© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 7, 626-628,
April 7, 1999
© 1999 Oxford University Press
REPORTS |
Comparison of Prophylactic Oophorectomy Specimens From Carriers and Noncarriers of a BRCA1 or BRCA2 Gene Mutation
Affiliations of authors: J. F. Stratton, Department of Obstetrics and Gynaecology, Rosie Maternity Hospital, Cambridge, U.K.; C. H. Buckley, Department of Reproductive Pathology, St. Mary's Hospital for Women and Children, Manchester, U.K.; D. Lowe, St. Bartholomew's Hospital, London, U.K.; B. A. J. Ponder, Department of Oncology, Addenbrooke's Hospital, Cambridge.
Correspondence to: Bruce A. J. Ponder, Ph.D., F.R.C.P., Cancer Research Campaign Department of Oncology, University of Cambridge, Cambridge Institute of Medical Research, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2XY, U.K.
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ABSTRACT |
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BACKGROUND: The natural history of ovarian cancer is not well understood and, to date, there is conflicting evidence as to whether or not there is a demonstrable precursor lesion. Some women at high risk of developing ovarian cancer because of their family history elect to have a prophylactic oophorectomy. To determine whether or not a recognizable premalignant lesion could be defined in familial ovarian carcinogenesis, we reviewed ovarian tissue specimens from women whose ovaries were removed prophylactically before gene testing became available and who were tested subsequently for BRCA1 or BRCA2 gene mutations. METHODS: We analyzed ovarian tissue specimens from 37 women. The specimens were examined for the presence of the following four features: inclusion cysts, clefts and fissures, ovarian epithelial metaplasia, and the presence of papillae on the ovarian surface epithelium. The specimens were also examined closely for the presence of dysplasia and occult neoplasia. Furthermore, the occurrence of endometriosis and benign ovarian tumors was documented in these women. The protein truncation test, nonradioactive single-stranded conformation polymorphism analysis, and heteroduplex analysis, followed by DNA sequencing, were used to identify BRCA1 or BRCA2 mutations in either blood samples or ovarian tissue specimens. RESULTS: Eleven women had inherited a mutated BRCA1 or BRCA2 gene; 26 women had not. There was no difference between these groups for any of the features studied. CONCLUSIONS: Our data suggest that many of the histologic "abnormalities" described in "normal" ovaries are, in fact, variations of the normal and are not associated with the development of cancer.
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INTRODUCTION |
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Many investigators have sought to define a premalignant lesion for epithelial ovarian cancer. Various histologic abnormalities in the ovarian surface epithelium have been described, including dysplastic and metaplastic changes and inclusion cysts (1-5); however, the clinical significance of these abnormalities has remained uncertain. The clinical significance of an abnormality might be determined by comparing putative preneoplastic lesions in ovaries from women who have a high risk of developing ovarian cancer with lesions in ovaries from women who have a normal risk. One such study was reported by Salazar et al. (6). In a nonblinded comparison, these authors found that various lesions occur with statistically significantly higher frequency in ovaries removed prophylactically because of a family history of ovarian cancer than in ovaries removed for other indications. They concluded that these changes were related to familial predisposition and may, therefore, represent early stages in the neoplastic process.
Herein, we report a study that is similar in conception to that of the study by Salazar et al. but in which the case and control groups are more closely matched and the study's pathologists were blinded to the case or control status of the specimen. We have scored histologic abnormalities in ovaries removed prophylactically from women who had a family history that suggested predisposing mutations, which were subsequently identified in either the BRCA1 or the BRCA2 gene. Some of these women have been shown to have inherited the predisposing mutation and, therefore, to have been at high risk of developing ovarian cancer. Others proved not to have inherited the mutation and were, therefore, at the same risk of developing ovarian cancer as the rest of the population.
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SUBJECTS AND METHODS |
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Study patients. We used the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Familial Ovarian Cancer Register to identify unaffected women from families with known BRCA1 or BRCA2 mutations who had undergone a prophylactic oophorectomy because of their familial risk before their genetic status could be determined. The UKCCCR study holds a national register of women with at least two affected first- or second-degree relatives with confirmed epithelial ovarian cancer. The register collects pathologic material (blood samples and tissue specimens) and clinical and epidemiologic information on consenting individuals from these families.
