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Microsatellite Instability and Epigenetic Inactivation of MLH1 and Outcome of Patients With Endometrial Carcinomas of the Endometrioid Type

Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

From the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology; Division of Endocrine and Oncologic Surgery, Department of Surgery; and the Division of Biostatistics, Washington University School of Medicine and Siteman Cancer Center, St Louis, MO

Address reprint requests to Israel Zighelboim, MD, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Washington University School of Medicine, 4911 Barnes Jewish Plaza, Box 8064, St Louis, MO 63110; e-mail: zighelboimi{at}wustl.edu

	ABSTRACT

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Purpose Most studies of microsatellite instability (MSI) and outcomes in endometrial cancer patients have included varied histologic subtypes. Nonetheless, MSI occurs almost exclusively in endometrioid tumors. The impact of MSI on outcomes in patients with endometrial cancer is controversial. We sought to determine whether MSI and MLH1 methylation are associated with clinicopathologic variables and survival outcomes in a large series of patients with endometrial carcinomas of the endometrioid type.

Patients and Methods Tumor samples, blood, and clinicopathologic data were prospectively collected and analyzed for 446 patients with endometrioid carcinomas. MSI was determined using five National Cancer Institute (NCI) consensus panel markers, and the methylation status of the MLH1 promoter was determined by combined bisulfite restriction analysis (COBRA). Associations with clinicopathologic variables and survival outcomes were evaluated.

Results MSI was identified in 147 cases (33%). MSI was associated with higher International Federation of Gynecology and Obstetrics (FIGO) grade (P < .0001). MSI+ tumors without MLH1 methylation were associated with younger age (P < .001). MSI was not associated with overall survival (OS; hazard ratio [HR], 1.011; 95% CI, 0.688 to 1.484; P = .96) or disease-free survival (DFS; HR 0.951; 95% CI, 0.554 to 1.635; P = .86). The combined MSI/MLH1 methylation status (treating MSI– as the reference) did not predict OS (MSI+/MLH1-U: HR, 0.62; 95% CI, 0.27 to 1.44; P = .26; MSI+/MLH1-M: HR, 0.95; 95% CI, 0.62 to 1.46; P = .82) or DFS (MSI+/MLH1-U: HR, 0.51; 95% CI, 0.22 to 1.19; P = .12; MSI+/MLH1-M: HR, 0.93; 95% CI, 0.62 to 1.40; P = .72).

Conclusion MSI is not associated with survival in patients with endometrioid endometrial cancer.

	INTRODUCTION

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Endometrial carcinoma is the most commonly diagnosed malignancy of the female reproductive tract in the United States. The majority of these cases are detected in early stages and cured with surgery alone. However, a subset of them is thought to be at high risk for recurrences and receive adjuvant therapies. Despite current efforts, it is estimated that 7,350 women will succumb to endometrial cancer in this country in 2006.¹

Several molecular alterations are associated with endometrial carcinogenesis. Mutations in KRAS2, CTNNB1, and TP53 occur at modest and variable frequencies. When histology and/or grade are taken into consideration, it becomes evident that some molecular defects are characteristic of definable subgroups of endometrial cancers. TP53 mutations are seen in the majority of serous endometrial cancers but are relatively uncommon among well- and moderately differentiated endometrioid tumors.² Among endometrioid endometrial cancer, loss of PTEN and DNA mismatch repair are the most frequently identified molecular alterations.^2-4

Loss of DNA mismatch repair causes genomic instability by facilitating the accumulation of somatic mutations in a wide range of sequences. Some of these changes are presumed to affect key regulatory genes related to cell growth and/or apoptosis. Microsatellite instability (MSI) is the hallmark of tumors with defects in DNA mismatch repair and occurs in approximately 20% to 30% of endometrial cancers. MSI is seen in endometrial tumors associated with the hereditary nonpolyposis colorectal cancer or Lynch syndrome, as well as sporadic endometrial cancers. In sporadic cases, MSI frequently results from epigenetic silencing of MLH1. Methylation accounts for the lack of functional immunodetectable MLH1 protein in the majority of MSI+ endometrial cancers.^2,5-8

Associations between defective DNA mismatch repair and particular clinicopathologic characteristics have long been recognized in colorectal carcinomas.^9-11 Moreover, MSI has prognostic implications for colorectal cancer patients. Colorectal tumors with MSI (MSI+) tend to have a better prognosis when compared with their DNA mismatch repair competent counterparts.^12-14 Multiple studies have attempted to determine the impact of MSI in the outcome of patients with endometrial cancer with conflicting results.^15-28 The apparently contradictory findings reported in different studies might be explained by small sample sizes, nonstandard definitions of the MSI phenotype, and more importantly inclusion or exclusion of histologic types inherently associated with aggressive disease and poor outcomes.

