritoneal carcinomas (13), and major failures of tumor DNA to pedigree. Tumor DNA was screened over exons 2–11 of the amplify in some PCR reactions (15). p53 gene by the PCR and single-strand confirmation poly- A growing body of evidence suggests that p53 and BRCA1 morphism analysis of the amplimers. Cycle-based DNA se- interact directly (16–18) and may play an important role in the quencing from separate reactions was used to confirm p53 DNA repair process (19–28). Other studies have demonstrated mutations. the potential for proliferative advantages to cells simultaneously Results: p53 gene mutations were detected in 42 of 86 null for both p53 and BRCA1 function (29, 30). Taken together, sporadic ovarian cancers, compared with 13 of 15 cancers these observations suggest that additional studies of p53 muta- and 16 of 20 tions associated with hereditary ovarian cancers may be useful (0.007 ؍ with somatic BRCA1 mutations (P p53 for several reasons: (a) if unique ovarian cancer p53 mutational .(0.01 ؍ cancers with germ-line BRCA1 mutations (P null mutations were found in 31.4% of BRCA1 mutant patterns do exist, their characterization may facilitate identifi- cancers, compared with only 9.3% of the sporadic cancers cation of potential germ-line BRCA1 carriers for targeted direct The p53 mutational spectrum of germ-line BRCA1 sequencing; and (b) there has been debate regarding .(0.002 ؍ P) BRCA1-related cancers was shifted toward transversions, survival of individuals with BRCA1-associated cancers (31–35). frameshifts, and non-CpG transitions relative to the spec- Because p53 mutations may also influence ovarian cancer sur- trum of sporadic ovarian cancers. Thirty-three unique ovar- vival (36), selection of control groups matched for p53 mutation ian cancer p53 mutations were sequenced. However, the status and potentially for p53 mutation type may be critical to specific p53 mutations in the BRCA1 mutant cancers were most appropriately address this issue. To these ends, we have no more unique to this cohort than the p53 mutations of the recently completed screening of ϳ150 ovarian, primary perito- sporadic cancers. neal, and fallopian tube carcinomas3 for BRCA1 null mutations using a protein truncation assay (37). The present study reports the relationship of p53 mutations in this group stratified for the presence or absence of BRCA1 mutation. Individuals with any Received 8/28/00; revised 1/3/01; accepted 1/3/01. family history of breast or ovarian cancer in which we did not The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked identify a BRCA1 mutation were excluded in the beginning to advertisement in accordance with 18 U.S.C. Section 1734 solely to minimize the confounding factors of BRCA2 or other, as yet indicate this fact. 1 This research was supported in part by the Florence and Marshall Schwid Award (to R. E. B.) from the Gynecological Cancer Foundation. 2 To whom requests for reprints should be addressed, at Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, 200 3 J. P. Geisler, M. A. Hatterman-Zogg, J. Rathe, T. Lallas, and R. E. Hawkins Drive, #4630 JCP, Iowa City, IA 52242-1009. Phone: Buller. Ovarian Cancer BRCA1 mutation detection: Protein truncation (319) 356-2015; Fax: (319) 353-8363; E-mail: richard-buller@uiowa. test (PTT) outperforms single strand conformation polymorphism (sscp) edu. analysis, submitted for publication.

