The molecular pathogenesis of hereditary ovarian carcinoma

Alterations in the tubal epithelium of women with BRCA1 and BRCA2 mutations

Authors


  • See editorial on pages 5119-21, this issue.

Abstract

BACKGROUND:

BRCA1 or BRCA2 (BRCA1/2)-mutated ovarian carcinomas may originate in the fallopian tube. The authors of this report investigated alterations in BRCA1/2 tubal epithelium to define the molecular pathogenesis of these carcinomas.

METHODS:

Tubal epithelium was evaluated from 31 BRCA1/2 mutation carriers with gynecologic carcinomas (BRCA CA), 89 mutation carriers who underwent risk-reducing salpingo-oophorectomy (RRSO), and 87 controls. Ki-67 expression and p53 foci (≥10 of 12 consecutive staining cells) were scored by 2 investigators who were blinded to patient designations. Expression levels of p27 and p21 were evaluated within p53 foci. Loss of heterozygosity at the BRCA1/2 mutation site was evaluated in microdissected p53 foci and tubal neoplasms.

RESULTS:

Background tubal proliferation as measured by Ki-67 staining was increased in the BRCA1 RRSO group (P = .005) compared with the control group. Women who had BRCA1/2 mutations had more p53 foci identified per tubal segment than women in the control group (P = .02). Levels of p27 were decreased in 12 of 28 p53 foci from women with BRCA1 mutations and in 0 of 16 p53 foci from controls (P = .002). There was no loss of the wild type BRCA1/2 allele in 5 tested p53 foci. Tubal neoplasia lost the wild type allele in 6 of 6 foci (P = .002).

CONCLUSIONS:

The current results suggested a model of tubal carcinogenesis in women with BRCA1/2 mutations. Increased proliferation occurred globally in at-risk tubal epithelium. A mutation in the tumor protein p53 gene TP53 with clonal proliferation and loss of p27 occurred before neoplastic proliferation. Loss of the wild type BRCA1/2 allele occurred with neoplastic proliferation and before invasion. Cancer 2010. © 2010 American Cancer Society.

The majority of epithelial ovarian carcinomas are of high-grade serous or undifferentiated histology and present with established metastatic disease. Early detection strategies have been hindered by an inability to define the molecular progression of a clear precursor lesion.

Approximately 10% to 15% of ovarian carcinomas occur in women with inherited mutations in the BRCA1 and BRCA2 breast cancer genes,1, 2 who have an estimated 20% to 50% lifetime risk of ovarian carcinoma.3, 4 Multiple studies have examined the prophylactically removed ovaries of these women in attempts to identify precursor lesions in ovarian epithelium. The majority of recent studies with pathologists who were blinded to patient status using both standard light microscopy and immunohistochemistry have been unable to identify a reproducible precursor (preneoplastic or noninvasive neoplastic) lesion in ovarian epithelium from high-risk women.5-7

Recently, several studies have considered the role of the fallopian tube in the pathogenesis of high-grade serous carcinomas of the ovary and peritoneum. Occult carcinomas identified at the time of risk-reducing salpingo-oophorectomy (RRSO) in women with BRCA1/2 mutations are found in 4.4% to 38% of high-risk women who undergo RRSO, and 57% to 100% of those lesions are found in the fallopian tube.8-15 These findings support the hypothesis that most hereditary carcinomas of the ovary and peritoneum are seeded by neoplastic cells from the fallopian tube.

Carcinomas in BRCA1/2 mutation carriers typically have lost the wild type BRCA1/2 allele.16-18 Loss of heterozygosity for BRCA1/2 also has been described in high-grade intraepithelial neoplasia (IEN) in the fallopian tubes of mutation carriers,19 indicating that loss of the wild type allele is an early step in BRCA1/2-related carcinogenesis.

High-grade serous pelvic carcinomas have a high rate of tumor protein p53 gene (TP53) mutations (range, 50%-80%).20-25 It was reported recently that fallopian tubes from women with and without BRCA1/2 mutations contained clusters of epithelial cells with immunostaining for p53, called “p53 signatures.”26, 27 These foci of p53-positive cells reportedly contained TP53 mutations,26 and it has been hypothesized that they represent precursor lesions to high-grade serous carcinomas.26-30 It is noteworthy that fallopian tubes from control women had a rate of p53 foci similar to that observed in BRCA1/2 mutation carriers despite their much lower risk of ovarian carcinoma.26, 27 If p53 foci are the site of later neoplastic proliferation, then there should be other molecular alterations that distinguish these foci in normal and high-risk women.