Pathologic analysis. Pathology departments were asked to send serial blocks or representative blocks from the ovaries for review by the reference pathologists. All pathologic material received was examined by two reference pathologists, both of whom were unaware of the mutation status of the individual. The amount of material received depended on the referring pathology department from the hospital where the oophorectomy was undertaken. In the mutation-positive group, blocks from both ovaries were available for review from seven (64%) of 11 women. In the mutation-negative group, both ovaries were reviewed from 16 (62%) of 26 women. Sections from one ovary were available for the remainder of the individuals in both groups. The average number of slides available for review from mutation-positive women was 11.5 (range, 1-43); the average number available from mutation-negative women was 6.7 (range, 1-18). This difference is not statistically significant. In all specimens, there was loss of some ovarian surface epithelium because of handling and processing. Sections were included in the analysis only if the pathologists judged there to be sufficient epithelium for examination. Because the ovaries were removed, processed, and analyzed blind to the mutation status, there should have been no bias with respect to mutation-positive or mutation-negative cases. The ovaries were scored jointly by each pathologist for the presence or absence of the following four features: inclusion cysts, clefts and fissures, ovarian epithelial metaplasia, and the presence of papillae on the ovarian surface. Ovarian epithelial metaplasia was subclassified into one of the following four categories: 1) columnar or cuboidal epithelium, 2) serous metaplasia, 3) endometrioid metaplasia, and 4) metaplasia not otherwise specified. The ovaries were examined closely for the presence of dysplasia and occult neoplasia. In addition, the occurrence of endometriosis, benign ovarian tumors, and other cancers was documented in these women.
Mutation analysis. We identified germline mutations in BRCA1 and BRCA2 by a combination of the protein truncation test, nonradioactive single-stranded conformation polymorphism analysis, and heteroduplex analysis, followed by sequencing (7-9). We confirmed the presence of a mutation in women who had undergone a prophylactic oophorectomy by use of genomic DNA extracted from blood or, in those cases where a blood sample was not available, from a paraffin section of ovary. Primers were selected to amplify the region of the gene known to contain the mutation in the relevant family. Single-stranded conformation analysis and heteroduplex analysis were performed on specimens from those individuals with frameshift mutations, and direct sequencing was used to identify missense and nonsense mutations (7,8).
Statistical methods. 
2 tests were used to test for differences in
the frequencies of histologic features between mutation carriers and noncarriers. The effect of a
number of positive histologic markers on mutation status was analyzed with a 2 test for trend.
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RESULTS |
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Pathologic material was available for review from prophylactic oophorectomy specimens obtained from 37 women belonging to families with a BRCA1 or a BRCA2 mutation. Of these 37 women, 11 had a mutation in either the BRCA1 gene (n = 9) or the BRCA2 gene (n = 2) and 26 had not inherited a mutated gene.
The average ages of oophorectomy in the groups of women who did and who did not have mutations were similar. Mutation-positive groups had a median age of 45 years (range, 33-66 years). Mutation-negative groups had a median age of 44 years (range, 33-63 years). Of the 11 women with mutations, 10 were parous compared with 20 of the 26 women who had no mutation.
The reference pathologists found no statistically significant difference in the frequency of any
feature analyzed in ovaries from women with BRCA mutations and from women without BRCA
mutations (Table 1).
Furthermore, they found no statistically significant
difference in the frequency of various pathologic features in the same individual (21 = 0.00 [Table 2]). Four of the 26
women with no mutation had endometriosis of the ovary compared with none of 11 with a
mutation. Two of the 26 women with no mutation had benign neoplasms of the ovary (a Brenner
tumor and cystic teratoma in one woman and an endometrioid adenofibroma in the second
woman). None of the 11 women with a mutation had a benign neoplasm of the ovary. These
differences were not statistically significant. One woman in whom a mutation was identified had
a microinvasive serous adenocarcinoma of the ovary. She was diagnosed as having primary
colonic adenocarcinoma 2 years after the prophylactic oophorectomy.
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DISCUSSION |
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In this study, we found changes in the ovarian surface epithelium similar to those described by others (1-6), but we found no difference between the ovaries from women predisposed or not predisposed to ovarian cancer by a BRCA1 or BRCA2 mutation. Because the families were originally ascertained to have at least two members with ovarian cancer who were close relatives, mutation carriers in these families are at a high risk of developing ovarian cancer.
