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Journal of Clinical Oncology, Vol 23, No 36 (December 20), 2005: pp. 9344-9350 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.5915 Women With Synchronous Primary Cancers of the Endometrium and Ovary: Do They Have Lynch Syndrome?From the Departments of Gynecologic Oncology, Pathology, and Clinical Cancer Genetics, the University of Texas M.D. Anderson Cancer Center, Houston, TX Address reprint requests to Karen H. Lu, MD, Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, 1155 Herman P. Pressler St, CPB 6.3244, Unit 1362, Houston, TX 77030; e-mail: khlu{at}mdanderson.org
PURPOSE: Lynch syndrome (hereditary nonpolyposis colorectal cancer; HNPCC) is an autosomal-dominant cancer predisposition syndrome that increases risk for multiple cancers, including colon, endometrial, and ovarian cancer. Revised Bethesda Criteria recommend that patients with two HNPCC-associated cancers undergo molecular evaluation to determine whether they have a mismatch repair (MMR) defect associated with HNPCC. The purpose of our study was to determine the likelihood of MMR defects (MSH2, MSH6, MLH1) in women with synchronous endometrial and ovarian cancer. PATIENTS AND METHODS: Between 1989 and 2004, 102 women with synchronous endometrial and ovarian cancers were identified; 59 patients had tumor blocks available for analysis. Patients were divided into risk groups based on family history: high (met Amsterdam criteria), medium (personal history or first-degree relative with an HNPCC-associated cancer), and low (all others). Protein expression for MSH2, MSH6, and MLH1 was evaluated by immunohistochemistry. Microsatellite instability and MLH1 promoter methylation analyses were performed on a subset of cases. RESULTS: Median age was 50 years. Two patients met Amsterdam criteria for HNPCC. Five additional patients, all medium-risk, had molecular findings consistent with a germline mutation of either MSH2 or MLH1. None of the low-risk patients had molecular results consistent with a germline mutation. CONCLUSION: Overall, 7% of women in our cohort met either clinical or molecular criteria for Lynch syndrome. All of these women had a prior history or a first-degree relative with an HNPCC-associated cancer. Limiting genetic evaluation to women with synchronous endometrial and ovarian cancer who have a family history suggestive of HNPCC may appropriately identify women with Lynch syndrome.
Lynch syndrome, or hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal-dominant hereditary cancer predisposition syndrome caused by a germline mutation in one of the mismatch repair (MMR) genes. It is often associated with early onset of cancer (age < 50 years) and the development of multiple cancer types, including colorectal, endometrial, and ovarian cancer.1-3 Mutations of MLH1, MSH2, MSH6, and PMS2 have been identified in families with Lynch syndrome, with mutations in the MLH1, MSH2, and MSH6 genes accounting for the majority of cases.4,5 These genetic defects in the DNA mismatch repair system result in microsatellite instability (MSI) and the absence of protein expression in the tumor. Currently, the diagnosis of Lynch syndrome is based on either clinical (revised Amsterdam criteria) or molecular criteria.6 To aid clinicians in identifying patients with a likelihood of Lynch syndrome, the Bethesda Guidelines were originally developed in 1996 to determine which individuals diagnosed with colorectal cancer should proceed with MSI or immunohistochemical (IHC) testing of their tumors.7 These guidelines were revised in 2004 to include extra-colonic tumors and to increase the sensitivity of detecting families with Lynch syndrome (HNPCC).8 According to these revised criteria (Table 1) patients with synchronous or metachronous colorectal or other HNPCC-associated tumors, regardless of age, should undergo MSI testing or IHC analysis for MMR gene expression.8 Although there is evidence to support the utility of testing all women with synchronous or metachronous colorectal and endometrial cancer, there is limited data available on the prevalence of Lynch syndrome in women with endometrial and ovarian cancer.9,10
The purpose of this study was to determine the likelihood of Lynch syndrome, based on clinical and molecular criteria, in women with synchronous primary cancers of the endometrium and ovary. This will provide evidence to determine whether there is utility in testing all women with synchronous endometrial and ovarian cancer, as recommended by the revised Bethesda Criteria.