The objective of the current study was to characterize the molecular events that differentiate fallopian tube epithelium in women with and without BRCA1/2 mutations. We examined expression of p53 and Ki-67 (a protein used as a marker for cellular proliferation)31 in histologically normal fallopian tube epithelium from normal-risk controls, from women with BRCA1/2 mutations who underwent RRSO, and from women with BRCA1/2 mutations who developed overt gynecologic carcinomas. Furthermore, we characterized the proliferation and expression of regulators of the cell cycle (Ki-67, p27, and p21) within p53 foci in tubal epithelium to define molecular alterations that could impact neoplastic potential in the p53 foci of high-risk women. We examined samples of tubal neoplasia and non-neoplastic p53 foci for loss of heterozygosity at the known BRCA1/2 mutation to define the molecular progression of these lesions. From these data, we are able to add significant detail to a model for the molecular pathogenesis of hereditary ovarian carcinoma.

MATERIALS AND METHODS

Formalin-fixed, paraffin-embedded specimens and clinical information were obtained through the University of Washington Gynecologic Oncology Tissue Bank, as approved by the Human Subjects Committee of the Institutional Review Board. One block from each fallopian tube was chosen that included distal fallopian tube. Tubal sections were obtained from 31 women with BRCA1 (n = 23) and BRCA2 (n = 8) associated ovarian, primary peritoneal, or tubal carcinoma (BRCA CA) and from 89 women with BRCA1 (n = 56) and BRCA2 (n = 33) mutations who underwent RRSO. The control group consisted of 61 women who underwent benign gynecologic surgery and 26 women who underwent salpingo-oophorectomy with negative testing for BRCA1/2 mutations (including full DNA sequencing and comprehensive rearrangement testing). Fallopian tubes from all groups except the BRCA CA group were completely serially sectioned as part of prospective studies that specifically examined the fallopian tube. Clinical data included age, presence of BRCA1/2 mutations, type of malignancy, disease stage, and tumor grade and histology.

Immunohistochemistry

Paraffin sections were deparaffinized and rehydrated, and endogenous peroxidases were blocked. Heat-mediated antigen retrieval was performed in a citrate buffer (Antigen Unmasking Solution; Vector Laboratories, Burlingame, Calif). Slides were treated with mouse monoclonal antibodies (Dako, Copenhagen, Denmark) against p53 (DO-7; diluted 1:500), Ki-67 (MIB-1; diluted 1:100), p27 (anti-Kip1; diluted 1:500; Transduction Laboratories, Lexington, Ky), and p21 (WAF1; diluted 1:100; Calbiochem, San Diego, Calif). After secondary antibody with horseradish peroxidase (antimouse; Vector Laboratories), sections were stained in 3,3′-diaminobenzidine and counterstained with hematoxylin. Negative and positive controls were assessed for each run.

Slides were scored by 2 independent observers who were blinded to the case designation for the number of p53 foci. In this study, p53 foci were defined as ≥10 of 12 consecutive cells that were stained strongly positive for p53 according to the definition of Shaw et al.27 For Ki-67, positive epithelial cells were scored as 0 (none), 1 (1% positive cells), 2 (2%-4% positive cells), 3 (5%-15% positive cells), or 4 (>15% positive cells). If bilateral tubes were assessed, then the highest score was recorded for the case. Immunostain findings are reported only for histologically normal fallopian tube and exclude p53 or Ki-67 staining of IEN or carcinoma.

Sections immediately adjacent to p53 foci were scored for Ki67, p27, and p21 expression as increased, decreased, or similar to expression levels in surrounding tubal epithelium. Discrepancies in the identification of p53 foci or in p27, p21, or Ki-67 within p53 foci were resolved by group review. Overall Ki-67 scores were analyzed separately for each observer, and P values are reported for Observer 1 and Observer 2. Because Ki-67 scores corresponded to unequal percentage values, they could not be combined accurately between the 2 observers.