The objective of our study was to determine whether DNA mismatch repair is associated with clinicopathologic variables including survival outcomes in a large, prospectively collected cohort of endometrioid endometrial cancer cases. A secondary objective was to determine whether there were differences among MSI+ cases when they were further classified with respect to MLH1 promoter methylation status.

	PATIENTS AND METHODS

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Study Participants and Clinical Data
Since 1991, we have prospectively acquired tumor samples from hysterectomy specimens from patients being treated for suspected uterine cancer by the Division of Gynecologic Oncology at Washington University Medical Center (St Louis, MO). All participants consented to molecular analyses and follow-up as part of Washington University Medical Center Human Studies Committee–approved protocols (HSC 91-507 and 93-0828).

A total of 932 specimens were collected between November 1991 and January 2005. All cases were evaluated and diagnoses confirmed by experienced gynecologic pathologists. Microsatellite analysis was successfully performed in 582 tumors with high neoplastic cellularity (> 70%). Tumors of nonendometrioid histology were excluded from this study (126 cases). Follow-up data were extracted from clinic charts, hospital records, and the Siteman Cancer Center's tumor registry surveillance database. Follow-up was not available for one endometrioid cancer patient. Similarly, we excluded nine cases of perioperative deaths (not cancer related and occurring within 30 days of surgical procedure). The remaining cases (n = 446) represented pure endometrioid endometrial carcinomas and were deemed eligible for this study.

None of the patients in our series received preoperative radiation or chemotherapy. Surgical staging and tumor grade was assigned on the basis of International Federation of Gynecology and Obstetrics (FIGO) 1988 criteria. Clinical and pathologic data for these cases were prospectively collected and stored in a computerized database. Specific initial treatments were individualized. At the completion of their initial treatment, these patients were routinely followed at 3-month intervals. Standard surveillance included physical examination and Pap smears for at least 2 years after initial treatment. Assessment with additional imaging studies and directed biopsies were performed as clinically indicated at the discretion of the treating physician. Recurrent disease was histologically confirmed as indicated.

MSI Typing and MLH1 Promoter Methylation Analysis
Tissue specimens and blood were obtained at the time of surgery, snap frozen, and stored at –70°C. DNA was then isolated from tumors using proteinase K and phenol extraction or with the DNeasy Tissue Kit (Qiagen Inc, Valencia, CA). DNA was extracted from peripheral blood leukocytes as previously described.^29-32 When blood was not available, normal DNA was extracted from uninvolved myometrium.

Microsatellite analysis was performed as previously described using five National Cancer Institute consensus microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250).^29,30,33 Microsatellite typing was repeated up to three times to resolve any uncertainties as to whether abnormal polymerase chain reaction (PCR) products suggestive of MSI were present for a given specimen. Tumors were designated as having high-level MSI (MSI+) if novel PCR bands were identified in at least two of the five consensus panel markers. Low-level MSI was diagnosed when a single marker demonstrated novel PCR product bands. Tumors were considered microsatellite stable if there was no evidence of MSI in any marker. Low-level MSI and microsatellite stable tumors were designated MSI negative (MSI–) for the purposes of this study.

