Zauber, N. Peter MD*; Denehy, Thad R. MD†; Taylor, Robert R. MD†; Ongcapin, Emelie H. MD‡; Marotta, Stephen P. PhD‡; Sabbath-Solitare, Marlene PhD‡; Kulkarni, Renuka MD‡; Pradhan, Tana Shah DO†; Hermelin, Daniela MD*; Bishop, D. Timothy PhD§
Endometrial carcinoma is the most common gynecologic malignancy in the United States, with 42,160 new cases in 2009.1 Several factors are well recognized as risks for the development of adenocarcinoma, including early menarche, nulliparity, late menopause, obesity, diabetes, hypertension, and hyperestrogenic states.2,3 Endometrial cancer is typically diagnosed in the sixth decade, frequently because of postmenopausal bleeding. However, it also occurs in younger women.
Endometrial cancer is a major component of the family cancer syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), also called Lynch syndrome. Whereas HNPCC is usually recognized by the high incidence of colorectal cancer among females, the risk for endometrial cancer (42% lifetime risk) may exceed the risk of colorectal cancer (30% risk).4 A feature of the colorectal tumors in HNPCC is the loss of expression of DNA mismatch repair (MMR) genes. Loss of expression of one of the MMR genes, hMLH1, is also found in a significant proportion of right-sided colorectal tumors outside HNPCC. The process by which the loss of expression occurs, however, differs between the 2 mechanisms: within HNPCC the loss of expression is predominantly by gene deletion, whereas outside HNPCC, the relevant change is epigenetic, resulting in acquired methylation of the hMLH1 promoter.5
Among colorectal tumors, the loss of expression of an MMR gene is usually reflected in a readily identifiable characteristic, the mutator phenotype, identified by the presence of multiple novel genetic alleles (microsatellite instability [MSI]) present within the tumor but not present within germ line tissue. Microsatellite instability, when present within a tumor suppressor gene, will have consequences for the development of neoplasms.6 Although MSI is a common feature of tumors, germ line inactivation of repair genes explains just a small proportion of cancer. The colorectal data suggest that at younger ages, the loss of expression of an MMR gene is more associated with inherited germ line mutations (with subsequent deletion of the wild-type copy), and patients showing this phenotype are more likely to have a family history.7
Studies involving molecular genetic changes of endometrial cancer have considered patients either younger than 50 years or of all age groups but with limited numbers of younger patients. Unfortunately, these studies frequently used different molecular tests, thereby making comparisons across studies difficult.8,9 Our study was designed to assess for MSI and DNA methylation in cancers from a large group of patients with cancer who are younger than 50 years at diagnosis, with a comparable group of older women. In addition, we extracted information about personal risk factors and medical histories of the patients as well as their family histories with respect to cancers. We wished to assess whether MSI and methylation were comparable between the 2 age groups and how the molecular changes correlated with the clinical information.
MATERIALS AND METHODS
Patient Population and Study Design
We selected all cases of endometrial cancer in women younger than 50 years identified from Department of Pathology computer files for the years 1995 to 2007 on whom surgery was performed by one hospital-based group. We then randomly selected a comparable number of cases of cancer occurring at age 50 years or older from the same time interval from the same gynecological surgery practice. Office and hospital medical records were available for review on all patients with the permission of the attending surgeons. Histories of cancers in the patients and their first-degree relatives were primarily obtained by retrospective chart review, and primary documentation was limited. Histological slides containing tumor tissue were examined under the supervision of the gynecologic pathologist (E.O.) to establish a uniform histological diagnosis according to current criteria. An area of normal tissue was paired with each tumor. Reference to all histological and molecular analyses was by coded numbers. The extended spectrum of HNPCC-related cancers is defined as tumors of the colorectum, endometrium, small bowel, ureter, kidney, stomach, ovary, pancreas, and brain. The hospital's institutional review board approved the project.