DNA Analysis

Loss of heterozygosity was analyzed in microdissected epithelium using DNA sequencing at the known BRCA1/2 mutation site in neoplastic tubal epithelium and in non-neoplastic p53 foci. Tubal epithelium was obtained by laser-capture microdissection with a Veritus system (Arcturus, Mountain View, Calif) from adjacent formalin-fixed sections. For each patient, normal epithelium from another tubal section also was analyzed. Lymphocyte DNA samples from the same patients were used as controls. DNA was extracted using the PicoPure DNA extraction kit (Arcturus), and genomic DNA was amplified by polymerase chain reaction (PCR) using primers specific to the patient's known mutation in BRCA1 or BRCA2. PCR products were purified and sequenced with BigDye Terminator version 3.1 (Applied Biosystems, Foster City, Calif) using the ABI 3100 Genetic Analyzer (Applied Biosystems). Sequences at the mutation site were analyzed for relative ratios of wild type and mutant sequences. All PCR and sequencing reactions were performed at least twice for each microdissected sample.

Statistical Analyses

Statistical analyses were performed using Prism or Instat software (Graphpad, Inc., San Diego, Calif), and Stata IC version 10.1 (Stata Corp., College Station, Tex). Comparisons of continuous variables were assessed using the Student t test for 2 variables. The Mann-Whitney t test was used for nonparametric data. All P values were 2-sided. Contingency tables were made for comparison of categorical variables, and P values were derived using the Fisher exact test or the chi-square test. Comparisons of independent and dependent variables were assessed using Spearman correlation.

RESULTS

Patient and Specimen Characteristics

Among the 31 women in the BRCA CA group (with BRCA1/2-associated, overt carcinomas), there were 23 ovarian carcinomas, 5 primary peritoneal carcinomas, and 3 fallopian tube carcinomas. Of the ovarian carcinomas, 20 were stage III/IV, and 3 were stage I (2 endometrioid, 1 serous). Histologically, the ovarian carcinomas were primarily serous carcinoma (n = 14) or undifferentiated carcinoma (n = 6), and 3 were endometrioid carcinoma. All primary peritoneal carcinomas were stage III/IV, including 4 serous carcinomas and 1 poorly differentiated adenocarcinoma. Of the fallopian tube carcinomas, 1 was stage IC, 1 was IIC, and 1 was unstaged, and all were of serous histology.

Of the 89 women in the BRCA1/2 RRSO group (women with BRCA1/2 mutations who underwent RRSO), 9 women had occult gynecologic neoplasms (6 high-grade intraepithelial neoplasms in the fallopian tube, 2 microinvasive stage IA tubal carcinomas, and 1 stage IA grade 1 endometrioid endometrial carcinoma). The women who had occult neoplasia identified at RRSO were included in the BRCA CA group in subsequent analyses. Many of these women were reported previously.12 Among the 26 women who underwent salpingo-oophorectomy and had negative genetic testing results, none had occult neoplasia.

The 61 women who underwent salpingo-oophorectomy for benign indications had the following pathologic diagnoses: benign ovarian lesions (n = 23), uterine leiomyomas (n = 14), endometriosis (n = 10), cervical IEN (n = 1), chronic pelvic inflammatory disease (n = 2), adenomyosis (n = 1), cervicitis (n = 2), and endometrial polyp (n = 1). Seven women had normal pathology.

Clinical characteristics of the study groups are listed in Table 1. The women with BRCA1/2-associated carcinomas (including occult neoplasia) were significantly older (BRCA CA group: median age, 49.5 years; range, 39-66 years) than the women with BRCA1/2 mutations who underwent RRSO (the BRCA1/2 RRSO group: median age, 45 years; range, 31-69 years; P = .0002; Mann-Whitney t test). Neither group differed significantly in age from the control group. The number of fallopian tube sections reviewed per patient differed across the groups. The BRCA CA group (including occult neoplasia) had significantly fewer fallopian tube sections per patient than the combined BRCA1 and BRCA2 RRSO groups (P < .0001; Mann-Whitney t test) and the control group (P = .004). Controls had fewer fallopian tube sections per patient than the BRCA1/2 RRSO group (P = .008).