undiscovered, hereditary ovarian cancer genes interacting with In each case when a tumor with a candidate BRCA1 mutation p53 as well as potential differences of p53 expression in familial was identified, genomic sequencing of the appropriately se- ovarian cancers. lected BRCA1 region was carried out. All mutations were ver- ified from repeat PCR reactions and bidirectional sequencing as MATERIALS AND METHODS we have done traditionally to detect p53 mutations (39). Once a All samples were procured in accordance with the Institu- tumor BRCA1 mutation was sequenced, a germ-line mononu- tional Committee for the Protection of Human Subjects. The clear cell DNA sample from the same individual was also methods used to isolate and screen genomic DNA as well as to sequenced over the identical region of the BRCA1 gene to characterize p53 mutations have been published previously (38– determine whether the tumor mutation was a hereditary germ- 40). Approximately 73% of the samples were from snap frozen line BRCA1 mutation or simply a somatic mutation, absent in samples, whereas 27% of the cancer DNA was isolated from the germ-line. PTT screening, of course, will not detect BRCA1 paraffin-embedded tissue. Briefly, each exon in the open read- missense mutations. However, until a functional BRCA1 assay ing frame is PCR amplified, and the amplimers were analyzed is developed, the significance of BRCA1 missense mutations is by SSCP4 analysis on 6% polyacrylamide gels. Each gel con- open to question. Furthermore, nearly 80% of the BRCA1 mu- tains positive and negative controls as well as a nondenatured tations reported in the Breast Cancer Information database are 5 sample. A mutation is inferred from shifts in banding patterns. null mutations. When a shift is found, a separate PCR reaction is used to Sporadic ovarian cancers were defined after a negative generate a product for cycle-based sequencing using the fmol BRCA1 screening assessment and by omitting any individuals system (Promega Corp., Madison, WI). Bidirectional sequenc- with a family history of breast or ovarian cancers in first-, ing identifies mutations that are subsequently resequenced from second-, or third-degree relatives. Complete pedigree analysis a third PCR reaction. Therefore, the evidence to support the with histological confirmation and/or death certificate review presence of each mutation is generated from three independent for the majority of the reported cancers among family members reactions. Tumor expression of p53 was determined by DO7 of study probands is part of the University of Iowa Hospital and antibody staining as we have described (41). However, we do Clinics Gynecological Oncology Tissue Bank record. For this Ͼ not use immunostaining to screen for p53 mutations because this assignment, we required a minimum of 10 female relatives 30 method does not detect most p53 null mutations (39). Overall, years of age in a three-generation pedigree (40). This method- ϳ10% of ovarian cancers overexpress p53 in the absence of ology was designed to eliminate familial ovarian cancers as a mutation, whereas 19% of mutations are D07 antibody negative. potential confounding variable for this study. All immunopositive, SSCP-negative tumors have been com- Statistical Analysis. Categorical variables were tested ␹2 pletely sequenced (36). Mutations are categorized as follows: by the statistic. Differences in means between continuous Ͻ transitions, purine 43 purine or pyrimidine 43 pyrimidine at variables were analyzed by the t-statistic (two tailed). P 0.05 CpG or non-CpG sites; transversions, purine 43 pyrimidine; was considered statistically significant. or insertion/deletion. Identification of tumor BRCA1 mutations was facilitated RESULTS both by modified SSCP screening of all exons and splice junc- Ovarian, primary peritoneal, and fallopian tube carcinomas tions as we have described (42) and more recently by the PTT. were analyzed for p53 mutations from 86 probands with spo- The latter technique required snap frozen tissue and only iden- radic ovarian cancer and 35 different individuals whose tumors tifies BRCA1 null mutations. Results of direct comparison of were found to harbor BRCA1 mutations. The BRCA1 mutant SSCP and PTT screening are the subject of a separate report.3 cancers included 20 germ-line and 15 somatic mutations. Table The BRCA1 mutant tumors included 6 with missense mutations 1 summarizes the characteristics of each study cohort with detected by SSCP screening (43) and 29 protein-truncating respect to age of diagnosis, distribution by cancer site, FIGO mutations, of which 16 have been reported by our group (43). stage (45) at presentation, histology, tumor grade, and DO7 Tumors were not consecutive. They were initially selected as we antibody staining. Although the mean age at diagnosis for car- have described (43) and more recently were selected on the riers of germ-line BRCA1 mutations, 54.9 years, was Ͼ5 years basis of available snap frozen tissue and loss of heterozygosity younger than for individuals with sporadic ovarian cancer, 60.2 at the BRCA1 locus determined with the intragenic markers years, a number of individuals with BRCA1-associated cancers D17S 855, D17S 1322, and D17S 1323 (44). This approach was were diagnosed in their 70s; therefore, this difference did not chosen because we and others have found that tumor BRCA1 achieve statistical significance. As might be expected, the mean mutations are almost universally accompanied by loss of het- age at diagnosis for individuals with somatic BRCA1 tumor erozygosity. All sporadic ovarian cancers in this study were mutations, 60.1 years, was identical to that of individuals with screened for BRCA1 mutations either by SSCP (paraffin- sporadic cancers. Most of the BRCA1 mutations were associated embedded samples) or both SSCP and PTT (snap frozen tissue). with ovarian rather than peritoneal or fallopian tube cancers. Several differences between these groups are noteworthy. Tu- mors with any BRCA1 mutation were less likely to be diagnosed

4 The abbreviations used are: SSCP, single-strand conformational polymorphism; PTT, protein truncation assay; FIGO, International Federa- tion of Gynecology and Obstetrics; ANOS, adenocarcinoma not other- 5 Internet address for the Breast Cancer Information Core: http:// wise specified. www.nchgr.nih.gov/intramural_research/lab_transfer/bic/index.html.

