Molecular pathology of endometrial hyperplasia and carcinoma.

Four different genetic abnormalities may occur in endometrioid adenocarcinomas of the endometrium (mircosatellite instability and mutations in the PTEN, k-RAS and beta-catenin genes), whereas nonendometrioid carcinomas of the endometrium often have p53 mutations and loss of heterozygosity on several chromosomes. Occasionally, a nonendometrioid carcinoma may develop as a result of dedifferentiation of a preexisting endometrioid carcinoma; in such a case, the tumor exhibits overlapping clinical, morphologic, immunohistochemical, and molecular features of the 2 types. The insaturation of microsatellite instability in endometrial carcinogenesis seems to occur late in the transition from complex hyperplasia to carcinoma, and it is preceded by progressive inactivation of MLH-1 by promoter hypermethylation. Moreover, the endometrioid adenocarcinomas that exhibit microsatellite instability show a stepwise progressive accumulation of secondary mutations in oncogenes and tumor suppressor genes that contain short-tandem repeats in their coding sequences. Mutations in the PTEN and k-RAS genes are also frequent in endometrioid adenocarcinomas of the endometrium, particularly in the tumors that exhibit microsatellite instability, whereas beta-catenin mutations do not seem to be associated with such a phenomenon.     

beta-Catenin expression pattern, beta-catenin gene mutations, and microsatellite instability in endometrioid ovarian carcinomas and synchronous endometrial carcinomas.

beta-Catenin gene mutations and microsatellite instability (MI) have been reported in endometrioid ovarian carcinomas. In colon but not endometrial cancer, beta-catenin gene mutations are associated with a replication error phenotype and MI. In this study the authors investigate whether beta-catenin mutations and MI are two independent oncogenic pathways in endometrioid ovarian carcinomas. They also evaluate the usefulness of these molecular markers in determining the primary origin of simultaneous tumors in the ovary and endometrium. This study was performed on 26 patients diagnosed with primary endometrioid ovarian carcinoma, five of whom also had pathologically diagnosed primary synchronous endometrioid endometrial carcinoma. Immunohistochemical and molecular analyses indicated that there were 25 primary ovarian tumors with four primary synchronous endometrial cancers and one ovarian metastasis of a primary endometrial carcinoma. All studies were performed on formalin-fixed, paraffin-embedded tissue samples. The beta-catenin expression pattern (nuclear vs. membranous) was analyzed immunohistochemically. Mutations in exon 3 of the beta-catenin gene were studied by polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing. MI status was established by studying BAT-26 and BAT-25 mononucleotide repeats. In the group with 21 single ovarian tumors, 18 (85%) had beta-catenin nuclear expression, eight (38%) had beta-catenin gene mutations (always associated with beta-catenin nuclear expression), and four (19%) had MI. Only one case (5%) had both beta-catenin gene mutations and MI. The mutations affected one of the serine/threonine residues targeted for phosphorylation by glycogen synthase kinase-3beta or adjacent residues. At codon 32, a GAC-to-TAC (D32Y) change was found; at codon 33, two TCT-to-TGT (S33C) changes were found; at codon 37, three TCT-to-TTT (S37F) changes and one TCT-to-TGT (S37C) change were found; and, lastly, one ACC-to-GCC change at codon 41 (T41A) was detected. Four of the 25 endometrioid ovarian carcinomas (16%) had an associated synchronous endometrial carcinoma. There was a higher percentage of beta-catenin mutations (n = 3, 75%) in synchronous ovarian carcinomas than in single ones, although with a similar percentage of MI (n = 1, 25%). beta-catenin mutations were S37C in two cases and D32G in one. One of the four endometrial carcinomas showed an S33C beta-catenin mutation, and two carcinomas had MI. None of the four tumors had both beta-catenin gene mutation and MI. beta-catenin gene mutations were always associated with a nuclear beta-catenin expression pattern, whereas MI was associated with a membranous pattern. In one patient both the ovarian and the endometrial carcinomas had beta-catenin gene mutations, in another patient both tumors showed MI, whereas in the remaining two patients the ovarian carcinomas showed beta-catenin gene mutations and the endometrial carcinomas showed MI. To summarize, the results of this study suggest that beta-catenin mutations and MI could represent two independent pathways in endometrioid ovarian carcinomas because they occur simultaneously very infrequently (in 5% of these cases). beta-catenin mutations are always associated with a nuclear beta-catenin expression pattern, whereas cases with a replication error -plus phenotype showed no abnormal beta-catenin subcellular localization. The study of the beta-catenin expression pattern, beta-catenin mutations, and MI, together with conventional clinicopathologic findings, could aid in distinguishing between the metastatic or independent origin of simultaneous endometrioid ovarian and endometrial carcinomas. Tumors with identical immunohistochemical and molecular features should therefore be considered to have a common origin.     