Table 1. Patient Characteristics
CharacteristicBRCA1/2 With Overt CarcinomaBRCA1/2 RRSO With Occult NeoplasiaBRCA1/2 RRSO With Negative PathologyControls
BRCA1BRCA2BRCA1BRCA2BRCA1BRCA2BenignNGT
  • BRCA1/2 indicates breast cancer genes 1 and 2; RRSO, risk-reducing salpingo-oophorectomy; NGT, negative genetic testing.

  • a

    The BRCA1/2 group with overt carcinoma and occult neoplasia were older than patients in the BRCA1/2 RRSO groups (median age, 49.5 years vs 45 years; P = .0002).

  • b

    The BRCA1/2 group with overt carcinoma and occult neoplasia had fewer tubal sections available per patient compared with patients in the BRCA1/2 RRSO group (P < .0001) and the control group (P = .004). Controls had fewer tubal sections per patient than women in the BRCA1/2 RRSO group, P = .008.

Total no. of patients2387249316126
Median age [range], ya49 [39-66]58 [47-76]46 [39-62]55.5 [46-65]44 [31-69]46 [35-69]48 [25-84]48 [33-61]
Median no. of tubal sections/case [range]b4.0 [2-13]4.5 [2-10]7.5 [3-19]9 [5-13]8.0 [3-19]9.0 [1-18]6.0 [2-15]8.0 [3-15]
No. of bilateral tubal sections available (%)14/23 (60.9)6/8 (75)4/7 (57.1)2/2 (100)41/49 (83.7)28/31 (90.3)40/61 (65.6)22/26 (84.6)

Immunohistochemistry

p53 foci in tubal epithelium

Patients from all risk groups had p53 foci (range, 26.4-47.5% of patients having any p53 foci) (Table 2). Fallopian tube specimens with p53 foci had more fallopian tube sections examined compared with specimens that did not have foci (median, 9 sections vs 7 sections; P = .003; Mann-Whitney t test), suggesting that the amount of tubal epithelium reviewed influenced how often p53 foci were identified. Because there were different numbers of tubal sections available among the groups (Table 1), it was difficult to compare positive results between groups based solely on the percentage with any identified p53 foci. For this reason, the number of p53 foci also was calculated relative to the total number of stained fallopian tube sections for each patient (Table 2). By this measure, p53 foci were more frequent in all BRCA1/2 mutation carriers compared with controls (P = .02; Mann-Whitney t test) and in mutation carriers with carcinomas (including occult neoplasias) compared with controls (P = .006). There was no difference in the frequency of p53 foci between BRCA1 and BRCA2 carriers (P = .83), between the BRCA CA group and the BRCA1/2 RRSO group (P = .08), or between the BRCA1/2 RRSO group and the control group (P = .1). There was no difference in age between women with and without p53 foci (P = .4) across all groups or within any particular group.

Table 2. p53 Foci
Case TypeBRCA1/2 With Overt CarcinomaBRCA1/2 RRSO With Occult NeoplasiaBRCA1/2 RRSO With Negative PathologyControls
BRCA1BRCA2BRCA1BRCA2BRCA1BRCA2BenignNGT
  • BRCA1/2 indicates breast cancer genes 1 and 2; RRSO, risk-reducing salpingo-oophorectomy; NGT, negative genetic testing.

  • a

    BRCA1/2 mutation carriers had a significantly higher frequency of p53 foci than controls (P = .02).

  • b

    Benign and NGT combined.

  • c

    BRCA1/2 mutation carriers combined.

No. with p53 foci (%)9/23 (39.1)5/8 (62.5)4/7 (57.1)1/2 (50)20/49 (40.8)11/31 (35.5)16/61 (26.2)7/26 (26.9)
Mean no. of p53 foci per tubal sections per patient (combined)a0.1020.2310.1350.1150.0650.0620.0420.059
 0.1350.1300.0640.054b
 0.134    
 0.087c  

Expression of p27, p21, and Ki-67 within p53 foci

Results for protein expression of p27 and p21 (cell cycle inhibitors) and of Ki-67 (a marker of cellular proliferation) within p53 foci are provided in Table 3 and Figure 1. An attempt was made to examine all p53 foci; however, the particular fold of fallopian tube epithelium that contained the focus was not always present in adjacent sections, and adjacent sections were not always available.

Figure 1.