Table 1 Histopathological features of tumors analyzed for p53 mutations Tumor classification Sporadic Somatic BRCA1 Germ-line BRCA1 Any BRCA1 (n ϭ 86) (n ϭ 15) (n ϭ 20) (n ϭ 35) Age at diagnosisa 60.2 [12.8] 60.1 [11.4] 54.9 [12.6] 57.2 [12.2] Site Ovary 78 (90.7%) 14 (93.3%) 18 (90.0%) 31 (88.6%) Fallopian tube 1 (1.1%) 1 (6.7%) 1 (5.0%) 2 (5.7%) Peritoneum 7 (8.1%) 1 (5.0%) 1 (2.9%) FIGO stageb I 16 (18.6%) 1 (6.7%) 1 (3.4%) II 5 (5.8%) 1 (6.7%) 3 (15.0%) 3 (10.3%) III 51 (59.3%) 10 (66.7%) 13 (65.0%) 19 (65.5%) IV 14 (16.3%) 3 (20.0%) 4 (20.0%) 6 (20.7%) Histology ANOS 17 (19.8%) 7 (46.7%) 3 (15.0%) 10 (28.6%) Clear cell 4 (4.7%) 1 (6.7%) 1 (2.9%) Endometroid 13 (15.1%) 3 (20.0%) 1 (5.0%) 4 (11.4%) Mucinous 8 (9.3%) 1 (6.7%) 1 (5.0%) 2 (5.7%) Papillary serous 42 (48.8%) 3 (20.0%) 13 (65.0%) 16 (45.7%) Transitional 2 (2.3%) 2 (10.0%) 2 (5.7%) Grade 1 11 (12.8%) 2 19 (22.1%) 7 (46.7%) 4 (20.0%) 11 (31.4%) 3 56 (65.1%) 8 (53.3%) 16 (80.0%) 24 (68.6%) D07 antibody positive 47 (52.7%) 11 (78.6%) 11 (55.0%) 22 (64.8%) a Numbers in brackets, SD. b FIGO staging corresponds to tumor confined to the ovary (stage I), confined to the pelvis (stage II), confined to the abdomen (stage III), and spread to the liver parenchyma or outside the abdomen (stage IV), when the disease is initially diagnosed (42).

Fig. 1 Left, representative SSCPs from exons 8 and 7 of the p53 gene. ND, nondenatured sample. Abnormalities are seen for tumors 25, 178, and 320 in exon 8 and for tumor 20 in exon 7. In each case, a mutation has been sequenced. Positive and negative controls for each exon are on a portion of the gel not shown here. Right, comparison of exon 8 sequence between normal (N) and tumor tissue (T, top label) for proband 15.R. The sequence is from an antisense sequencing primer and reads forward from top to bottom. Arrow, G 3 C base change in the tumor rendering codon 281 GAC to CAC or Asp to His.

as FIGO stage I disease (␹2, 4.13; P ϭ 0.04). They were also cancers (P Ͼ 0.05). There were no differences in the frequency more often of higher histological grade, i.e., grade 2 or 3 (␹2, of DO7 antibody-positive cells between groups. 4.92; P ϭ 0.03). The highest incidence of grade 3 cancers (80%) Fig. 1, left, shows a representative SSCP analysis. Fig. 1, was found in the germ-line mutant BRCA1 cohort. Comparison right, shows the sequence of a representative p53 mutation from of all histological subtypes simultaneously showed no differ- an individual carrying a germ-line BRCA1 mutation (2457 ences between groups. However, grouping the more prevalent C3T stop 780). In this case, a p53 GAC to CAC transition types such as ANOS versus other histologies, or ANOS versus mutation is seen in tumor DN but not in germ-line DNA. The papillary serous, versus all other histologies revealed that ANOS specific p53 mutations associated with the study cancers are was more prevalent in tumors with somatic BRCA1 mutations listed in Table 2. Overall, only 42 of the 86 sporadic cancers (␹2, 5.10; P ϭ 0.02). In general, transitional carcinomas of the (48.8%) contained p53 mutations. In contrast, 13 of 15 cancers ovary are relatively uncommon. Nonetheless, two cases were with somatic BRCA1 mutations (86.7%; ␹2, 7.37; P ϭ 0.007) reported in germ-line BRCA1 carriers (2 of 20; 10%) versus two and 16 of 20 cancers in germ-line BRCA1 carriers (80%; ␹2, cases (2 of 86; 2.3%) in individuals with sporadic ovarian 6.36; P ϭ 0.01) contained p53 mutations. The frequency distri-