After obtaining approval from the institutional review board at the University of Texas M.D. Anderson Cancer Center, our study population was identified through a search of the Tumor Registry database maintained by the Department of Medical Informatics. Between 1989 and 2004, 102 women with synchronous endometrial and ovarian cancer were identified. Clinical and pathologic information was obtained from the medical record. Patients were categorized into risk groups based on reported family history: high risk (family history that met revised Amsterdam criteria for HNPCC), medium risk (personal history or at least one first-degree relative with an HNPCC-related cancer), and low risk (all others). Paraffin-embedded tumor blocks were available for 59 patients. Fifty-four patients (92%) had both endometrial and ovarian tumor specimens available for analysis. In the remaining five patients, only the endometrial tumor was evaluated. A gynecologic pathologist (R.R.B.) reviewed the hematoxylin and eosin–stained slides from all tumors to confirm the diagnosis of synchronous primary cancers. IHC analysis was performed on all tumor specimens, endometrial and ovarian, to determine the protein expression of MSH2, MSH6, and MLH1. MSI and MLH1 promoter methylation studies were then performed in a subset of cases. For IHC analysis, mouse monoclonal antibodies against MSH2 (Oncogene, La Jolla, CA), MSH6 (Transduction Laboratories, San Diego, CA), and MLH1 (Pharmingen, San Diego, CA) were used. The immunostaining was performed with the LSAB horseradish peroxidase kit (DAKO, Carpinteria, CA) for MSH2 and the Envision horseradish peroxidase kit (DAKO) for MLH1, using diaminobenzidine as a chromogen. Sections were then counterstained with hematoxylin. The normal staining patterns for MSH2, MSH6, and MLH1 are nuclear. Loss of expression of MSH2, MSH6, and MLH1 in cancer cells was demonstrated by the total absence of nuclear staining in the tumor. Adjacent normal stroma or infiltrating lymphocytes served as an internal positive control for each case. Nuclear staining of the tumor was scored as either positive or negative compared with the corresponding internal control. MSI testing was performed in tumors that were found to have loss of expression of MSH2, MSH6, or MLH1 by IHC. For these cases, tumor cells were hand microdissected from adjacent normal tissue to insure that each tumor sample contained at least 70% neoplastic cells. Normal tissue from the same patients was also analyzed as a control. DNA was extracted from both tumor and normal tissue for each sample. After DNA amplification using fluorescent labeled primers, a panel of six microsatellite markers, including the five National Cancer Institute recommended markers (BAT25, BAT26, D71S250, D2S125, and D5S346) and an additional mononucleotide marker (BAT40), were analyzed for allelic shift using a multiplex fluorescence-based polymerase chain reaction (PCR) assay.11 Amplified PCR products were then analyzed on an ABI Genetic Analyzer (PE/Applied Biosystems, Foster City, CA), using the GeneScan Analysis software provided by the manufacturer. Tumors showing allelic shift at two or more markers were considered MSI-high, whereas tumors showing allelic shift at only one marker were classified as MSI-low. Tumors with no allelic shift were classified as microsatellite stable. Tumors with loss of MLH1 protein expression were evaluated using the MLH1 promoter methylation assay to determine whether the loss of MLH1 was due to an epigenetic event or the result of a germline mutation. DNA was isolated from paraffin-embedded tissue specimens that were microdissected to provide relatively pure tumor samples. After treatment with bisulfite, the DNA was amplified using PCR primers that were specific for methylated (M) or the unmethylated (U) versions of MLH1 (MLH1-M forward, 5'-gatagcgatttttaacgc-3' and MLH1-M reverse, 5'-tctataaatactaaatctcttcg-3'; MLH1-U forward, 5'-agagtggatagtgatttttaatgt-3' and MLH1-U reverse, 5'-actctataaattactaaatctcttca-3'). PCR products were then separated on 6% polyacrylamide gels and visualized after staining with ethidium bromide. DNA extracted from the RKO colon carcinoma cell line was used as a positive control. Once all data were collected, patients were considered to have a high likelihood of having Lynch syndrome/HNPCC based on a combination of clinical and molecular criteria. These criteria included (1) a family history that met revised Amsterdam criteria, (2) loss of MSH2 or MSH6 protein expression associated with an MSI-high tumor, or (3) loss of MLH1 protein expression associated with an MSI-high tumor with no evidence of MLH1 promoter methylation (Table 2).
Data were analyzed using SPSS 12.0 software (SPSS Inc, Chicago, IL). The  2 test was used to assess the significance of differences in clinical and pathologic variables. Continuous variables were analyzed using t tests. A P value less than .05 was considered significant.