DNA Extraction and Purification, PCR, and Gel Electrophoresis
All tissue specimens were formalin-fixed and paraffin-embedded. Relevant areas were identified on hematoxylin and eosin slides, and comparable areas from unstained sections were isolated using a blade and transferred to an Eppendorf tube. Paraffin wax was removed by xylene; this was followed by ethanol washes. Cellular material was lysed in a proteinase potassium buffer solution, and DNA was isolated and purified using the Qiagen QIAmp Tissue Kit (Qiagen Inc, Santa Clara, Calif). DNA was stored at 4°C in 10-mmol/L TE buffer, pH 9.0, and subsequently augmented with standard polymerase chain reaction (PCR) techniques.
The Bethesda panel of markers used for the detection of MSI included 2 mononucleotide markers, BAT25 and BAT26, and 3 dinucleotide markers, D2S123, D5S346, and D17S250. Each tumor DNA specimen was paired with its corresponding normal DNA specimen from the same patient for a total of 10 separate PCR reactions. Separation and detection of amplified fragments were performed on an ABI 3130 Genetic Analyzer using Genemapper software. Microsatellite instability for a given primer set was defined as a change in the allele pattern, with the appearance of one or more new PCR products relative to those produced by the normal DNA. A tumor was defined as MSI high if 2 or more of the 5 markers revealed MSI.
Methylation status of MMRs was ascertained using the methylation-specific DNA detection system SALSA MS-MLPA ME011 (MRC-Holland, Amsterdam, The Netherlands). This kit uses 21 probes containing an Hha1 recognition site to detect aberrant methylation in 7 MMR genes: MLH1 (5 probes), MSH2 (3), MHSH6 (3), MSH3 (3), MLH3 (1), PMS2 (3), and MGMT (3). Eleven additional reference probes are included that do not include an HhaI site. Two hundred nanograms of genomic DNA was hybridized overnight with the 32-probe mix and then divided for 2 separate reactions. In the first reaction, those probes hybridized at the potential methylation sites are ligated. Ligation enables subsequent PCR amplification of the probes. In the second reaction, there is a similar ligation followed by an HhaI restriction enzyme cutting of the probes at only the unmethylated HhaI sites. Thus, only those HhaI probes that are bound at a methylated site were subsequently PCR amplified. All of the probes contain universal PCR primer recognition sites, and a single PCR reaction can therefore amplify all of the ligation products in both the ligation reaction (uncut) and ligation/HhaI reaction (cut) sample sets. The methylation status for each sample was then determined by comparing the respective PCR product ratios from both reaction sets. The ligation and PCR reactions were performed in an ABI 9700 thermal cycler (Applied Biosystems, Foster City, Calif). The ME011 kit contained all of the necessary reagents except for the HhaI enzyme, which was purchased from Promega (Madison, Wis). Polymerase chain reactions were analyzed on an ABI 3130 Genetic Analyzer with Genemapper software (Applied Biosystems). Genemapper data were subsequently exported for methylation analysis using Coffalyser software available at the MRC-Holland website (www.mlpa.com). As a control, 10 tumors with no MSI, 5 from each age group, were evaluated for methylation status, and all were found to be unmethylated.
We conducted a case-case analysis of women with endometrial cancer diagnosed at age younger than 50 years and at 50 years or older. We estimated the odds ratio (ORs) for younger versus older age at diagnosis for risk factors and used Breslow test of homogeneity to assess whether the ORs differed significantly across subgroups. The t test was used to compare mean values between the 2 groups. The study was designed to be able to detect ORs of 2.4 or greater, with the power of 0.80 when the prevalence of the risk factor was 20% to 50% at the 0.05 (2-sided level of significance).10 All analyses were performed using SAS 9.1 (SAS, Cary, NC).