CTNNB1 mutations and beta-catenin expression in endometrial carcinomas

Mutations in the beta-catenin gene (CTNNB 1) with abnormal nuclear accumulation of beta-catenin have recently been identified in endometrial carcinoma (EC). Their relationship with microsatellite instability (MI) is unclear. It has been suggested that matrix metalloproteinase-7 (MMP-7) and cyclin D1 (cD) genes are targets for beta-catenin activation. DNA from 73 patients with EC was obtained from tumor and normal tissue (59 endometrioid and 14 nonendometrioid). CTNNB 1 mutations in exon 3 were assessed by single-strand conformation polymorphism and DNA sequencing. The results were correlated with immunostaining for beta-catenin, MMP-7, and cD. Three (CA)n repeats and mononucleotide tracts BAT 25 and BAT 26 had been previously used for MI analysis. CTNNB1 mutations were identified in 15 ECs (20.5%), all of them endometrioid carcinomas (15 of 59; 25.4%). They occurred in 6 of 19 MI-positive ECs (31.5%) and in 9 of 54 MI-negative ECs (16.6%). Eleven of the 15 CTNNB 1-mutated ECs showed beta-catenin nuclear immunostaining (P <.05). MMP-7 expression (>50% cells) was observed in 23 ECs, with 7 of these showing CTNNB 1 mutations. Significant expression of cD (>50% cells) was detected in 8 ECs, with 5 of these exhibiting CTNNB 1 mutations (P <.05). The results confirm that beta-catenin plays a role in endometrial carcinogenesis, particularly in endometrioid carcinomas. The results also suggest that MMP-7 and particularly cD may be targets of beta-catenin activation in ECs.     

PTEN mutations do not cause nuclear beta-catenin accumulation in endometrial carcinomas

PTEN: and beta-catenin mutations constitute the predominant genetic alterations in endometrioid carcinomas of the endometrium. PTEN encodes a dual-specificity phosphatase with lipid phosphatase and protein tyrosine phosphatase activities that regulate both apoptosis and interactions with the extracellular matrix. Recent studies have associated PTEN mutations with tumorigenesis of prostate carcinoma via the Wnt signaling pathway, leading to nuclear beta-catenin accumulation. To elucidate the potential interaction of PTEN and beta-catenin in endometrial cancer, we performed mutation analyses of the entire PTEN gene and of exon 3 of the beta-catenin gene that is most frequently targeted by mutations. A total of 82 endometrial carcinomas comprising 62 type I endometrioid carcinomas and 20 type II high-grade carcinomas were investigated. In addition in a subset of 22 carcinomas, the intracellular beta-catenin distribution was analyzed by immunohistochemistry. Overall, 20 (24.4%) of 82 tumors revealed mutations in the PTEN gene, and 16 (19.5%) of 82, in the beta-catenin gene. Six tumors (7.3%) showed mutations in both the PTEN and beta-catenin gene. Mutations were mainly detected in endometrioid carcinomas of the endometrium. As expected, a striking nuclear accumulation of beta-catenin could be shown in tumors with beta-catenin mutations. In the vast majority of tumors with PTEN mutations, a regular staining pattern of the cytoplasmic and membranous compartments was found. We therefore conclude that, in contrast to prostate cancer, mutations in the PTEN gene seem not to affect cellular distribution of the beta-catenin protein in endometrial carcinomas.     