Demographic characteristics for the 102 patients are listed in Table 3. The median age at diagnosis was 50 years (range, 28 to 89 years). The median body-mass index was 27.0 kg/m2 (range, 17.7 to 64.4 kg/m2). Based on reported family history, two patients (2%) were considered high risk, 14 patients (14%) were considered medium risk, and 81 patients (79%) were considered low risk for HNPCC. Family history information was not available for five women.
Ninety-six percent of the endometrial tumors were endometrioid histology. The remaining 4% were mixed endometrioid with either a component of serous or clear-cell carcinoma. Sixty-five percent of patients (67 of 102) had endometrioid tumors of both the endometrium and ovary. Other ovarian histologies included mixed endometrioid (n = 11), serous (n = 11), mucinous (n = 6), granulosa cell (n = 2), carcinoid (n = 1), carcinosarcoma (n = 1), and undifferentiated (n = 3). Eighty percent of patients had stage I endometrial disease, and 62% had stage I ovarian disease at the time of diagnosis. Forty-nine percent had stage I disease of the endometrium and ovary. Among the 59 cases whose tumors were available for molecular evaluation, 12 patients (20%) had loss of MSH2, MSH6, or MLH1 associated with an MSI-high tumor (Table 4). Six of these women had molecular findings consistent with a suspected mutation and six women had findings consistent with methylation of the MLH1 promoter. Cases 7 and 8 had clinical mutational analysis for MLH1, which confirmed the absence of a germline mutation. In the patients who were found to have MSI-high tumors at both sites, the patterns of expression at each microsatellite locus were different between the endometrial and ovarian tumors, supporting the diagnosis of synchronous primary cancers (Fig 1).
Two women met Amsterdam criteria for Lynch syndrome (HNPCC). Fourteen women had a personal history or a first-degree relative with an HNPCC-associated cancer (medium risk). A detailed family history and the molecular findings for these 14 women are shown in Table 5. Four women had findings consistent with an MSH2 mutation, and one woman had findings consistent with an MLH1 mutation. The remaining six cases that had tumor available had findings that did not suggest HNPCC (methylation of the MLH1 promoter or normal expression of MSH2, MSH6, and MLH1). There was a significant association between family history risk group and suspected MMR mutation (P < .001).
Figure 2 provides a summary of our findings. A majority of patients (81 [84%] of 97 patients) did not have a family history suggestive of HNPCC. Among these low-risk women, we were able to obtain 46 tumor specimens. None of these women had molecular findings suggestive of a germline MMR defect. All of the cases with a suspected germline MMR defect based on molecular findings were among the high/medium-risk subgroup.
A majority (five of seven) of the women with a high likelihood of Lynch syndrome/HNPCC were younger than 50 years at the time of diagnosis. Although this is an interesting finding, there was not a statistically significant difference in mean age between women with normal molecular findings and those with a suspected mutation. Overall, 49 women (48%) were younger than 50 years at the time of diagnosis.
Synchronous primary cancers of the endometrium and ovary have been identified in 7% to 29% of young women diagnosed with endometrial cancer.12-15 Although the etiology of these cancers has not been clearly determined, familial cancer syndromes often account for patients diagnosed with multiple cancers or patients diagnosed with cancer at a young age. Lynch syndrome (HNPCC) is an autosomal-dominant cancer predisposition syndrome that increases risk for multiple cancers, including colorectal, endometrial, and ovarian cancer. According to the recently revised Bethesda Criteria, women with two HNPCC-associated cancers should undergo further molecular evaluation, including MSI or IHC.8 Colorectal and endometrial cancer are the two most common cancers that develop in women with Lynch syndrome, with a lifetime risk of up to 54% and 60%, respectively.16,17 There have been several studies that have reported a high incidence of MMR defects in women with synchronous or metachronous colorectal and endometrial cancers. Millar et al9 evaluated tumor specimens from 40 women with colon and endometrial cancer and identified seven mutation carriers (17.5%). Plank et al10 evaluated a cohort of women who were diagnosed with their first cancer before age 50 years. A germline mutation was suspected in 36% of patients who had concordant loss of the same MMR protein in both the endometrial and colon cancer. Women with Lynch syndrome also have a 12% lifetime risk of developing ovarian cancer.16,17 To date, there are limited published data available on the prevalence of MMR defects in women diagnosed with both endometrial and ovarian cancer. Shannon et al18 performed MSI testing on samples from 45 women with synchronous endometrial and ovarian cancers. Only 3% of the tumors they tested were found to be MSI-high. They concluded that synchronous