The COBRA method was used to evaluate methylation of the MLH1 promoter.³⁴ Briefly, tumor DNA was bisulfite converted using commercially available kits (CpGenome DNA Modification Kit, Intergen, Purchase, NY; and EZ DNA Methylation Gold Kit, Zymo Research, Orange, CA) following manufacturers’ recommended protocols. Target CpG-rich sequences in the MLH1 promoter region were amplified using two rounds of PCR. Restriction digest of PCR products was then undertaken using enzymes that recognize sequences potentially altered by methylation. PCR primers and conditions, as well as restriction enzymes for this assay, have previously been published by our group.³⁵

Statistical and Survival Analyses
A recent report suggested that MSI has a favorable effect on survival outcomes in patients with endometrial cancer with a hazard ratio (HR) of 0.3 for overall survival (OS) and 0.4 for disease-free survival (DFS).²⁸ Assuming survival times follow exponential distributions, a total number of 38 events are needed to be observed to detect an HR of 0.4 with 80% power and a two-sided significance level of .05. Estimating an event rate of approximately 30%, we anticipated that 128 cases would be required to demonstrate an improved or worsened outcome as defined previously.

The relationship between MSI status and/or MLH1 status and relevant covariates was performed using ² and t tests. OS was defined as the time from diagnosis to the date of death from any cause. Survivors were excluded at the date of last contact. DFS was defined as the time from surgery to recurrence or progression. The Kaplan-Meier product limit method was used to estimate survival. Both univariate and multivariate Cox proportional hazard models were fitted to check the possible predictive effects of the covariates on OS and DFS. In the analysis of DFS, Gray's competing risk methods were used to account for the potential competing effect of death.³⁶ All analyses were two-sided, and significance was set at a P value of .05.

Statistical analyses were performed using SAS (SAS Institutes, Cary, NC) and cmprsk R (http://biowww.dfci.harvard.edu/gray) statistical packages.

	RESULTS

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Our study population consisted of 446 patients with endometrioid endometrial carcinoma. The median follow-up time was 54.8 months (range, 0.7 to 171.6). Demographics and clinicopathologic characteristics of the cohort are presented in Table 1. MSI was identified in 147 (33%) of 446 cases. Representative examples of three National Cancer Institute consensus markers demonstrating MSI+ in one of our cases are depicted in Figure 1A. We evaluated the associations between MSI and clinicopathologic variables (Table 1). The MSI+ phenotype was strongly associated with higher FIGO grade (P < .0001). Patients carrying MSI+ tumors tended to be slightly older at the time of diagnosis and to be diagnosed at earlier stages. However, these differences were not statistically significant (P = .07 and .06, respectively). Microsatellite status was not associated with race or use of adjuvant treatment.

View larger version (51K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Representative example of microsatellite instability (MSI) and MLH1 methylation analyses. (A) Novel polymerase chain reaction (PCR) bands (arrows) in MSI+ tumor 1730 DNA. (B) On the left, combined bisulfite restriction analysis (COBRA) demonstrates restriction with BstU I (CGCG) and Mbo I (GATC) indicating MLH1 methylation (tumor 1730). On the right, immunohistochemistry demonstrates lack of immunodetectable protein in this MLH1-methylated specimen. Immunohistochemical confirmation of COBRA findings was not performed for all specimens. T, tumor; N, normal; bp, base pairs. (*), case 1730 MSI+/MLH1-methylated.

We also evaluated potential associations between clinicopathologic variables and DFS. Again, older age had a significant but small impact on DFS (HR, 1.034; 95% CI, 1.010 to 1.058; P = .005) whereas advanced stage (stage III/IV; HR, 2.757; 95% CI, 2.169 to 3.504; P < .0001), higher FIGO grade (grade 2: HR, 2.898; 95% CI, 1.481 to 5.670; P = .002; grade 3: HR, 6.862; 95% CI, 3.520 to 13.378; P < .0001) and the use of adjuvant treatment (HR, 3.047; 95% CI, 1.842 to 5.038; P < .0001) were strong predictors of poor DFS. After multivariate analysis, the effects of age (HR, 1.023; 95% CI, 1.0006 to 1.047; P = .04), stage (stage II: not estimable; stage III/IV: HR, 2.790; 95% CI, 1.961 to 3.970; P < .0001) and grade (grade 2: HR, 2.378; 95% CI, 1.194 to 4.736; P = .01; grade 3: HR, 3.796; 95% CI, 1.855 to 7.771; P < .0001) on DFS remained statistically significant.

When the potential of MSI as a predictor of outcome in patients with endometrioid endometrial cancer was evaluated, we found that MSI status was not associated with OS (HR, 1.011; 95% CI, 0.688 to 1.484; P = .96) or with DFS (HR, 0.951; 95% CI, 0.554 to 1.635; P = .86). Kaplan-Meier survival analyses for OS and DFS according to MSI status are presented in Figure 2.