A total of 213 endometrial carcinoma cases were identified. There were 101 patients (47%) younger than 50 years at diagnosis and 112 patients (53%) 50 years or older. For the younger group, 91 cancers (90%) were endometrioid, 4 (4%) were adenosquamous, 4 (4%) were adenocarcinomas with benign squamous, 1 (1%) was villoglandular, and 1 (1%) was clear cell. For the older group, 100 cancers (89.3%) were endometrioid, 1 (1%) was adenosquamous, 3 (3%) were villoglandular, 1 (1%) was undifferentiated, 1 (1%) was mixed mesodermal, 5 (5%) were serous, and 1 (1%) was mucinous. Race was recorded for only 21 cases, with 17 whites and 4 African Americans. The baseline characteristics and risk factors are shown in Table 1. The mean body mass index (BMI) of our patients younger than 50 years (total, 97) was 32.7 kg/m2, whereas the mean BMI for the older women (total, 109) was 29.8 kg/m2. This is a significant difference, with P = 0.02, and it reflects a difference between the 2 age groups only, with women markedly obese, with a BMI of 40 or greater. This was found in only 10% of the older women but in 25% of the younger women (P = 0.02). However, the 2 age groups did not differ in the overall percentage of patients who had a BMI of greater than 30 kg/m2, had diabetes, or had a previous different cancer. The younger patients were more likely to be nulliparous attributable at least in part to the age differences between the 2 groups. No further information was available to investigate this difference in more detail, whereas the older patients were more likely to have hypertension. History of cigarette use could not be determined from the retrospective chart review. Information on estrogen use was available for 191 patients and was used infrequently, but slightly more often in the older age group.
Pathological tumor (T) stage was similar for both groups as was the likelihood of local organ involvement as determined by pathological evaluation. Local organ involvement was detected pathologically for 28 (13.1%) cases only; 22 cases had one site involved (lymph node, 9, or ovary, 13), and 6 cases had 2 sites. However, nodal status information was absent from 54% of the surgical pathological reports, and status of metastases also could not be determined for 95% of the cases (Table 1). Five cases (2%) were identified as stage MO and 5 (2%) as stage M1.
High microsatellite instability (MSI high) was found in tumors from a total of 56 patients (26.3%). Among the younger group, 21 (20.8%) tumors had MSI, compared with 35 of the older group (31.2%). This difference approaches significance (P = 0.08). For all 213 patients, the percentage of tumors with MSI was similar for those with no other organ involvement (46 [25%]) and those with other organ involvement (10 [36%]) (P = 0.22; OR, 1.68; CI, 0.72-3.90). Microsatellite instability did not vary by decade of age (P = 0.46 for trend). Despite the difference between the young and the old groups with respect to parity, we did not find any difference in the frequency of MSI in their tumors when controlling for nulliparity (Breslow test, P = 0.24). There was no difference between the younger and the older groups with respect to obesity (BMI >30) and MSI, nor with the added variable of the methylation status (Table 2).
A total of 37 (66.1%) of the 56 MSI tumors contained methylated DNA. Thirty-two tumors were methylated at all 5 hMLH1 sites, one tumor was methylated at 4 hMLH1 sites, one tumor was methylated at 3 hMLH1 sites, and one tumor was methylated at 2 hMLH1 sites. One additional tumor was methylated at 6 sites, 5 hMLH1 sites and 1 MGMT site, and one additional tumor was methylated at 8 sites, 5 hMLH1 sites and 3 PMS2 sites. Thus, all tumors with methylated DNA involved some or all of the sites for the hMLH1 gene, and we imply hMLH1 methylation when referring to a methylated tumor (Figs. 1 and 2).
A total of 19 (8.9%) of the 213 tumors were both unstable and unmethylated. Thirteen of the 19 patients were of the younger age group (12.9% of the total 101), whereas 6 were from the older age group (5.3% of the total 112). This almost reached statistical significance, (P = 0.0547; OR, 2.61; CI, 0.95-7.15; Fig. 3).
