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The High Frequency of De novo Promoter Methylation in Synchronous Primary Endometrial and Ovarian Carcinomas

Authors' Affiliation: Anatomic Pathology Unit, Department of Human Morphology, University of Insubria, Varese, Italy

Requests for reprints: Daniela Furlan, Department of Pathology, Ospedale di Circolo, Viale Borri, 57, 21100 Varese, Italy. Phone: 39-332270601; Fax: 39-332270600; E-mail: daniela.furlan{at}uninsubria.it.

	Abstract

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Purpose: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers.

Experimental Design: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5' regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status.

Results: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases.

Conclusions: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.

In the last two decades, the main challenge regarding coexisting carcinomas of the ovary and endometrium has been to make a differential diagnosis between metastatic disease and independent primaries because there will be quite different clinical implications and prognosis in each case. Thus far, this discrimination has largely relied on the conventional clinicopathologic criteria proposed by Ulbright and Roth (3) and more recently revised by Scully et al. (13). On the contrary, immunohistochemistry, DNA flow cytometry (14), and clonality analyses using molecular pathology techniques have been employed with very limited efficacy in cases showing inconclusive clinicopathologic features (15, 16). Surprisingly, in some cases, the tumor molecular profiles led to the opposite conclusion of the clinicopathologic diagnosis, by showing either identical genetic patterns in cases fulfilling the clinicopathologic criteria for independent primary tumors (17, 18) or different profiles in metastatic carcinomas (18).

In this work, we selected a panel of synchronous ovarian and endometrial carcinomas fulfilling the clinicopathologic criteria for independent primary tumors and investigated whether de novo promoter methylation may be a frequent genetic marker of these tumors, as already shown for endometrial carcinomas of endometrioid histologic type (19). Moreover, we evaluated the frequency of identical molecular profiles in tumors from the same patient to consider the hypothesis that a preneoplastic field may really be a common event underlying the pathogenesis of these tumors.

	Materials and Methods

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Patients and samples. Thirteen cases of synchronous carcinomas of the uterine corpus and ovary were retrieved with informed consent from Surgical Pathology files at the Varese hospital. These cases had been collected during a 29-year period ranging from 1976 to 2005. The series consisted of 31 neoplasms, including 13 endometrial and 18 ovarian carcinomas (five patients showed bilateral ovarian neoplasia), simultaneously detected during surgery. In all cases, a total abdominal hysterectomy and bilateral salpingoophorectomy had been done. The use and the type of adjuvant therapy showed a great heterogeneity because our series had been collected during a 29-year period. Clinical information, therapy, and patient follow-up data were obtained from the hospital charts, the tumor registry of the province of Varese, and from communication with gynecologic oncologists. The cases were classified as synchronous carcinomas based on conventional clinicopathologic findings and patient follow-up according to the criteria proposed by Eifel et al. (2), Ulbright and Roth (3), Prat et al. (14), and Scully and Clement (13). The histopathologic study included a complete revision of all cases done separately by two senior pathologists. The following variables were evaluated: histologic type, grade, stage, size, the presence/extent of blood vessels, tubal and miometrial invasion, bilaterality and pattern of ovarian involvement, and coexistence with endometriosis or endometrial hyperplasia. The immunoperoxidase study was done on formalin-fixed paraffin sections that were dewaxed and rehydrated using Bio-Clear (Bio-Optica, Milan, Italy) and alcohol.

Endogenous peroxidase was blocked by dipping sections in 3% aqueous H₂O₂ for 10 minutes, and antigen retrieval was done with a 10-minute microwave treatment in 10 mmol/L citrate buffer (pH 6). The immunostaining was done with the avidin-biotin peroxidase complex technique (20) using diaminobenzidine as chromogen. Sections were incubated overnight at 4°C with a mouse monoclonal antibody against full-length hMLH1 protein (G168-15, PharMingen, San Diego, CA) at 1:100 dilution. Sections were lightly counterstained with hematoxylin. The normal staining pattern for hMLH1 is nuclear, and a case was considered positive only in the presence of nuclear staining of neoplastic cells. A case was considered negative for expression of hMLH1 only when there was a complete absence of nuclear staining of neoplastic cells in the presence of an unquestionable internal positive control represented by normal epithelial, stromal and muscle cells, or lymphocytes.

DNA extraction and methylation-specific PCR. Genomic DNA was extracted from representative 8-µm sections of each endometrial and ovarian carcinoma and from matched normal lymph nodes of all the patients included in this study. Three sections of every specimen were cut from paraffin-embedded tissue, treated twice with xylene, and then washed twice with ethanol. DNA was extracted using a Dneasy Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Neoplastic areas were manually microdissected for DNA extraction and contained at least 70% of tumor cells, to minimize contamination by normal cells (21).