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Kaplan-Meier curves for (A) overall survival and (B) disease-free survival by microsatellite instability (MSI) status. Vertical bars represent excluded cases.

The associations between MSI/MLH1 methylation status and OS and DFS are illustrated in Figure 3. The combined MSI/MLH1 methylation status (treating MSI– as the reference) was not a significant predictor of OS (MSI+/MLH1-U: HR, 0.62; 95% CI, 0.27 to 1.44; P = .26; MSI+/MLH1-M: HR, 0.95; 95% CI, 0.62 to 1.46; P = .82). Similarly, there was no significant association between MSI/MLH1 methylation status and DFS (MSI+/MLH1-U: HR, 0.51; 95% CI, 0.22 to 1.19; P = .12; MSI+/MLH1-M: HR, 0.93; 95% CI, 0.62 to 1.40; P = .72). A subgroup outcome analysis was performed to determine whether there were outcome differences among MSI+ cases when classified according to MLH1 methylation status. MSI+/MLH1-U cases are biologically distinct given the relatively high frequency of inherited disease in this group. In MSI+/MLH1-U cases, the differences were not significant for OS (HR, 1.634; 95% CI, 0.750 to 3.559; P = .22) or DFS (HR, 3.072; 95% CI, 0.707 to 13.347; P = .13).

View larger version (24K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Kaplan-Meier curves for (A) overall survival and (B) disease-free survival by microsatellite instability (MSI)/MLH1 status. Vertical bars represent excluded cases.

	DISCUSSION

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Several studies have tried to better define clinicopathologic variables associated with the risk of recurrence with the goal of being better able to implement effective therapeutic strategies in patients with early-stage "high-risk" endometrial cancer.^38,39 Risk stratification based on clinicopathologic criteria and the use of radiation- and/or chemotherapy-based adjuvant treatments has proven unsuccessful.⁴⁰ Therefore, molecular characterization represents an attractive alternative that could also potentially direct conventional adjuvant therapies and promote the development of novel targeted treatments for this malignancy.

Defective DNA mismatch repair represents one of the most common molecular defects in endometrioid endometrial cancers, and tumors with these defects are readily identifiable through MSI analysis.^2-4 The discovery of prognostic implications for MSI in colorectal cancer^12-14 prompted multiple DNA mismatch repair studies in gynecologic malignancies.

Studies in endometrial cancer have yielded conflicting results regarding the clinicopathologic significance of MSI in these malignancies. Possible explanations for the inconsistent results that have been reported include cohort differences, lack of statistical power, differences in overall study design, and/or technical limitations. Differences in the molecular techniques used and variations in neoplastic cellularity of the tumor specimens evaluated could explain, for example, the wide range of MSI frequencies encountered in the literature (9% to 45%).^15-28 Despite the inception of a consensus panel for the determination of microsatellite instability in colorectal cancer in 1998,³³ many uterine cancer studies have used nonstandard MSI definitions and genetic markers. Importantly, most studies of DNA mismatch repair in endometrial cancer have included different histotypes.^{15-21,24,25,28} Aggressive histotypes such as papillary serous, clear-cell and undifferentiated carcinoma are not only per se associated with worse outcomes but also demonstrate much lower frequencies of MSI. MSI has, in fact, been demonstrated to be limited primarily to endometrioid type endometrial cancer.^{2,17,18,20,41}

To our knowledge, ours represents the largest DNA mismatch repair study in a cohort of surgically staged patients with endometrioid endometrial cancer. Because MSI is rare in nonendometrioid tumors, and patients with these histotypes have a worse outcome overall, we focused our analyses on endometrioid tumors. We defined MSI by standard criteria.³³ We also determined the MLH1 methylation status in MSI+ tumors. The assessment of MLH1 methylation has important implications in the setting of MSI+ endometrial cancer. Epigenetic inactivation of MLH1 accounts for the vast majority (75% to 92%) of sporadic MSI+ endometrial cancers.^6,7 Furthermore, in the absence of MLH1 methylation, MSI+ tumors are much more likely to be associated with germline mutations in DNA mismatch repair genes.^7,8,42,43,44 As has been previously shown for colorectal cancer,¹³ MSI+ endometrial tumors might be associated with particular characteristics and improved prognosis when they arise in a background of inherited cancer susceptibility.

