Lluis Catasus

hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.

Endometrial carcinoma: pathology and genetics

In the Western world, endometrial carcinoma is the most common malignant tumour of the female genital tract and the fourth most common cancer in women after carcinomas of breast, colorectum, and lung. The annual incidence has been estimated at 10-20 per 100 000 women. In the United States, endometrial carcinoma accounts for approximately 6000 deaths per year. Two different clinicopathological subtypes are recognised: the oestrogen-related (type I, endometrioid) and the non-oestrogen related (type II, non-endometrioid). The clinicopathological differences are parallelled by specific genetic alterations, with type I showing microsatellite instability and mutations in PTEN, PIK3CA, K-Ras, and CTNNB1 (beta-catenin), and type II exhibiting p53 mutations and chromosomal instability. This article reviews the genetic changes of endometrial carcinogenesis in the light of morphological features of the tumours and their precursors.

Microsatellite instability in endometrial carcinomas: clinicopathologic correlations in a series of 42 cases.

Instability at microsatellite repeat sequences (MI) has been observed in endometrial carcinomas (EC) arising sporadically or in association with the hereditary colon cancer syndrome. However, the clinical and pathological features of the EC with MI have not been characterized. DNA of 42 patients with EC was extracted from blood and from fresh-frozen and paraffin-embedded tumor tissue. Microsatellite loci on chromosomes 4, 5, 10, 12, 17, and 18 were amplified by polymerase chain reaction. MI was defined by a mobility shift in the tumor DNA as compared with normal DNA. Results were correlated with the clinical and pathological features of the tumors. MI at three or more loci was detected in 12 of 42 cases (28%). There were no significant differences between EC with and without MI with regard to age of presentation, stage, evidence of estrogenic stimulation, mucinous differentiation, estrogen receptor, c-erbB2, or p53 immunostaining. However, MI was more frequent in endometrioid (11/33, 33.3%) than in nonendometrioid (1/9, 11%) carcinomas. Only one papillary serous carcinomas showed MI. MI was found in one of two cases of endometrial hyperplasia adjacent to EC. It was concluded that MI is a common genetic abnormality of endometrial carcinoma and appears to be more frequent in endometrioid than in nonendometrioid tumors.

K-ras mutations in endometrial carcinomas with microsatellite instability.

K‐ras mutations are known to occur in hyperplasias and carcinomas of the endometrium. No clear correlation has been found yet between K‐ras mutations and microsatellite instability (MI) in these lesions. Fifty‐eight endometrial carcinomas (ECs) and 22 endometrial hyperplasias (EHs) were analysed for K‐ras mutation by single‐strand conformational polymorphism analysis (SSCP), restriction analysis, and DNA sequencing. MI status had been established previously at five dinucleotide loci and was reconfirmed with markers BAT‐25 and BAT‐26 by SSCP. K‐ras mutations were detected in 11 ECs (18.9%). All 11 tumours were endometrioid carcinomas. K‐ras mutations were more frequent in MI‐positive (6/14, 42.8%) than in MI‐negative tumours (5/44, 11.3%) (p=0.017). Methylation‐related transitions were detected in five of the six MI‐positive tumours but in only one of the five MI‐negative carcinomas. K‐ras mutation was identified in only one atypical EH (1/22, 4.5%); in this case, the EH co‐existed with EC and both lesions exhibited MI. The results support a close relationship between K‐ras mutations and the phenomenon of MI in endometrial carcinomas. The frequent occurrence of methylation‐related transitions in these tumours may indicate a cause–effect relationship with the altered methylation status which has been described in association with MI. Copyright

PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters

Mutations of the oncogene PIK3CA occur frequently in endometrial carcinomas, but their prognostic significance is unclear. To determine the clinicopathological and molecular implications of these mutations, PIK3CA status was investigated in 109 endometrial (102 endometrioid and 7 mixed) carcinomas and the results were compared with clinicopathological parameters associated with prognosis. Tumors were also investigated for microsatellite instability and PTEN, β-catenin gene (CTNNB1), K-RAS, and B-RAF mutations. We found 35 PIK3CA somatic missense mutations in 32 (29%) endometrial carcinomas. Eighteen mutations occurred in exon 20 (kinase domain), and 17 in exon 9 (helical domain). Almost all mutated tumors were pure endometrioid adenocarcinomas. All tumors with PIK3CA mutations exhibited myometrial invasion (P=0.032). Lymphovascular invasion was found more frequently in mutated (28%) than nonmutated carcinomas (18%). Histological grade varied significantly according to the location of the PIK3CA mutations whether in exon 9 or exon 20 (P=0.033). The frequency of exon 9 mutations was higher in grade 1 carcinomas (57%) than in grade 2 (29%) or grade 3 (14%) tumors. Conversely, mutations in exon 20 were more common in grade 3 (60%) than in grade 2 (20%) or grade 1 (20%) carcinomas. None of the tumors confined to the endometrium (stage IA) had PIK3CA mutations. Furthermore, whereas 64% of adenocarcinomas with exon 9 mutations had invaded ≤½ of the myometrial thickness (stage IB), 73% of tumors with exon 20 mutations had either deeper myometrial invasion (stage IC) or cervical involvement (stage II) (P=0.045). PIK3CA mutations coexisted with microsatellite instability and mutations in PTEN, CTNNB1, K-RAS, and B-RAF genes. These results favor that PIK3CA mutations are associated with myometrial invasion and, moreover, that tumors harboring PIK3CA mutations in exon 20 are frequently high-grade, deeply invasive endometrial carcinomas that tend to exhibit lymphovascular invasion.

Concomitant PI3K–AKT and p53 alterations in endometrial carcinomas are associated with poor prognosis

The status of p53 and the phosphatidylinositol 3-kinase–AKT (PI3K–AKT) signaling pathway was investigated in 132 endometrial carcinomas, including endometrioid endometrial carcinomas, non-endometrioid endometrial carcinomas, and mixed endometrioid adenocarcinomas-non-endometrioid adenocarcinomas. Results were compared with the clinicopathologic parameters associated with prognosis, patients’ follow-up, and other genetic alterations found frequently in these tumors. Molecular genetic differences between low-grade and high-grade endometrioid adenocarcinomas were encountered; ie, PIK3CA mutations were detected in 26 and 34% of cases, respectively. We found p53 alterations in only 17% of high-grade endometrioid adenocarcinomas. In contrast, non-endometrioid adenocarcinomas had a higher frequency of p53 alterations (54%), PIK3CA mRNA overexpression (45%), and exon 20 PIK3CA mutations (21%). In the mixed endometrioid adenocarcinomas-non-endometrioid adenocarcinomas, the most frequent alterations were p53 (50%) and PIK3CA (44%) mutations, followed by PTEN mutations (38%). In some cases, p53 and PIK3CA alterations coexisted, but they rarely coexisted with the PTEN mutations. Our findings suggest that the PIK3CA mutations are frequent events in endometrial carcinomas of any histological type. However, location of the PIK3CA mutations, either in exon 9 or exon 20, varies significantly according to the histologic grade and type of carcinoma. Carcinomas with exon 20 PIK3CA mutations or PIK3CA mRNA overexpression were often high-grade carcinomas associated with myometrial invasion; in contrast, tumors that carried exon 9 mutations were more likely to be low-grade carcinomas. The Kaplan–Meier analysis suggested that p53 alterations (strong immunoexpression or mutations) conferred a worse prognosis (P=0.000). Although alterations in the PI3K–AKT signaling pathway alone did not influence overall survival, patients with deregulated PI3K–AKT pathway (PIK3CA and/or PTEN alterations) and p53 alterations had shorter survival (P=0.000) than patients with only p53 alterations. Such a relationship was lost when we considered exon 9 PIK3CA mutations. Our results contribute to further characterize the molecular genetic model for endometrial carcinogenesis.