primary tumors of the endometrium and ovary were unlikely a part of the Lynch syndrome. In our series, 7% of patients (seven of 102) were found to have a high likelihood of Lynch syndrome based on a combination of clinical and molecular criteria (Table 2). All of these women had either a personal history or a first-degree relative with an HNPCC-associated cancer. There were a total of 81 patients who were considered low risk based on family history. None of the women who had tumor tissue available for analysis had molecular findings that were suggestive of a germline MMR defect. Six (13%) of these cases had loss of MLH1 expression that was likely due to a somatic change associated with methylation of the MLH1 promoter. This finding is consistent with other studies that have identified MLH1 promoter methylation in up to 23% of sporadic endometrial cancers and up to 77% of MSI high endometrial tumors.19,20 Our findings suggest that testing all women diagnosed with synchronous primary cancers of the endometrium and ovary is unlikely to yield a high percentage of women with Lynch syndrome. If only the patients with a personal history or at least one first-degree relative with an HNPCC-associated cancer were tested, all of the patients with a high likelihood of Lynch syndrome would have been captured. In addition to multiple cancers, the diagnosis of cancer at a young age is often associated with hereditary cancer syndromes. In women with synchronous or metachronous endometrial and colon cancer, mutation carriers have been shown to be younger than nonmutation carriers (45 v 55 years).9 In our study, we did not find that diagnosis at a young age was significantly associated with a suspected mutation. The median age among those with a suspected mutation and those with no suspected mutation was 47 and 49 years, respectively. Previous reports have shown that a large proportion of women with synchronous primary cancers of the endometrium and ovary are less than 50 years old at the time of diagnosis.21,22 We also found that these women tended to be premenopausal, obese, and nulliparous, suggesting that synchronous primary cancers of the endometrium and ovary may reflect a hormonal field defect.22 Further investigation into the etiology of these tumors is required to evaluate this question. The diagnosis of synchronous primary cancers of the endometrium and ovary can be difficult to make. Gynecologic pathologists at our institution use guidelines established by Scully et al22,23 to determine whether tumors of the endometrium and ovary represent metastatic disease or independent primary tumors. At the present time, there are no clear molecular markers used to distinguish synchronous primary cancers from metastatic disease. Of note, in the 10 cases that had MSI analysis of both the endometrial and ovarian tumors, the pattern of expression at each MSI locus was different between the two cancers. This is highly suggestive that the tumors in these women are indeed synchronous primary cancers and not metastasis from one site to the other. Although our cohort represents a large group of women diagnosed with synchronous primary cancers of the endometrium and ovary, the data are from a single, tertiary care institution. In addition, the molecular analysis was limited to a subset of cases. Paraffin-embedded tumor blocks were available in a high percentage of patients (13 [81%] of 16) with a concerning family history, and we found that a significant proportion of these cases (six [46%] of 13) had molecular findings consistent with a germline MMR defect. Genetic testing, however, would need to be performed to confirm the diagnosis of Lynch syndrome. In the group of women with a low-risk family history, we were only able to perform molecular studies on approximately one half of cases (46 [57%] of 81); among these, we did not identify a single case that had molecular findings suggestive of a germline mutation. In summary, 7% of women with synchronous endometrial and ovarian cancer had either clinical or molecular criteria suggestive of Lynch syndrome. This was considerably lower than that seen in women with synchronous or metachronous colon and endometrial cancer. All of the patients in our study who had a high likelihood of Lynch syndrome had a personal history or at least one first-degree relative with an HNPCC-associated cancer. Therefore, we believe that limiting genetic evaluation to women with synchronous endometrial and ovarian cancer who have a prior history of or at least one first-degree relative with an HNPCC-associated cancer may appropriately identify women with Lynch syndrome.
The authors indicated no potential conflicts of interest.
Supported by National Institutes of Health Grant No. N01-CN-05127 (Chemoprevention of Endometrial Cancer in Hereditary Nonpolyposis Colorectal Cancer) and 1P50CA098258-01 (Specialized Program of Research Excellence in Uterine Cancer). Presented at the Annual Meeting for The Society of Gynecologic Oncologists, Miami, FL, March 19-23, 2005. Authors’ disclosures of potential conflicts of interest are found at the end of this article.