We observed a marked difference between the younger and older women regarding MSI and the methylation status of their tumors. Of the 21 patients in the younger group with MSI tumors, 13 (61.9%) had unmethylated tumors, whereas in the older group, only 6 (17.1%) of the 35 women had unmethylated MSI tumors. This difference is statistically significant (P = 0.007; Table 2). These 6 older women ranged in age from 56 to 71, with a mean age of 59 years. Five had an endometrioid carcinoma and one had a serous adenocarcinoma.
Thirty-nine (18.3%) patients had an additional cancer. A total of 14 (14.4%) of 97 younger women for whom personal history was known had an additional tumor. These were ovarian primary (8), breast cancer (3), and one each for cervical cancer, brain tumor, and melanoma. Four of these 14 patients had microsatellite (MS) unstable uterine cancers, and their second primaries were ovary (2), breast (1), and melanoma (1). A total of 25 (22.3%) of 112 older women reported an ovarian primary (5), breast cancer (13), and one each for melanoma, leukemia, lymphoma, and cancers of the stomach, colon, thyroid, and pharynx. Six of these 25 older patients with any prior cancer had MS unstable uterine cancers, and their second primaries were breast cancer (3) and one each with colon cancer, thyroid cancer, and leukemia. A personal history of an additional cancer did not differ significantly between the 2 groups of women with cancers, both unstable and unmethylated, or unstable and methylated; however, the number of individuals in each group was small (data not shown).
Of the 3 patients younger than 50 years who had prior breast cancer, only one had taken tamoxifen. She had an endometrioid carcinoma that was MS stable. Of the 13 older patients who had prior breast cancer, only 2 patients had taken tamoxifen. Both had endometrioid carcinoma: one was MS stable and the other was unstable and methylated. Two of these 13 patients had taken raloxifene, both with MS-stable endometrioid tumors, and the other 9 older aged patients had no exposure to antiestrogens.
For the entire cohort, there was no difference between the 2 age groups in the family history of at least one HNPCC-spectrum cancer. Among all the younger patients, 33 (32.7%) reported a family history of one or more relatives with an HNPCC cancer, with similar findings among all the older patients (37 [33.0%], (P = 0.96; Table 1).
For the 13 younger women with uterine cancers demonstrating MSI and no methylation, 8 patients (61.5%) had a family history of an HNPCC-related cancer in any relative. Of the remaining 88 younger women (with MS stable or unstable/methylated tumors), only 25 (28.4%) had a positive family history in any relative, and this difference is statistically significant, with P = 0.02 (OR, 4.0; CI, 1.2-13.5). A similar difference was seen when relatives were limited to just first-degree (data not shown). For the older women, however, only 1 of 6 (16.7%) with cancers demonstrating MSI and no methylation had a positive family history in any relative, while 36 of the other 106 (34.0%) older women (with MS stable or unstable/methylated tumors) had a positive family history. For the older women, this difference is not significant, with P = 0.38 (OR, 0.39; CI, 0.04-3.4) and trends in the opposite direction from the young women. A similar result was found when family history was limited to first-degree relatives only (data not shown). Analyses for the 2 age groups of patients specifically for only relatives with colon cancer, or only endometrial cancer, or for a more restricted definition of HNPCC-related cancers, yielded similar trends when analyzed, controlling for all relatives or for only first-degree relatives (data not shown). A Breslow-Day test for homogeneity of the ORs indicates that the results for the 2 age groups are truly different.