Microsatellite instability, MLH‐1 promoter hypermethylation, and frameshift mutations at coding mononucleotide repeat microsatellites in ovarian tumors

Microsatellite instability (MI) is frequent in endometrial carcinomas (ECs), but its occurrence in ovarian tumors is uncertain. Microsatellite instability positive ECs frequently are associated with frameshift mutations in coding mononucleotide tracts in IGFIIR, BAX, hMSH6, and hMSH3.

Microsatellite instability and immunostaining for MSH‐2 and MLH‐1 in cutaneous and internal tumors from patients with the Muir–Torre syndrome

Background:  Muir‐Torre syndrome (MTS) is characterized by the co‐existence of sebaceous gland tumors of the skin and internal malignancies. Currently, MTS is regarded as a variant of the hereditary non‐polyposis colon cancer syndrome (HNPCC). Both MTS and HNPCC are secondary to germline mutations in DNA mismatch repair genes (mainly MSH‐2 and MLH‐1).

Repression of E-cadherin by SNAIL, ZEB1, and TWIST in invasive ductal carcinomas of the breast: a cooperative effort?

It has been suggested that down-regulation of E-cadherin in invasive breast ductal carcinomas is mediated by the aberrant expression of several of its transcriptional repressors, but their inhibitory role and clinical importance are not yet well established. We investigated gene and protein expression patterns of the E-cadherin repressors SNAIL, ZEB1, and TWIST in relation to clinicopathologic parameters, in a series of 88 patients with invasive breast ductal carcinomas. Up-regulation of SNAIL messenger RNA (P = .008) and down-regulation of TWIST (P = .022) were associated with triple-negative tumors, whereas ZEB1 gene expression was more frequent in hormone-positive tumors (P = .004). Loss of E-cadherin was found in 19% of the tumors, but it did not correlate with aberrant expression of any of the repressors investigated herein. Nonetheless, we found that ZEB-1 protein overexpression inversely correlated with high tumor grade (P = .018), nuclear grade (P = .002), and presence of lymph nodes (P = .001), and these data were consistent with the gene expression data for ZEB1. Clinically, down-regulation of ZEB1 messenger RNA was associated with poor overall survival (P = .011) and disease-free survival (P = .053), whereas patients with TWIST negative tumors had a worse overall survival (P = .008) and disease-free survival (P = .006). Our data indicate that deregulation of TWIST is somehow important in the aggressiveness of triple-negative carcinomas and poor patient outcome, whereas down-regulation of ZEB1 seems to play a role in tumor spread, metastases, and poor survival.

Prognostic significance of FOXL2 mutation and mRNA expression in adult and juvenile granulosa cell tumors of the ovary

Recently, mutation of the FOXL2 gene has been consistently identified in adult granulosa cell tumors of the ovary. The purpose of this study is to investigate whether the FOXL2 mutation and mRNA expression have a role in the pathogenesis of juvenile and adult granulosa cell tumors and influence tumor progression. Thirty-four adult granulosa cell tumors and 20 juvenile granulosa cell tumors were examined for the presence of the FOXL2 (C402G) mutation. Expression levels were studied by quantitative PCR and immunohistochemistry. We found that FOXL2 (C402G) mutation was present in 19/27 (70%) of the adult type tumors but in none of the juvenile granulosa cell tumors (0/18). No correlation was encountered between the presence of FOXL2 mutation and various clinicopathologic parameters except for the presence of a different sex-cord component, which was more frequently found in the subgroup of wild-type adult granulosa cell tumors than in the mutated tumors. Patients with tumors harboring the FOXL2 (C402G) mutation had a worse disease-free survival than those with the wild-type gene. Expression levels of FOXL2 mRNA had an impact on disease-free survival in both adult and juvenile granulosa cell tumors. We also found that the mutated tumors had a higher immunohistochemical expression of the FOXL2 protein, and there was a linear correlation between mRNA and immunohistochemical FOXL2 expression in both adult and juvenile granulosa cell tumors. Patients with juvenile granulosa cell tumors and higher FOXL2 protein expression had worse overall survival and disease-free survival than those with negative or weakly immunoreactive tumors. Our data suggest that FOXL2 mutation and mRNA expression are of prognostic importance in both adult and juvenile granulosa cell tumors.