Examples of p53 foci and staining for p27, p21, and Ki-67 are shown within foci and carcinoma. Scale bars = 10 μm. (A-D) Results are shown from a woman aged 62 years with a breast cancer 1 gene (BRCA1) mutation who underwent risk-reducing salpingo-oophorectomy (RRSO) and had normal pathologic findings. Images show (A) a large p53 focus, (B) decreased p27 expression within the focus, (C) unchanged p21 expression in the p53 focus compared with the nearby epithelial staining, and (D) increased Ki-67 expression in the p53 focus relative to nearby epithelial staining. (E-H) Results are shown from a woman aged 48 years with BRCA1 mutation who underwent RRSO and had normal pathologic findings. Images show (E) a p53 focus, (F) decreased p27 expression within the focus, (G) unchanged p21 expression in the p53 focus compared with nearby epithelial staining, and (H) unchanged Ki-67 expression compared with nearby epithelial staining. (I-L) Fallopian tube cross-sections are shown from a woman aged 39 years with endometriosis (benign control), including (I) a p53 focus, (J) unaltered p27 expression within the p53 focus relative to nearby tubal epithelium, (K) decreased p21 expression in the p53 focus, and (L) Ki-67 expression similar to that observed in the p53 focus and nearby tubal epithelium. (M-P) Images of microinvasive tubal carcinoma from a BRCA1 mutation carrier include (M) carcinoma that is stained positive for p53, (N) p27 that appears decreased relative to nearby benign epithelium, (O) p21 staining that is patchy and unchanged from surrounding benign epithelium, and (P) diffusely positive Ki-67 staining in carcinoma.

Table 3. Alterations in p27, p21, and Ki-67 in p53 Foci
VariableBRCA1/2 With Carcinoma and Occult NeoplasiaBRCA1/2 RRSO With Negative PathologyControls
BRCA1BRCA2BRCA1BRCA2BenignNGT
  • BRCA1/2 indicates breast cancer genes 1 and 2; RRSO, risk-reducing salpingo-oophorectomy; NGT, negative genetic testing.

  • a

    Levels of p27 were decreased in 12 of 28 p53 foci from BRCA1 mutation carriers but never in controls (P = .002). Levels of p27 were increased in 1 patient in the BRCA1 RRSO group, 1 patient in the BRCA2 RRSO group, 1 patient in the benign control group, and 1 patient in the NGT control group. Levels of p21 were increased in 1 patient in the benign control group.

p274/9a1/118/19a2/120/8a0/8a
p211/80/121/191/121/72/8
Ki-673/120/132/220/131/151/8

Levels of p27 were decreased in 12 of 28 p53 foci (42.9%) from BRCA1 mutation carriers compared with 0 of 16 control foci (P = .002; Fisher exact test). These 44 foci were from 40 women, and 11 of 24 (45.8%) BRCA1 mutation carriers had at least 1 p53 focus with decreased p27 expression compared with 0 of 16 controls (P = .001). The expression of p27 was decreased in 3 of 23 p53 foci (13%) from BRCA2 mutation carriers, which was fewer than in BRCA1 p53 foci (P = .03) and nonsignificantly more than in control foci (P = .26). The 23 p53 foci from BRCA2 mutation carriers were from 14 women, and 3 of those 14 women (21.4%) had at least 1 p53 focus with decreased p27 compared with 11 of 24 BRCA1 mutation carriers (P = .18) and 0 of 16 controls (P = .09). Levels of p21 most commonly were unchanged within foci (59 of 66 foci), and p21 was expressed similarly in p53 foci from all groups.

Ki-67 expression was increased in 5 of 34 p53 foci from BRCA1 mutation carriers, in 0 of 26 p53 foci from BRCA2 mutation carriers, and in 2 of 23 p53 foci from controls. The percentage of p53 foci with increased Ki-67 expression did not differ significantly between study groups.