Our results are at odds with results reported by Salazar et al. (6), who found striking differences between the ovaries of women with a family history of ovarian cancer and those with no family history. The explanation of this difference is not clear. In our study, the "case" and "control" subjects were more closely matched and were precisely defined with respect to risk status. Furthermore, the examination of ovarian specimens was carried out by pathologists who were blinded to the mutation status of the subjects. A possible weakness of our study is that, because the oophorectomies were carried out over the space of several years at different hospitals, the amount of material available for pathologic review was variable. In our study, however, if differences in the extent of tissue sampled obscured true differences between carriers and noncarriers of BRCA1 or BRCA2 mutations, then there should have been a bias in the amount of material sampled that favored noncarriers. This was not the case. Now that mutation testing before oophorectomy is widely available, it will be difficult to set up a prospective study that compares mutation carriers with noncarriers in which these issues of sample processing could be addressed.
A further difference between our study and the study by Salazar et al. (6) is that Salazar et al. semiquantitatively assessed the frequencies of the different
histologic features of the ovarian epithelium; we recorded simply the presence or absence of each
feature examined. However, the differences reported by Salazar et al. were also statistically
significant even if scored simply for presence or absence. Salazar et al. found a statistically
significant association between risk status and the presence of multiple different abnormal
features in the same individual, but a similar analysis of our data showed no such effect (Table 2
).
Our results suggest that none of the histologic features described is specifically associated with a strong predisposition to ovarian cancer and, therefore, that there is no reason to regard them as preneoplastic. Furthermore, in the 11 ovaries that were taken from women who had a strong predisposition, apart from the single case with a focus of microinvasive disease, we did not identify any features that could be recognized as clearly preneoplastic. The median age of these women at oophorectomy was 45 years, well into the risk period for ovarian cancer development (10-14). It does remain possible, however, that an apparently normal ovary removed from a woman at high risk will harbor a nonapparent primary cancer and, notwithstanding our data, careful pathologic examination of ovaries removed prophylactically for familial risk should be mandatory.
It should be remembered that these findings relate specifically to mutations in BRCA1 and BRCA2, which may be a special case. Probably only 5% or so of all ovarian cancers are related to mutations in these genes (15). Current data suggest that mutations in BRCA1 and BRCA2 are associated with defects in DNA repair (16,17). If this is the basis of cancer predisposition by these mutations, arguably BRCA1 and BRCA2 should be regarded as "caretaker" genes rather than as "gatekeeper" genes (18). In the case of predisposition by a caretaker gene mutation, the limiting step in carcinogenesis may be the initial gatekeeper step, although progression through subsequent preneoplastic stages (accelerated by the caretaker mutation) may be relatively rapid. Recognizable preneoplastic stages, therefore, may be uncommon. This distinction is exemplified by mutation in the gatekeeper gene APC, which results in multiple polyps of the colon, and by mutations in the caretaker genes MLH1 and MSH2, which result in far fewer polyps in hereditary nonpolyposis colon cancer syndrome. Similarly, women with germline BRCA1 and BRCA2 mutations have a lower frequency of premalignant changes (ductal carcinoma in situ and lobular carcinoma in situ) in the breast epithelium adjacent to invasive breast cancers than women with sporadic breast cancers (19). By extrapolation, preneoplastic lesions in the ovarian surface epithelium may also be infrequent in carriers of a BRCA1 or BRCA2 mutation.
Our findings suggest that the lesions of the ovarian surface epithelium that have been proposed most often as preneoplastic may not in fact have preneoplastic significance. For the strongly predisposing mutations in BRCA1 and BRCA2, a frequent morphologically recognizable preneoplastic lesion may not exist. This, however, does not exclude the possibility that molecular or serologic changes may occur without a recognizable histologic change that might be used for early detection.
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NOTES |
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Funded by programme grants from WellBeing and the Cancer Research Campaign (CRC). J. F. Stratton is a WellBeing Research Fellow; B. A. J. Ponder is a Gibb Fellow of the CRC.
The study was based on the UKCCCR Familial Ovarian Cancer Register. The steering group was as follows: B. A. J. Ponder (Chairman), T. D. Bishop (St. James University Hospital, Leeds, U.K.), W. P. Collins (Kings College Hospital, London, U.K.), D. F. Easton (Strangeways Research Laboratories, Cambridge), G. Fraser (Imperial Cancer Research Fund, Oxford, U.K.), I. Jacobs (St. Bartholomew's Hospital, London, U.K.), D. Lowe, J. Shepherd (St. Bartholomew's Hospital), and C. M. Steel (University of St. Andrew's, U.K.).
We thank Dr. Easton for statistical advice.
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