Methylation-specific PCR was carried out following the original method developed by Herman et al. (22) with the protocol modifications reported by Boyd and Zon (23) to improve DNA recovery after bisulfite treatment. Briefly, 300 to 600 ng of genomic DNA were subjected to sodium bisulfite treatment, then purified using Microcon 100 cartridges (Millipore, Billerica, MA), and stored at –20°C. The methylation analysis was assessed for the CpG-rich 5' regions of three genes (i.e., hMLH1, CDKN2A, and MGMT). These genes were selected because of their high frequency of promoter hypermethylation in cancers with a methylator phenotype of the colon-rectum and endometrium (19, 24, 25). Bisulfite-converted DNA was amplified separately using the published methylation-specific PCR primers specific for methylated and unmethylated sequences of hMLH1, CDKN2A, and MGMT CpG sites (22, 24, 26). Forward primers were synthesized with a fluorescent tag (FAM or HEX) on the 5'end and purified using standard high-performance liquid chromatography. A single round of fluorescent PCR was done using 5 µL of the bisulfite-converted DNA (30-60 ng assuming 100% yield) in a 15-µL reaction containing 1.5 µL of 10x buffer (Roche, Mannheim, Germany), 0.3 µmol/L primer pairs, 200 µmol/L deoxynucleotide triphosphates, and 2 units DNA polymerase (Roche). Annealing temperatures and MgCl₂ buffer concentrations optimized for each primer mix are reported in Table 1 together with the specific size and the fluorescent tag of each amplicon. Thermal cycling conditions were 5 minutes at 95°C, 10 cycles of 94°C/50 seconds, specific T annealing/50 seconds, and 72°C/50 seconds and 25 cycles of 89°C/30 seconds, specific T annealing/30 seconds, and 72°C/30 seconds. All PCRs were done with positive controls for both unmethylated and methylated alleles and no DNA control. DNA from normal lymphocytes treated in vitro with SssI methyltransferase (New England Biolabs, Ipswich, MA) was used as a positive control for methylated alleles, whereas the same DNA without any enzymatic treatment served as a positive control for unmethylated alleles. The fluorescently labeled PCR products were electrophoresed on an Applied Biosystems 310 automated DNA sequencer (Applied Biosystems, Milan, Italy), and the fluorescent signals from the differently sized alleles were recorded and analyzed using Genescan software (version 2.1; Applied Biosystems). All methylation-specific PCR data have been obtained from at least two independent modifications of DNA, and we scored as positive only signals detectable in repeated experiments.

Statistical analysis. Statistical analysis was assessed using Fisher's exact test and ² test with Yates' correction (SPSS 7.5 software).

	Results

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Clinicopathologic profile of the tumors. The main clinicopathologic features and details of therapy of coexisting endometrial and ovarian tumors are reported in Table 2 . The ages of patients at diagnosis ranged from 40 to 76 years, with a median age of 58 years. Endometrial tumors showed sizes ranging from 0.5 to 7 cm (median size, 3.4 cm). In 12 of 13 cases (92.3%), they showed an endometrioid histologic type; two cases were associated with a mucinous component; 10 of 12 cases were well-differentiated (G1) neoplasms. One case displayed a clear cell histologic type. Foci of atypical hyperplasia were observed near the carcinoma in two patients (cases 5 and 10).

Synchronous neoplasms showed an identical histologic type in nine cases, including eight endometrioid carcinomas and one clear cell carcinoma. On the contrary, the histologic type was discordant in four cases comprising two endometrioid carcinomas of the endometrium associated with ovarian mucinous carcinomas and two endometrioid carcinomas of the endometrium associated with ovarian serous papillary carcinomas.

De novo promoter hypermethylation and microsatellite instability in synchronous endometrial and ovarian tumors. Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. No PCR product using methylation-specific primers was observed for normal DNA from lymph nodes of each patient. Overall, at least one methylated gene was observed in tumors of 12 of 13 (92%) patients for a total of 24 of 31 (77%) carcinomas included in this study. A diagram of the neoplasm arrangement based on the number of methylated loci per tumor of each patient is shown in Fig. 1 . Methylation of hMLH1 was observed in all synchronous tumors of five patients (five endometrial and seven ovarian carcinomas), whereas CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of the cases, respectively) and ovarian carcinomas (39% and 39% of the cases, respectively). Moreover, concordant methylation at two or three genes was observed in 35% of cases.

