Original articles
Microsatellite Instability and Expression of MLH1 and MSH2 in Normal and Malignant Endometrial and Ovarian Epithelium in Hereditary Nonpolyposis Colorectal Cancer Family Members

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Abstract

Mismatch repair deficiency is a characteristic molecular finding in hereditary nonpolyposis colorectal cancer (HNPCC), and has been demonstrated in both colorectal cancers and benign adenomas. Endometrial and ovarian cancers are common extracolonic tumors in this syndrome; however, few studies have investigated whether genetic changes occur in histologically normal endometrial and ovarian epithelia from HNPCC family members. If early genetic changes exist, they might be used as molecular markers to detect susceptibility to endometrial and ovarian cancers. In this study, we analyzed microsatellite instability (MSI) and MLH1 and MSH2 immunohistochemical expression in 20 histologically normal epithelia (12 endometrial and 8 ovarian) and 8 cancers (4 endometrial and 4 ovarian) obtained from 20 individuals representing 7 unrelated HNPCC families. While MSI was observed in endometrial (75%) and ovarian (100%) cancers, no case was determined to exhibit MSI in histologically normal epithelia of the endometrium or ovary. Similarly, in immunohistochemical expressions for MLH1 and MSH2, histologically normal epithelia had no genetic changes predisposing to malignancy. In cancer cases, a correlation existed between the expression of MLH1 and MSH2, the presence of germline mutations in the hMLH1 and hMSH2 genes, and the presence of tumor MSI. These data suggest that MSI and MLH1 and MSH2 expression are not useful biomarkers for the early detection of endometrial and ovarian malignancy in cancer-unaffected HNPCC germline mutation carriers. Further studies of other genetic changes in normal and premalignant precursor lesions are needed.

Introduction

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disorder which is characterized by an early onset of colorectal cancer, and is associated with extracolonic tumors of the endometrium, ovary, stomach, small bowel, renal pelvis, and ureter [1]. Endometrial cancer is the second most common malignancy in female HNPCC patients 2, 3, 4, 5, 6, and ovarian cancer is also a frequent component of this syndrome 3, 4, 5.

Previous studies have identified and characterized mismatch repair deficiency as the molecular basis of HNPCC. The culprit germline mutation in HNPCC involves one of at least four genes which participate in DNA mismatch repair, namely, hMLH1, hMSH2, hPMS1, and hPMS2 [1]. Germline mutations in hMLH1 and hMSH2 account for the majority (more than 70%) of HNPCC families identified to date, whereas hPMS1 and hPMS2 mutations are thought to account for only rare families. Microsatellite instability (MSI) is considered to be a marker of the defective mismatch repair mechanisms. In fact, MSI has been reported in essentially all of the tumors that comprise the HNPCC phenotype [1].

Aaltonen et al. found MSI in 8 of 14 (57%) colorectal adenomas from HNPCC patients, and concluded that studying colon adenomas for MSI may be useful in the diagnosis of HNPCC, especially in kindreds without known germline mutations [7]. This raises the question of whether other tissues may show evidence of MSI. The clinical significance of this issue stems from the perspective that identification facilitates early detection and potentially, the prevention of cancer in high-risk individuals. However, no investigations to date have been focused on the analysis of MSI and other genetic changes in histologically normal epithelia of endometrium and ovary in high-risk individuals from HNPCC kindreds. To investigate whether there may be genetic changes which are useful as molecular markers of susceptibility to endometrial and ovarian cancers, we evaluated MSI and MLH1 and MSH2 expression in both histologically normal epithelia and cancers of the endometrium and ovary in individuals belonging to HNPCC families.

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Patients and Tissue Samples

Seven unrelated HNPCC families, referred to the Creighton University/Hereditary Cancer Institute, constituted the family resource for this investigation. Of these families, the characteristic mutations of the faulty genes have been identified at the base pair level in 4 kindreds and detected by protein truncation analysis in 3 kindreds. All gene mutation analyses were performed by OncorMed (Gaithersburg, MD, USA) as previously described 8, 9. Twenty histologically normal epithelia (12

MSI Analysis

We have used 17 microsatellite loci including mono-, di-, tri-, and tetranucleotide repeats to investigate MSI in a sample of 20 normal epithelia and 8 cancers obtained from HNPCC family members.

In normal epithelia of endometrium and ovary, some of which were from individuals with germline mutations in hMLH1 or hMSH2, 8 (40%) cases showed MSI at one microsatellite locus, but there was no case that exhibited MSI in at least 3 loci (Table 1). On the other hand, MSI was observed in at least 3 loci

Discussion

An increased risk for endometrial and ovarian cancers in HNPCC family members is well established 2, 3, 4, 5. Dunlop et al. have demonstrated that women with germline mutations in hMLH1 or hMSH2 have up to a 50% higher risk to age 70 of endometrial cancer compared to colorectal cancer [6]. These data clearly indicate that gynecologic cancers play an important role in HNPCC family members [14]. However, early molecular events in the development of HNPCC gynecologic cancers are not well

Acknowledgements

This work was financially supported by a grant from the Health Future Foundation.We thank Lisa Linder-Stephenson, Stephen Kelly, Denise Hoover, Susan Slominski-Caster, and Yulia Kinarsky for excellent technical assistance. We are grateful to Beth Karr for collecting specimens.

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