In this study, 147 (33%) tumors were MSI+. This high frequency of MSI could be explained in part by the strict inclusion of endometrioid tumors of high neoplastic cellularity. Cases with MSI were associated with higher FIGO grade. Fiumicino et al²² reported a similar observation in their study of 65 early-stage endometrioid endometrial cancers. The reason for such association remains elusive. The mutator phenotype (MSI) might confer a selective advantage as a consequence of mutations in specific genes or through abnormalities in signaling mechanisms leading to deregulated apoptosis. We hypothesize that these changes might select for particularly aggressive clones with aggressive appearing histology (eg, higher FIGO grade).

MSI+ endometrial cancers in the absence of MLH1 methylation are often associated with a germline mutation in one of the DNA mismatch repair genes.^8,44 MSI+ tumors without MLH1 methylation occurred most commonly in younger women. On average, these patients were 10 years younger than their MLH1-methylated counterparts at the time of diagnosis (P < .001). Similar findings have been previously reported for colorectal cancer.¹⁴

As anticipated, surgical stage and FIGO grade were significant predictors of OS and DFS in our patient population. Recently, Black et al²⁸ reported that MSI was a significant predictor of favorable OS and disease-specific survival (HR, 0.3 for both) in a study of 473 patients with endometrioid, serous, clear-cell, and undifferentiated endometrial carcinomas. Despite adequate statistical power to demonstrate even smaller survival differences than those reported by Black et al,²⁸ there was no difference in survival attributable to MSI in our cohort. A number of factors can explain this apparently contradictory finding. First, even with sophisticated statistical models for the survival analysis, it may not be possible to control for the effects the nonendometrioid cases have in recurrence and mortality. As previously noted, nonendometrioid tumors are associated with bad outcomes and rarely demonstrate MSI. Another confounding factor could be differences in the fraction of cases with inherited cancer susceptibility. If this group were over-represented in Black's cohort,²⁸ overall survival in MSI+ might have been influenced by this subpopulation. Studies that demonstrated better outcomes in MSI+ colorectal cancer despite more advanced disease included relatively younger patients.¹⁴ Younger age at presentation in both colorectal and endometrial cancer has been associated with familial disease. Because patients were not uniformly classified as either sporadic or familial cases, it is not possible to speculate as to whether improved outcomes are, in fact, related to the DNA mismatch repair or if they rather represent a yet unexplained characteristic of familial cancer susceptibility syndromes. Lack of MLH1 methylation is a feature of inherited endometrial cancers and/or disease associated with mismatch repair gene mutations rather than epigenetic silencing. Our analysis was underpowered to demonstrate such differences in survival among MSI+ MLH1 methylated and MSI+ MLH1 unmethylated cases.

The ability to identify patients with endometrioid carcinomas at high risk for recurrence is currently limited to the use of defined clinicopathologic features.^38,39 Molecular characterization of tumors could complement current histopathologic approaches. Our study clearly demonstrates that MSI status lacks prognostic value in women with endometrioid endometrial carcinoma. The Gynecologic Oncology Group is currently conducting a study on molecular characterization of endometrial cancer (GOG210). This study might provide the opportunity to develop optimal screening strategies aimed at identifying patients with inherited cancer syndromes and to test the exact impact of epigenetic inactivation versus specific mismatch repair gene mutations in the pathogenesis and behavior of endometrial cancers.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Conception and design: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Financial support: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Administrative support: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Provision of study materials or patients: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Collection and assembly of data: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Data analysis and interpretation: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Manuscript writing: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

Final approval of manuscript: Israel Zighelboim, Paul J. Goodfellow, Feng Gao, Randall K. Gibb, Matthew A. Powell, Janet S. Rader, David G. Mutch

	NOTES

 
Supported by RO1 CA71754 (P.J.G.) and Barnes-Jewish Foundation 00161-0205. The Siteman Cancer Center is supported by National Cancer Institute Cancer Center Support Grant No. P30 CA91842.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted July 10, 2006; accepted February 26, 2007.

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