Ki-67 staining in tubal epithelium

Overall, Ki-67 expression in tubal epithelium was analyzed separately by both 2 observers, who were blinded to the patient's designation. Tubal Ki-67 expression was significantly greater in women with BRCA1 mutations who underwent RRSO than in controls (Observer 1, P = .003; Observer 2, P = .005; chi-square test for trend) (Fig. 2). Women in the BRCA1 RRSO group also had higher Ki-67 expression than women in the BRCA2 RRSO group (Observer 1, P = .03; Observer 2, P = .08). In contrast, Women in the BRCA2 RRSO group did not have different Ki-67 expression compared with controls (Observer 1, P = 1.0; Observer 2, P = .5) (Fig. 2). Within the BRCA1 RRSO group, those with p53 foci had higher Ki-67 expression than those without p53 foci (Observer 1, P = .02; Observer 2, P = .04; chi-square test for trend). In controls and in the BRCA2 RRSO group, there was no difference in Ki-67 expression between those with and without p53 foci. Among controls, age had a highly significant inverse correlation with Ki-67 expression (Observer 1, Spearman correlation [r] = −0.35; P = .001; Observer 2, r = −0.32; P = .002). This inverse correlation still was present but was less significant in the BRCA1/2 RRSO groups (Observer 1, r = −0.24; P = .02; Observer 2, r = −0.20; P = .06). Ages were similar between women in the BRCA1 RRSO group and controls and did not explain the differences observed in Ki-67 expression.

Figure 2.

Distribution of Ki-67 scores in the breast cancer 1 gene (BRCA1) risk-reducing salpingo-oophorectomy (RRSO) group and the BRCA2 RRSO group versus the control group. (A) The proportion of patients with each Ki-67 score (range, 0-4) is illustrated comparing the BRCA1 RRSO group with the control group. Patients in the BRCA1 RRSO group had significantly higher Ki-67 scores than controls (Observer [Obs] 1, P = .003; Obs 2, P = .005; chi-square test for trend). (B) Ki-67 scores in the BRCA2 RRSO group were not significantly different from the scores in the control group.

DNA Analysis Within p53 Foci and Intraepithelial Neoplasia

Nine p53 foci and paired tubal epithelium from the same patient distant from the p53 foci were isolated by laser capture microdissection of adjacent sections, and DNA was extracted. For 5 of these 9 p53 foci, DNA samples were amplified successfully by PCR, sequenced for the known BRCA1 or BRCA2 mutation, and compared with germline DNA as well as tubal epithelium from the same patient without p53 staining. All 5 of these p53 foci had demonstrated decreased p27 protein expression. The mutations in these patients included 4 BRCA1 mutations (cytosine [C] deletion at position 2576 [2576delC], a C-to-thymine [T] 5214 variant [5214C→T], a guanine [G]-to-adenine [A] 1224 variant [1224G→A], and 1246delA) and 1 BRCA2 mutation (5358del4). There was no evidence of loss of heterozygosity at the mutation site in any sample. Similarly, microdissected fallopian tube epithelium from the same patients in areas without p53 staining consistently revealed heterozygosity for the known mutation. In contrast, 6 of 6 intraepithelial or microinvasive neoplasms from the fallopian tubes of BRCA1 mutation carriers had loss of the wild type allele (P = .002; Fisher exact test). Examples of sequencing data for p53 foci and tubal IEN are provided in Figure 3.

Figure 3.

Sequencing results from p53 foci and tubal neoplasia are shown. Laser-capture photomicrographs and hematoxylin and eosin (H + E)-stained slides are included for reference. (A) Sequencing results are shown for lymphocyte DNA, p53 focus, and control benign fallopian tube (FT) epithelium from patient F56. All specimens had the heterozygous mutation in the breast cancer gene 1 (BRCA1) cytosine (C) to thymine (T) 5214 variant (5214C→T). G indicates guanine; A, adenine; Y, pyrimidine. (B) Sequencing results for lymphocyte DNA and FT intraepithelial neoplasia (IEN) are shown from patient F23. FT IEN has lost the wild type allele and predominantly revealed the BRCA1 T to G 300 variant (300T→G). (C) Sequencing results from lymphocyte DNA and microinvasive FT carcinoma (ca) from patient F25 are shown. The carcinoma has lost the wild type allele. 3109insAA indicates frameshift AA insertion at 3109.

