Among multiple genetic pathways involved in endometrial cancer (EC), the mutator pathway is characterized by defective mismatch repair (MMR) causing microsatellite instability (MSI+). Inactivation of MMR genes allows elevation of mutation rates in key target genes involved in important cellular pathways, providing a selective growth advantage. Our aim was to investigate apoptotic and growth regulatory target genes in young endometrioid adenocarcinoma patients and their association with stepwise neoplastic progression through distinct stages of hyperplasia and cancer. Screening of 184 ECs revealed 38 microsatellite high (MSI-H), 10 microsatellite low (MSI-L) and 136 microsatellite stable (MSS) tumors. We observed somatic frameshift mutations in the coding region repeats of the target genes in 12/38 MSI-H tumors (T) and in 3/8 of available associated hyperplasias (HY). Mutations were detected in FAS (T=1, HY=1), BAX (T=6, HY=1), CASP5 (T=2) and IGFIIR (T=3, HY=1) genes. None of the MSI-L or MSS tumors showed alterations in these coding repeats. Increased mutation frequency in apoptotic and growth regulatory genes demonstrated a significant relationship with advancing tumor grade (p=0.02) by Fisher's exact test. Furthermore, a significant trend was found by Bartholomew's test (P<0.05) for the apoptotic pathway and close to significant (p approximately 0.06) for the overall mutation status for both pathways combined. Our results suggest that genes implicated in apoptosis may serve as targets in the progression of MSI+ EC in young patients.

Differential methylation is an important epigenetic control mechanism, which has been implicated in the development of a variety of cancers. Methylation of promoter regions of normally unmethylated tumor suppressor genes leads to transcriptional inactivation and ultimately to tumor formation. We hypothesized that epigenetic inactivation of adenomatous polyposis coli (APC), a key player in the suppressor pathway, may contribute to the development of endometrial cancer. We investigated APC methylation in endometrial adenocarcinoma specimens obtained from a series of patients (n = 114) and compared methylation profiles with microsatellite instability (MSI+) status. DNA microdissected from formalin-fixed, paraffin-embedded matched normal and tumor specimens, and a subset of associated endometrial hyperplasia was subjected to methylation-specific PCR of the APC promoter 1A region. Tumor-specific hypermethylation of APC with corresponding unmethylated normal endometrial tissue occurred in 43% (17 of 40) of MSI+ cases (P = 0.0007) and 16% (12 of 74) of microsatellite stable cases (P = 0:04). Interestingly, tumor tissue was unmethylated with normal tissue displaying APC methylation in 4% (5 of 114, 2MSI+ and 3 microsatellite stable) of cases. Endometrial cell lines AN3CA, RL95-2, and HEC-1B all displayed exclusive methylation of promoter 1A, and treatment of the AN3CA cell line with the demethylating agent 5-aza-2'-deoxycytidine exhibited re-expression of APC as confirmed by RT-PCR analysis. Our results demonstrate APC methylation in endometrial cancer for the first time and show that APC hypermethylation occurs at an increased frequency in MSI+ endometrial tumors (P = 0.01).

Microsatellite instability (MSI) is observed in a subset of endometrial cancers (ECs) and is attributed to defects in mismatch repair. Mismatch repair deficiency allows for accumulation of mutations in the coding repeats of key target genes, which may be involved in the initiation and progression of MSI+ EC. We examined genes implicated in DNA repair pathways in 38 MSI-high (MSI-H), 10 MSI-low, 25 microsatellite stable ECs, and a selected panel of associated premalignant hyperplasias. Genetic alterations were correlated to histopathological data, including tumor grade and stage. Somatic frameshift mutations were observed in hMLH3, hMSH3, hMSH6, CHK1, and BAX genes in MSI-H endometrial hyperplasias and cancers, whereas mutations in ATR and CDC25C were observed only in MSI-H ECs. Increased mutation frequency in DNA damage response pathway genes including ATR, CHK1, and BAX demonstrated a significant trend with advancing tumor grade (P < 0.05). Our observations of the same mutations at short coding mononucleotide repeats in both premalignant lesions and tumors and association of increased frequency of mutation accumulation with advancing tumor grade suggest that these alterations may play a role in the development and progression of MSI+ EC.

Mismatch repair (MMR) gene mutations cause hereditary nonpolyposis colorectal cancer (HNPCC), a common form of familial colorectal cancer. Among MMR genes, germline MSH6 mutations are often observed in HNPCC-like families with an increased frequency of endometrial cancer. We have previously shown that a proportion of women affected with double primary cancers of the colorectum and endometrium carry germline MSH2 or MLH1 mutations and, thus, belong to HNPCC families. In this study, we have investigated the specific contribution of MSH6 defects to such double primary patients. By sequence analysis of the entire coding region of MSH6 , three putative missense mutations were identified in patients with atypical family histories that do not meet HNPCC criteria. Moreover, one of these mutations, a novel substitution Arg901His, was found in a patient previously shown to carry a truncating germline MLH1 mutation. Thus, MSH6 mutations are likely to contribute to the etiology of double primary cancers of the colorectum and endometrium.