View larger version (9K): [in this window] [in a new window]   Fig. 1. De novo methylation and microsatellite instability (MSI) phenotype in synchronous endometrial and ovarian carcinomas. , high-frequency microsatellite unstable tumors; , presence of promoter hypermethylation; , microsatellite stable tumors or absence of promoter hypermethylation; –, not available. E, endometrium; Lt, left ovary; Rt, right ovary.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Coexistence of hMLH1 promoter methylation, loss of hMLH1 immunohistochemical expression, and microsatellite instability in the endometrial and both the ovarian carcinomas of the same patient (case 3 in Table 1). hMLH1 MET, specific primers for hMLH1-methylated sequences; hMLH1 UNMET, specific primers for hMLH1-unmethylated sequences.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Microsatellite instability (MSI) status in synchronous endometrial and ovarian tumors. , presence of microsatellite instability; , absence of microsatellite instability; E, endometrium; Rt ov, right ovary; Lt ov, left ovary; MSS, microsatellite stable.

No association between clinicopathologic features (tumor size, histotype, grade, bilaterality of ovarian involvement, stage, and follow-up) and CpG island methylation or high-frequency microsatellite instability phenotype was observed.

	Discussion

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During the last two decades, many epidemiologic and clinicopathologic data have shown that women with synchronous primary tumors of the endometrium and ovary represent a distinct subgroup of patients with early-stage and low-grade cancers in both organs and an overall prognosis surprisingly better than if their disease were classified as a single organ carcinoma with metastasis (2, 5, 9, 28). Although the pathogenesis of these tumors is completely unknown, there are several lines of evidence which suggest that these tumors may share both the etiologic and histopathologic features of estrogen-dependent neoplasms of the endometrium (10, 29, 30).

For the first time, this work has shown a high frequency of de novo hMLH1, CDKN2A, and MGMT promoter methylation in a panel of 31 synchronous ovarian and endometrial carcinomas, fulfilling the clinicopathologic criteria for independent primary tumors.

Considering the methylation status of each gene separately, 39% of tumors (5 of 13 endometrial carcinomas and 7 of 18 ovarian carcinomas) showed hMLH1 promoter hypermethylation invariably associated with the absence of immunohistochemical expression of the protein and a microsatellite instability phenotype. This frequency of microsatellite instability is almost double that reported for singly occurring sporadic endometrioid carcinomas of both the endometrium (19, 31–33) and ovary (34). On the contrary, it is consistent with the frequency of hMLH1 hypermethylation and/or microsatellite instability percentage recently observed by two different groups analyzing the synchronous ovarian and endometrial carcinomas of a total of 29 patients (35, 36). To date, only one study has reported a very low incidence of microsatellite instability in a series of 45 patients with synchronous ovarian and endometrial carcinomas (37). This discrepancy may be explained by the fact that these authors did microsatellite instability analysis using conventional methods involving polyacrylamide gel and autoradiography, whereas our study and the studies by Kaneki et al. (35) and Irving et al. (36) used a high-resolution method involving fluorescence-labeled PCR and laser scanning. This is a critical point especially taking into account the small shortenings usually observed in mononucleotide markers when microsatellite instability is present in endometrial cancers (27). Second, almost 40% of the ovarian carcinomas analyzed by Shannon et al. were unusually non endometrioid tumors in contrast with those examined in our study and in the studies of Kaneki et al. (35) and Irving et al. (36).

Overall, these data suggest that epigenetic silencing of hMLH1 may play a more significant role in synchronous than in singly occurring carcinomas of the endometrium and ovary, especially when endometrioid histology is present at both sites. In addition, because it has been shown that hMLH1 hypermethylation is an early event in human endometrial tumorigenesis, being present in 10% of typical and in 33% of atypical endometrial hyperplasia (38), as well as being involved in the malignant transformation of endometriosis (11), it is reasonable to infer that abnormal methylation of hMLH1 may be an early indicator of tumorigenesis for a large fraction of synchronous ovarian and endometrial carcinomas. In this respect, further investigations of hMLH1 hypermethylation status in normal-appearing cells and preneoplastic lesions adjacent to neoplastic tissues of these tumors could be useful.