Concern for a possible familial cancer syndrome such as HNPCC in women with cancer is usually directed toward those of the younger age. However, in a study of 130 confirmed HNPCC families, the median age for endometrial cancer was 49 years, indicating that many patients were diagnosed with uterine cancer after the age of 50 years.11 A large study of sporadic cancers found 28 (6.8%) of 413 lesions to be MSI positive/MLH1 unmethylated, supporting a diagnosis of HNPCC. The average age at the time of diagnosis for these 28 cases was 55.4 years.12 It has been suggested that most of the women diagnosed with HNPCC will present with a gynecologic malignancy as the sentinel cancer.8
The true incidence of germ line mutations in DNA mismatch repair genes for endometrial cancer patients is unknown. A few studies have evaluated women younger than 50 years but did not consider a concurrent group of patients older than 50 years.9,13,14 Gene testing was incomplete in several. One report concluded that at least 9% of patients younger than 50 years carry a germ line mutation.13 Only one study of younger patients incorporated family history and identified 5 patients from their cohort of 58 (8.6%) with a relevant family history, with 4 of the 5 patients carrying an identified germ line mutation.15 One study compared women younger than 50 years of age (n = 55) with those older than 50 years (n = 368) but focused on detecting MSH6 gene carriers.16 Another study detected MSI in 29 of 120 endometrial cancers diagnosed at an age younger than 50 years, and based on methylation studies, the authors concluded that 6 patients (4.1%) were presumptive Lynch syndrome.9 Our results are comparable to these studies.
Kauff17 has questioned the best method to evaluate women with cancer for HNPCC, as various analytical techniques provide a number of paths for identifying these patients.
Immunohistochemical analysis of mismatch repair protein expression is limited by variable interpretation and may be normal with germ line mutations of MLH6.18 Direct testing for germ line genetic mutations is feasible but expensive and time consuming and assumes that the patient's particular mutation is one previously characterized.
Microsatellite instability is a necessary finding, but it is not independently sufficient, as MSI may result from somatic as well as germ line changes. However, assessment of tumor DNA for the presence of methylation is then useful to exclude those patients with a somatic epigenetic cause for the observed MSI, thereby suggesting that the cases without methylation represent germ line mutations of a DNA repair gene.
We believe that MSI testing followed by methylation assay is an effective method to identify patients with endometrial cancer as possible HNPCC. Our results indicate that 62% of women younger than 50 years of age with MSI cancers are unmethylated, whereas only 17% of such cancers from women older than 50 years are unmethylated. For the older women, acquired methylation is clearly the major cause of MSI in their uterine cancers as has been previously demonstrated.5
In addition, a positive family history of an HNPCC-related cancer in a first-degree relative further separates the 2 age groups. For the younger age group, 62% of patients with tumors both unstable and unmethylated had a positive family history. However, family history was positive for only 17% of the older women with tumors both unstable and unmethylated. It is unclear why there is this difference. We would not expect variation in history taking to be a factor because the same physicians cared for all patients. It would seem likely that older individuals would potentially have longer-lived relatives with more opportunity for the development of cancers, not less. This difference probably reflects the limitations of family history information and/or patients' recall but might suggest other, as of yet, undefined molecular mechanisms.
The major weakness of our study results from the fact that it is not a prospective study. However, we believe this is minimized by the consistency of the source of the patients and the available information. Our study is strengthened by the comparison of a large group of younger women with older patients. A further important feature is the application of a new, novel methodology for the detection of DNA methylation that is direct and highly reproducible.
In conclusion, recognition of the association between endometrial carcinoma and HNPCC is important both for the individual patient and her first-degree relatives. The patients and their family members are at risk of multiple cancers and should receive appropriate surveillance. It is helpful to identify a particular assay or combination of analyses that effectively narrows the pool of patients requiring germ line testing. There are strengths and weaknesses to all available screening modalities.19
We have shown that the combination of MSI testing and DNA methylation studies results in the identification of presumptive HNPCC in approximately 13% of women with endometrial cancer presenting at younger than 50 years and in approximately 5% of women 50 years or older. Family history of an HNPCC associated cancer in a relative is further supportive of the molecular evaluation in younger women. We believe the 2 tests-for MSI and methylation-combined with family history are an excellent method for identifying patients at high risk of a cancer-family syndrome. We recommend that women with uterine cancers that are MS unstable and do not have DNA methylation are candidates for further testing for specific germ line mutations.
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