Table 2 Spectrum of p53 mutations in ovarian cancers Sporadic ovarian cancers: Absent BRCA1 truncating mutations Tumora Cell typeb Stagec Grade Exon Residue p53 mutationd Mutation typee 170 PS IIIC 3 2 11 GAG/AAG Glu3Lys CpG 215 ANOS IIIC 3 4 91 TGG/TAG Trp3Stop Non-CpG 316 PS IIIC 2 4 95 12210 Ins 17 Stop 128 Insertion 112 PS IIIC 3 5 126 13053 agTA3ggTA splice site Non-CpG 122 ANOS IIIC 3 5 131 AAC/CAC Asn3Hisf Tv 293 M IV 3 5 132 AAG/AAT Lys3Asnf Tv 145 E IIIC 3 5 135 TGC/TAC CYS3Tyr Non-CpG 318P PS IIIC 3 5 141 TGC/TAC CYS3Tyrf Non-CpG 303 M IA 2 5 154 CGC/GCC Pro3Alaf Tv 110 E IIIA 3 5 158 CGC/CAC Arg3Hisf CpG 212 PS IIIC 3 5 161 GCC/ACC Ala3Thrf Non-CpG 79 PS IV 2 5 175 CGC/CAC Arg3His CpG 210 PS IIIC 3 5 175 CGC/CAC Arg3Hisf CpG 308 E IIIC 3 5 175 CGC/CAC Arg3Hisf CpG 87 C IIIC 3 5 179 CAT/AAT His3Asn Non-CpG 419 PS IIIC 3 6 193 CAT/CGT His3Argf Non-CpG 252 PS IIIC 2 6 195 ATC/ACC Ile3Thrf Tv 270 E IIC 3 6 214 CAT/CGT His3Argf Non-CpG 264 PS IIC 3 6 220 TAT/TGT Tyr3Cys Non-CpG 155 ANOS IIIC 3 6 220 TAT/AAT Tyr3Asnf Tv 37 E IIIC 2 7 237 ATG/ATA Met3Ilef Non-CpG 353 PS IV 3 7 238 TGT/GGT Cys3Glyf Tv 202 ANOS IIIC 3 7 241 14049 Del C Stop 246 Deletion 436CCG/CCA Pro3Pro CpG 189 E IIC 2 7 245 GGC/AGC Gly3Ser CpG 247P ANOS IV 3 7 245 GGC/GAC Gly3Aspf Non-CpG 116 PS IIIC 2 7 257 CTG/CGG Leu3Argf Tv 340 PS IIIC 3 7 258 GAA/TAA Glu3Stop Tv 253 PS IIIC 3 7 261 14451 agTG—ϾatTG splice sitef Tv 190 M IC 2 8 266 GGA/AGA Gly3Arg Non-CpG 70 PS IIIC 3 8 270 TTT/TCT Phe3Serf Non-CpG 25 PS IIIB 3 8 273 CGT GGT Arg3Glyf Tv 103 ANOS IIIC 3 8 273 CGT/CTT Arg3Leu Tv 178 PS IIIB 3 8 273 CGT/CAT Arg3His CpG 205 PS IIIC 3 8 273 CGT/TGT Arg3Cysf CpG 357 PS IIIC 2 8 273 CGT/CAT Arg3Hisf CpG 100 PS IIIC 3 8 280 AGA/GGA Arg3Gly Non-CpG 123P ANOS IIIC 3 8 282 CGG/TGG Arg3Trpf CpG 321 E IIIC 2 8 285 GAG/AAG Glu3Lysf Non-CpG 159 E IIIC 3 8 300 14571 Del C Stop 322 Deletion 317 PS IIIC 2 9 330 14751 Del TCStop 335 Deletion 76 E IIIC 3 9 331 14755 AGgt3Agac splice site Non-CpG 199P PS IIIC 3 10 342 CGA/TGA Arg3Stop CpG Germ-line BRCA1 mutant ovarian cancers 4 PS IIC 3 None 203 M IIIC 3 None 288 ANOS IIIC 3 None 15.H T IIIB 3 None 53 PS II 2 4 62 12108 Del 4 Stop 121 Deletion 533 PS IV 3 5 126 13055 Del 21 (in frame) Deletion 271T ANOS IV 3 5 151 CCC/GCC Pro3Alaf Tv 22 PS IIIB 3 5 165 CAG/TAG Gln3Stop Non-CpG 685 PS IIIC 3 6 190 CCT/CTT Pro3Leuf Non-CpG 195 PS IIIC 3 6 194 CTT/CCT Leu3Prof Non-CpG 213 PS IV 3 6 200 13362 Ins T Stop 208 Insertion 279 ANOS IIIC 3 6 214 CAT/CGT His3Argf Non-CpG 8 274–5 14490 Del 6 (in frame) Deletion 174 PS III 3 7 225 13959 agGT3acGT splice site Tv 17 PS IIIC 3 7 238 TGT/TTT Cys3Phe Tv 452 E IIIC 2 7 244 GGC/AGC Gly3Serf Non-CpG 535 PS IV 3 7 248 CGG/CAG Arg3Glnf CpG 273 T IIIC 2 8 272 GTG/ATG Val3Metf Non-CpG 15.R PS IIIC 3 8 281 GAC/CAC Asp3His Tv 85P PS IIIC 2 8 298 GAG/TAG Glu3Stop Tv 306 PS IIB 3 9 332 14755 AGgt-Ͼ Agtt Splice site Tv