Besides microsatellite instability and hMLH1 hypermethylation, synchronous endometrial and ovarian carcinomas often exhibit other genetic alterations known to be early events in the development of endometrioid carcinoma of the endometrium, such as PTEN, ß-catenin/CTNNB1, and K-ras abnormalities (36, 39). Until now, mutation tests for these genes as well as microsatellite instability analysis were mainly employed in these tumors to support a differential clinicopathologic diagnosis between metastatic disease and independent primaries. Surprisingly, many discrepancies between the two approaches were observed. In particular, most cases fulfilling the clinicopathologic criteria for independent primaries were found to exhibit identical gene mutations or similar microsatellite instability patterns in both endometrial and ovarian carcinomas (36, 40). In agreement with these findings, our study showed that when hMLH1 hypermethylation was observed in one of the synchronous primary cancers the other cancers of the same patient also exhibited hypermethylation of the gene, accompanied by the loss of the immunohistochemical expression of the protein and the presence of a microsatellite instability phenotype (Figs. 1 and 2). As already suggested by other investigators, one explanation for these results may be the hypothesis of the existence of a field effect in the upper genital tract and the ovaries. As in other sites, a field cancerization phenotype could be the result of either independent molecular events affecting multiple cells separately under the action of a common carcinogenic agent, or one molecular event in a single clonal progenitor that gives rise to multiple foci of tumorigenesis via mechanisms of widespread clonal expansion (41). In this connection, many histologic, epidemiologic, and molecular observations directly support the hypothesis that endometrioid and clear cell ovarian carcinomas may arise through malignant transformation of multiple foci of atypical endometriosis (12). Although the molecular basis of a field defect remains elusive, accumulating evidence suggests that aberrant methylation of promoter associated CpG islands seem to be early events in tumorigenesis (42) and may mark the field defect that precedes and predisposes to the development of cancer (43). Very recently, Shen et al. (44) showed that MGMT methylation could be one of the mediators of field cancerization in the colon mucosa, being present not only in 46% of the colorectal cancers examined but also in over 70% of normal-appearing mucosal specimens that were located 1 and 10 cm away from the tumors with MGMT methylation. In this connection, a new and interesting result of our work concerns the unexpectedly high frequency of MGMT and CDKN2A hypermethylation, observed in 48% and 41% of tumors, respectively. This finding supports the hypothesis that the epigenetic inactivation of specific genes is a common and early event in the development of synchronous primary endometrial and ovarian tumors and may qualify as a marker of a field defect in these tumors.

CDKN2A is one of the cycle-dependent kinase inhibitors and acts as a negative cell cycle regulator (45). It is thought to be a potent tumor suppressor gene because of its negative effect on the cell cycle; indeed, frequent mutations, deletions, or promoter hypermethylation were shown in a variety of human tumors (46). In agreement with our data, Tsuda et al. (47) suggested that promoter hypermethylation of CDKN2A is a very common event in endometrial tumorigenesis, causing loss of p16 protein in 50% of endometrioid endometrial carcinomas and 44% of endometrial hyperplasia. Analogously, Katsaros et al. (48) found CDKN2A promoter methylation in 40% of epithelial ovarian cancers analyzing a wide series well representative of all histologic types. Other groups have reported a lower rate of CDKN2A methylation in singly occurring ovarian and endometrial carcinomas (19, 49–52) as well as in atypical endometriosis (11). The reason for this discrepancy might partly rest on the series of tumors analyzed, often small or heterogeneous for the histologic type. Our findings clearly show that the silencing of this gene is a frequent feature of synchronous primary endometrial and ovarian tumors and may be used as an additional marker of the field defect in these tumors.

MGMT is a DNA repair gene and plays a key role in protecting cells against the cytotoxic, mutagenic, and clastogenic effects of agents inducing O⁶-alkylguanine in DNA. Moreover, it is a major determinant of the resistance of cells to various alkylating cytostatic drugs, such as procarbazine, decarbazine, and temozolomide and chloroethylating agents, such as carmustine, nimustine, elmustine fotemustine, and others (53). Although low frequency of MGMT hypermethylation in singly occurring endometrial and ovarian cancers was reported (24, 51, 52), a positive correlation was shown between the protein activity and the histologic grade and the International Federation of Gynaecology and Obstetrics stage of primary ovarian tumors (54). Interestingly, MGMT activity was significantly higher in serous epithelial tumors compared with nonserous epithelial tumors (687 ± 53 versus 479 ± 45 fmol/mg, P = 0.010) defining, among this second group, a fraction of potentially chemosensitive ovarian carcinomas (about 5% of cases) characterized by very low MGMT activity.

In conclusion, our data suggest that synchronous primary endometrial and ovarian carcinomas show a high frequency of de novo hMLH1, CDKN2A, and MGMT methylation; in addition, they may comprise a set of gynaecologic malignancies with a specific sensitivity to alkylating chemotherapy. Prospective clinical trials are required to verify whether MGMT methylation with subsequent gene silencing may play a role in modulating the clinical response and prognosis in these patients and to test if the detection of hMLH1, CDKN2A, and MGMT hypermethylation may be useful in the risk assessment for these tumors.

	Footnotes

 
Grant support: University of Insubria, Varese, Italy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This study has been carried out using instruments from the Centro Grandi Strumenti of the University of Insubria.

Received 12/ 6/05; revised 2/ 8/06; accepted 3/23/06.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Furlan, D. Articles by Riva, C. Search for Related Content PubMed PubMed Citation Articles by Furlan, D. Articles by Riva, C.