1. Lynch HT, Smyrk T: Hereditary nonpolyposis colorectal cancer (Lynch syndrome): An updated review. Cancer 78:1149-1167, 1996[CrossRef][Medline] 2. Marra G, Boland CR: Hereditary nonpolyposis colorectal cancer: The syndrome, the genes, and historical perspectives. J Natl Cancer Inst 87:1114-1125, 1995 3. Aarnio M, Mecklin JP, Aaltonen LA, et al: Life-time risk of different cancers in hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Int J Cancer 64:430-433, 1995[Medline] 4. Fishel R, Lescoe MK, Rao MR, et al: The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75:1027-1038, 1993[CrossRef][Medline] 5. Leach FS, Nicolaides NC, Papadopoulos N, et al: Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 75:1215-1225, 1993[CrossRef][Medline] 6. Vasen HF, Watson P, Mecklin JP, et al: New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 116:1453-1456, 1999[CrossRef][Medline] 7. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al: A National Cancer Institute workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: Meeting highlights and Bethesda guidelines. J Natl Cancer Inst 89:1758-1762, 1997 8. Umar A, Boland CR, Terdiman JP, et al: Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 96:261-268, 2004 9. Millar AL, Pal T, Madlensky L, et al: Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium. Hum Mol Genet 8:823-829, 1999 10. Planck M, Rambech E, Moslein G, et al: High frequency of microsatellite instability and loss of mismatch-repair protein expression in patients with double primary tumors of the endometrium and colorectum. Cancer 94:2502-2510, 2002[CrossRef][Medline] 11. Boland CR, Thibodeau SN, Hamilton SR, et al: A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58:5248-5257, 1998 12. Evans-Metcalf ER, Brooks SE, Reale FR, et al: Profile of women 45 years of age and younger with endometrial cancer. Obstet Gynecol 91:349-354, 1998[CrossRef][Medline] 13. Tran BN, Connell PP, Waggoner S, et al: Characteristics and outcome of endometrial carcinoma patients age 45 years and younger. Am J Clin Oncol 23:476-480, 2000[Medline] 14. Gitsch G, Hanzal E, Jensen D, et al: Endometrial cancer in premenopausal women 45 years and younger. Obstet Gynecol 85:504-508, 1995[CrossRef][Medline] 15. Soliman PT, Oh JC, Schmeler KM, et al: Risk factors for young premenopausal women with endometrial cancer. Obstet Gynecol 105:575-580, 2005[Medline] 16. Dunlop MG, Farrington SM, Carothers AD, et al: Cancer risk associated with germline DNA mismatch repair gene mutations. Hum Mol Genet 6:105-110, 1997 17. Aarnio M, Sankila R, Pukkala E, et al: Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J Cancer 81:214-218, 1999[CrossRef][Medline] 18. Shannon C, Kirk J, Barnetson R, et al: Incidence of microsatellite instability in synchronous tumors of the ovary and endometrium. Clin Cancer Res 9:1387-1392, 2003 19. Salvesen HB, MacDonald N, Ryan A, et al: Methylation of hMLH1 in a population-based series of endometrial carcinomas. Clin Cancer Res 6:3607-3613, 2000 20. Simpkins SB, Bocker T, Swisher EM, et al: MLH1 promoter methylation and gene silencing is the primary cause of microsatellite instability in sporadic endometrial cancers. Hum Mol Genet 8:661-666, 1999 21. Zaino R, Whitney C, Brady MF, et al: Simultaneously detected endometrial and ovarian carcinomas: A prospective clinicopathologic study of 74 cases—A Gynecologic Oncology Group study. Gynecol Oncol 83:355-362, 2001[CrossRef][Medline] 22. Soliman PT, Slomovitz BM, Broaddus RR, et al: Synchronous primary cancers of the endometrium and ovary: A single institution review of 84 cases. Gynecol Oncol 94:456-462, 2004[CrossRef][Medline] 23. Scully RE YR, Clement PB: Tumors of the Ovary, Maldeveloped Gonads, Fallopian Tube, and Broad Ligament: Atlas of Tumor Pathology. Bethesda, MD, Armed Forces Institute of Pathology, 1998 Submitted July 23, 2005; accepted September 27, 2005.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