Table 2 Continued Sporadic ovarian cancers: Absent BRCA1 truncating mutations Tumora Cell typeb Stagec Grade Exon Residue p53 mutationd Mutation typee Somatic BRCA1 mutant ovarian cancer 29 PS IIIC 2 None 201 ANOS IIIC 3 None 398 ANOS IIIC 3 5 132 AAG/AGG Lys3Argf Non-CpG 52 E IIIC 2 5 135 TGC/TAC Cys3Tyrf Non-CpG 149 E IIIC 2 5 155 ACC/AAC Thr3Asnf Tv 458F ANOS IV 3 Ј5 176 13207 Del C Stop 206 Deletion 88 ANOS IIIC 3 5 184 13231 Del TA Stop 207 Deletion 55 PS IIIC 2 6 195 ATC/ACC Ile3Thrf Non-CpG 89 C IA 3 7 236 TAC/TAG Tyr3Stopf Tv 9 290 CGC/TGC Arg3Cys CpG 83 M IV 3 7 241 TCC/TTC Ser3Phe Non-CpG 133 ANOS IIIC 2 7 234 TAC/TGC Tyr3Cysf Non-CpG 235 PS IIIC 3 7 245 GGC/AGC Gly3Serf CpG 544 PS IIIC 2 7 245 GGC/GAC Gly3Aspf Non-CpG 220 E IIA 3 7 248 CGG/CAG Arg3Glyf CpG 408 ANOS IV 3 8 282 CGG/GGG Arg3Glyf Tv a All cancers are ovarian unless indicated by: T, fallopian tube; or P, peritoneal. b Cell types are: E, endometrioid; C, clear cell; M, mucinous; PS, papillary serous; and T, transitional. c FIGO staging system. d Base where occurs is bold. Uppercase is coding sequence; lower case is splice site at intron/exon boundary. e Mutation types are CpG transitions, non-CpG transitions, transversions (Tv), or frameshift (insertion/deletion) that result in premature chain termination unless they are in the reading frame (multiples of three). f p53 mutation not reported previously by our laboratory.