DISCUSSION

The relatively high rate of p53 foci in tubal epithelium from normal-risk women in this study and others,26, 27 compared with the relative infrequency of ovarian carcinoma in these women, suggests that other alterations are necessary if these foci serve as potential sites of malignant transformation. To define differences in neoplastic potential between p53 foci in high-risk and normal-risk women, we examined protein expression of the cell cycle inhibitors p27 and p21 and the proliferation marker Ki-67 within p53 foci. Expression of p27 was decreased frequently in p53 foci from BRCA1 mutation carriers (and in some BRCA2 mutation carriers) but was never decreased in control foci (P = .002). To our knowledge, these data are the first to demonstrate a difference between p53 foci in high-risk women and p53 foci that occur in women who are unlikely to develop ovarian carcinoma. Consequently, these data support the neoplastic potential of a significant number of the p53 foci that arise in tubal epithelium of BRCA1 mutation carriers. Conversely, the retention of normal cell cycle checkpoints in p53 foci in normal-risk women may explain the rarity of neoplastic progression of these lesions in women at normal risk for ovarian or tubal carcinoma.

It has been suggested that alterations in TP53 are a prerequisite to BRCA1-associated carcinogenesis.21, 32TP53 mutations have been identified in a small number of tested p53 foci (from women with and without BRCA1/2 mutations); and, in 1 patient, the identical TP53 mutation was identified in a coexisting IEN (also called tubal intraepithelial carcinoma).26 Similarly, Kindelberger and colleagues identified the same TP53 mutation in tubal IEN and coexistent sporadic pelvic serous carcinomas.30 These data and ours suggest that alterations of TP53 not only are important for neoplastic progression but may precede a histologically identifiable neoplasm in at-risk epithelium. It is noteworthy that a lower percentage of p53 foci in BRCA2 mutation carriers had loss of p27 compared with BRCA1 mutation carriers. If loss of p27 contributes to neoplastic transformation of p53 foci, then the decreased rate of p27 loss in BRCA2 p53 foci may be related to the lower lifetime risk of ovarian carcinoma in women who have BRCA2 mutations compared with women who have BRCA1 mutations. Alternatively, the lower rate of p27 loss may indicate that alterations in cyclin-dependent kinase inhibitor 1B (CDKN1B) (which encodes p27) plays a less prominent role in BRCA2 tubal carcinogenesis compared with BRCA1 tubal carcinogenesis.

CDKN1B acts as a tumor suppressor by negatively regulating the transition from G0-phase to S-phase through the inhibition of cyclin E-CDK2.33 Studies in human breast cancer cell lines have demonstrated that BRCA1 is a transcriptional activator of the CDKN1B promoter.34, 35 Breast cancers in women with BRCA1/2 mutations characteristically have decreased p27 expression compared with sporadic carcinomas and normal breast tissue.36, 37

BRCA1/2 carcinomas typically have lost the wild type allele.16-18 Similarly, we demonstrated loss of the wild type allele in all 6 early tubal neoplasms in BRCA1 mutation carriers. However, in 5 non-neoplastic p53 foci, we did not find evidence for loss of heterozygosity of BRCA1/2. Thus, TP53 mutations and decreased p27 expression appear to occur before loss of the wild type allele in the pathogenesis of BRCA1 tubal carcinomas. Haploinsufficiency of BRCA1/2 in combination with a TP53 mutation may have contributed to the loss of p27 in p53 foci of non-neoplastic tubal epithelium. The decrease in p27 expression and the resultant loss of cell cycle inhibition may result in increased cell proliferation and neoplastic potential. The 6 patients who had early tubal neoplasia all had loss of the wild type allele. Therefore, both TP53 mutation and p27 loss precede loss of the wild type allele, which probably is the rate-limiting step for neoplastic transformation. By the time loss of the wild type BRCA1/2 allele occurs, neoplastic proliferation is histologically evident.

Ki-67 levels were elevated in a minority of p53 foci from both normal-risk women and BRCA1/2 mutation carriers. These data are similar to findings recently reported by Shaw and colleagues.27 Most pathologically recognized IENs (in situ or early invasive carcinoma) have both increased Ki-67 and p53.26 However, the coexpression of Ki-67 and p53 does not define IEN in the absence of severe cytologic or architectural atypia. Conversely, Shaw and colleagues demonstrated that 21% of high-grade IENs will not over express p53, and 1 tubal carcinoma also lacked p53 expression.27 Therefore, it is important to define clear histopathologic criteria for diagnosing tubal neoplasms that do not rely on the p53 and Ki-67 expression pattern. It is critical to avoid both under and over diagnosing tubal IEN, because some patients receive chemotherapy for these lesions.8, 38