A single tumor in each group was found to contain two different p53 mutations. Table 3 summarizes the p53 status of the cancers analyzed. Mutations were classified as transition at CpG sites or non-CpG sites, transversion, or frameshift. Tumors with wild-type p53 status were subclassified on the basis of their DO7 antibody staining characteristics. When the three cohorts were analyzed pairwise in contingency tables, significant differences between sporadic versus germ-line BRCA1 mutant (␹2, 13.7; P ϭ 0.02) and sporadic versus any BRCA1 mutation (␹2, 15.9; P ϭ 0.009) groups were found. In addition, if DO7 antibody-positive, p53 mutation-negative tumors are combined with all p53 mutation-positive tumors, the frequency of any p53 dysfunction is estimated at only 62.8% for sporadic cancers but 93.4% for somatic BRCA1 mutant ␹2 ϭ ␹2 ϭ Fig. 2 Distribution of p53 mutations by exon for the sporadic (n ϭ 43), cancers ( , 5.40; P 0.02) and 90.0% ( , 5.50; P 0.02) somatic BRCA1 mutant (n ϭ 14), and germ-line BRCA1 mutant ovarian when there is a concomitant germ-line BRCA1 mutation. cancer (n ϭ 19). For the percentage calculation, the number of exon- Eighty % of the DO7 antibody-positive tumors with wild- specific mutations is divided by n, the number of mutations in each type p53 sequence based upon complete sequencing was cohort. ϫ100. found in individuals without any family history of breast or ovarian cancer. Thus, the mechanism that gives rise to overexpression of wild-type p53 may be relatively unique to sporadic ovarian cancers. Non-CpG transitions, transver- bution of p53 mutations by exon is listed for each cohort in Fig. sions, and frameshift mutations were 1.6 (P ϭ not signifi- 2. p53 mutations in sporadic ovarian cancers were found cant), 2.3 (␹2, 5.21; P ϭ 0.02), and 4.3 (␹2, 5.48; P ϭ 0.02) throughout the open reading frame except within exons 3 and times more common in the ovarian cancers of germ-line 11. More than half of the mutations were concentrated in exons BRCA1 carriers than in sporadic ovarian cancers. The inci- 5 and 8. p53 mutations in somatic BRCA1 mutant cancers were dence of CpG transitions was only 5% for germ-line BRCA1 found in exons 5–9 with Ͼ78% concentrated in exons 5 and 7. mutant cancers compared with a 20% rate in somatic BRCA1 In contrast, the p53 mutations found in individuals carrying mutant cancers and a 14% rate in sporadic ovarian cancers germ-line BRCA1 mutations were more uniformly distributed with wild-type BRCA1. These differences did not reach sta- throughout exons 4–9. Slightly Ͼ70% occurred in exons 6–8. tistical significance. p53 null mutations can result either from

Table 3 Classification of p53 mutation status Tumor classificationa Sporadic Somatic BRCA1 Germ-line BRCA1 Any BRCA1 p53 mutation status (n ϭ 86) (n ϭ 15) (n ϭ 20) (n ϭ 35) None, DO7 negative 32 (37.2%) 1 (6.6%) 2 (10.0%) 5 (14.3%) None, DO7 positive 12 (14.0%) 1 (6.6%) 2 (10.0%) 3 (8.6%) CpG transition 12 (14.0%) 3 (20.0%) 1 (5.0%) 4 (11.4%) Non-CpG transition 16 (18.6%) 6 (40.0%) 6 (30.0%) 12 (34.3%) Tranversion 11 (12.8%) 3 (20.0%) 6 (30.0%) 9 (25.7%) Frameshift 4 (4.7%) 2 (13.3%) 4 (20%) 6 (17.1%) a Number of tumors (% of tumors analyzed). The total percentage exceeds 100 because 3 tumors contained two mutations each.

Table 4 Evaluation of specific p53 mutations and their frequencies Iowa ovarian cancers Frequency in world literaturea Sporadic Somatic BRCA1 Germ-line BRCA1 Any BRCA1 Ͼ5 11 (25.6%) 5 (35.7%) 1 (5.9%) 5 (16.1%) 2–5 8 (18.6%) 4 (28.6%) 4 (28.5%) 9 (29.0%) Never or 1 24 (55.8%) 5 (35.7%) 12 (70.6%) 17 (54.8%) a The number of times a specific mutation in our series has been reported in the Soussi Database (April 1999 release, http://perso.curie.fr/ Thierry-Soussi/index.html.).

insertion/deletion (frameshift) mutations or from nonsense along with an increasing tendency to develop cancer as one point mutations. Overall, 8 of 86 (9.3%) sporadic ovarian ages. These observations would lead one to predict that sporadic cancers contained p53 null mutations. However, 11 of 35 ovarian cancers would be more likely to harbor CpG transition (31.4%) of the BRCA1 mutant cancers and 8 of 20 (40%) of mutations and would occur at an older age than BRCA1-related the germ-line BRCA1 cancers contained p53 null mutations cancers. Indeed, our germ-line BRCA1 ovarian cancers were (␹2, 9.20; P ϭ 0.002 and ␹2, 11.93; P ϭ 0.0006, respective- diagnosed at an earlier age than the sporadic or somatic BRCA1 ly). Thus, the combination of simultaneous p53 and BRCA1 cancers, both of which were more likely to have p53 CpG null ovarian cancers was found to be particularly prevalent. transition mutations than were the germ-line BRCA1 cancers. To determine whether the p53 mutations associated with However, the differences were not statistically significant, prob- BRCA1 mutant cancers were unique, we evaluated the frequency ably because of relatively small sample sizes when analyzed on of the specific p53 mutations we sequenced relative to the the basis of individual mutation types. Non-CpG transitions, frequency that each individual mutation has been reported to transversions, and frameshift p53 mutations were seen to occur 6 occur in ovarian cancer according to the Soussi database. Table at a higher frequency in the ovarian cancers of BRCA1 carriers. 4 summarizes this analysis. Overall, 41 (55%) of the p53 mu- These differences did approach or achieve statistical signifi- ϭ tations were either unique (n 33) to our study or had only cance. All three types of mutations were more common in ϭ been reported once before (n 8). This was true for 71% of the ovarian cancers with concomitant BRCA1 mutation. p53 muta- p53 mutations associated with germ-line BRCA1 tumors but also tions in general were more likely to occur in BRCA1 mutant true for 55.6% of the sporadic ovarian cancers. Only one p53 ovarian cancer (␹2, 11.9; P ϭ 0.0006) than in sporadic cancers. mutation in the germ-line BRCA1 group had been reported as The forces responsible for such shifts in the p53 mutational many as five times before, which contrasts with 11, or 26%, of spectra of BRCA1-associated disease need to be elucidated to the sporadic cancer p53 mutations and 5, or 35.7%, of the better understand the carcinogenic process in hereditary ovarian somatic mutant BRCA1 cancers. cancer. To date, only four other studies have reported analysis of DISCUSSION p53 mutations in BRCA1-related ovarian or peritoneal cancers Mutations in cancer arise through multiple mechanisms. (12–15). Similar studies of BRCA1 mutant breast cancers have Spontaneous deamination of methylcytosine at CpG sites gives also been sparse (9–11). Most of these studies suffer from a rise to classical transition mutations (2, 46, 47). These mutations variety of limitations, such as evaluating only the Ashkenazi are thought to occur at random throughout the genome. An population (10, 12), failure to include a comparable control accumulation of such events over an individual’s lifetime goes population (12), and small numbers of BRCA1 mutations pre- cluding statistical analysis (14). Only a single tumor with a somatic BRCA1 mutation had its p53 status determined (14). Although the frequency of p53 mutations in BRCA1-related 6 Internet address: http://perso.curie.fr/Thierry.Soussi/index.htm. primary peritoneal carcinoma is as high as we have found (13),

the general consensus is to be careful in comparing mechanisms ACKNOWLEDGMENTS between ovarian and peritoneal carcinomas. Major p53 SSCP We thank Marisa Dolan, Sara McClain, Jenny Rathe, and Matthew screening problems secondary to failure to amplify all or at least J. Buller for expert technical assistance. Rebecca Sandersfeld and Linda 50% of the exons limits the only other sizable study with an Sanders provided assistance with manuscript preparation. appropriate control population of sporadic ovarian cancers (15). Nonetheless, there is a clear consensus between these studies, REFERENCES including the present work, that ovarian cancer BRCA1 mutation 1. Levine, A. J., Momand, J., and Finlay, C. A. The p53 tumour suppressor gene. Nature (Lond.)., 351: 453–456, 1991. is nearly uniformly accompanied by p53 dysfunction. It is 2. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. 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Richard E. Buller, Thomas A. Lallas, Mark S. Shahin, et al.

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