Ki-67 expression was increased globally in tubal epithelium from women with BRCA1 mutations who underwent RRSO compared with normal-risk women. Similarly, Piek and colleagues reported a higher proportion of Ki-67–expressing tubal epithelial cells in morphologically normal tissues that were removed for risk-reduction compared with the proportion in controls from a small series of 12 high-risk women that included 7 women with confirmed BRCA1 mutations.19 Burga and colleagues recently demonstrated that human mammary epithelial cells heterozygous for a BRCA1 mutation had a higher proliferative rate in cell culture compared with wild type mammary cells.39 Together, these data suggest that haploinsufficiency of BRCA1 influences proliferation in breast and tubal epithelium, which are the tissues most at risk for malignant transformation in BRCA1 mutation carriers. In our series, the women with BRCA1 mutations who underwent RRSO and had p53 foci had more tubal epithelial proliferation compared with women who had no p53 foci, suggesting that the conditions of increased proliferation may contribute to the formation of p53 foci. In normal-risk women, we identified a highly significant inverse correlation between tubal epithelial proliferation and age. It is noteworthy that the correlation between decreasing proliferation and advancing age was less prominent in the tubal epithelium from high-risk women. We speculate that the increased proliferation in tubal epithelium from BRCA1 mutation carriers, particularly as they age, sets the stage for neoplastic transformation.

We observed a significant increase in the frequency of p53 foci in women with BRCA1/2 mutations compared with controls, particularly in BRCA1/2 mutation carriers who had overt or occult gynecologic carcinoma. In the current study, the percentage of women with any p53 foci was consistent with data reported by Crum et al from Brigham and Women's Hospital that first described the existence of p53 foci in tubal epithelium,26 and the percentage in our study was higher than the rate reported by Shaw et al.27 However, neither Shaw and colleagues nor Crum and colleagues observed a difference in the number of p53 foci between cases and controls, and neither group evaluated BRCA1/2 mutation carriers with overt carcinomas. Because p53 foci are a relatively rare event, the identification of p53 foci is likely to depend on the volume of tubal epithelium evaluated. To account for variable tubal epithelial volumes between patients, we analyzed the number of p53 foci per number of tubal sections evaluated. Fewer tubal sections were available from the women in our study with overt carcinoma, but the number of p53 foci per tubal section was highest in these patients (Tables 1 and 2). We had a sufficient number of women who underwent RRSO to analyze the data separately for women with BRCA1 and BRCA2 mutations, and the rate of p53 foci was similar for tubal epithelium with mutations in either gene (Table 2). Although the patients with BRCA1/2-related carcinomas tended to be older, there was no significant difference in age between those with and without p53 foci, a finding that was confirmed by Saleemuddin et al.40

Previous authors have suggested a model for BRCA1/2 tubal carcinogenesis based on the progression of p53 foci to overt neoplasia.29 Our data allowed us to expand this model and add potential molecular details (Fig. 4). We propose that haploinsufficiency of BRCA1 leads to increased tubal epithelial proliferation (as evidenced by increased Ki-67 expression). This tubal proliferation does not decrease appropriately with advancing age (as it does in normal-risk women). These conditions support the clonal expansion of tubal cells with random TP53 mutations leading to the formation of p53 foci in BRCA1-haploinsufficient epithelium. A decrease in p27 expression occurs in cells that are TP53 mutant and BRCA1 haploinsufficient, leading to loss of cell cycle inhibition and increased neoplastic potential. Finally, loss of heterozygosity for BRCA1 occurs at the time of development of histologically identifiable IEN but before invasion.

Figure 4.

This is a proposed model for the molecular pathogenesis of hereditary ovarian carcinoma. Haploinsufficiency of the breast cancer 1 gene (BRCA1) in mutation carriers leads to increased tubal epithelial proliferation, as demonstrated by increased Ki-67 staining. Increased proliferation could increase the likelihood of TP53 mutations, leading to p53 foci. Loss of cell cycle inhibition through down-regulation of p27 followed by loss of DNA repair by loss of the wild type BRCA1 allele then leads to neoplastic proliferation. Tubal neoplasia can then become invasive with or without (+/−) metastasis and can seed the ovary and peritoneal cavity.

CONFLICT OF INTEREST DISCLOSURES

Supported by the Yvonne Betson Trust and R01CA131965 (to E.M.S.).

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