PIK3CA gene mutations in endometrial carcinoma: correlation with PTEN and K-RAS alterations

Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway are common in endometrial carcinoma. Inactivation of the tumor suppressor gene PTEN leads to a constitutively active PI3K pathway, which plays a role in the early steps of endometrial tumorigenesis. Other alterations in the PI3K/AKT pathway are mutations in the PIK3CA gene, which encode the p110alpha catalytic subunit of PI3K. PIK3CA mutations cluster to the helical (exon 9) and the kinase (exon 20) domains of the gene. In endometrial carcinomas, PIK3CA mutations have been found to coexist frequently with PTEN mutations, but it is not clear whether they occur in cells with monoallelic or biallelic inactivation of PTEN. In the present study we have evaluated PIK3CA mutational status in a series of 33 endometrial carcinomas, previously screened for microsatellite instability and mutations in PTEN, K-RAS, and CTNNB-1. The tumors were also evaluated for loss of heterozygosity on 10q23 and hypermethylation of the promoter region of PTEN/psiPTEN to assess the monoallelic or biallelic inactivation status of PTEN. PIK3CA mutations were detected in 8 (24%) of the 33 cases. Seven mutations were located in exon 20 and 1 in exon 9. PTEN alterations were found in 19 cases (57%). Biallelic inactivation of PTEN was demonstrated in 11 tumors, whereas 8 tumors exhibited alteration in only 1 of the 2 alleles. PIK3CA mutations coexisted with monoallelic alterations of PTEN in 4 cases (2 mutations and 2 allelic imbalances), with biallelic PTEN inactivation in 1 case (mutation and promoter methylation), and 3 tumors showed PIK3CA mutations in association with wild-type PTEN. PIK3CA mutations did not correlate with microsatellite instability or mutations in CTNNB-1. However, PIK3CA and K-RAS mutations (8 cases) were mutually exclusive alterations. In summary, the results confirm that PIK3CA mutations are frequent in endometrial carcinoma and support the hypothesis that PIK3CA mutations may have an additive effect to PTEN monoallelic inactivation in endometrial carcinoma.     

Molecular genetic alterations in endometrioid carcinomas of the ovary: similar frequency of beta-catenin abnormalities but lower rate of microsatellite instability and PTEN alterations than in uterine endometrioid carcinomas

Endometrioid carcinomas of the ovary closely resemble their uterine counterparts. It has been suggested that the former tumors have the same molecular alterations (microsatellite instability [MSI], PTEN, and beta-catenin) described in endometrioid carcinomas of the uterus. We analyzed 55 ovarian carcinomas, including 22 endometrioid, 18 clear cell, and 15 mixed types. MSI was detected in 5 of 39 cases (13%). MLH1 promoter hypermethylation was identified in 2 of the 5 MSI-positive tumors. PTEN was mutated in 5 of 54 cases (9%); of these, 3 had MSI and exhibited frameshift mutations in short-coding mononucleotide repeats. Beta-catenin nuclear expression was detected in 11 of 54 cases (20%) by immunostaining; of these, 7 exhibited CTNNB1 gene mutations. These alterations were found more frequently in endometrioid carcinomas than in tumors of the other 2 groups. Among the former tumors, MSI was detected in 3 of 17 cases (17.5%); PTEN mutations, in 3 of 21 (14%); and beta-catenin, in 8 of 21 (38%). The molecular alterations were found more often in tumors associated with endometriosis than in tumors without endometriosis. Six endometrioid tumors demonstrating matrix metalloproteinase-7 (MMP-7) immunoreactivity with nuclear accumulation of beta-catenin had good outcomes, in contrast to poor outcomes in 7 of 9 predominantly nonendometrioid tumors demonstrating expression of MMP-7 only. We found a similar frequency of beta-catenin abnormalities but lower rates of MSI and PTEN alterations than in uterine endometrioid carcinomas. Alterations in beta-catenin and PTEN genes, as well as MSI, are frequent in low-stage ovarian carcinomas of endometrioid type that have a favorable prognosis.     

Expression of cyclin D1 in normal, metaplastic, hyperplastic endometrium and endometrioid carcinoma suggests a role in endometrial carcinogenesis

CONTEXT: Endometrioid carcinoma is often preceded by characteristic histopathologic lesions known as endometrial hyperplasia. Estrogen appears to be involved in the development of endometrioid carcinoma. Other mechanisms of endometrial carcinogenesis include mutations in p53 and PTEN tumor suppressor genes and overexpression of cyclin D1. However, the pattern of cyclin D1 expression is not well defined in normal, hyperplastic, neoplastic, and metaplastic endometrium. DESIGN: Cyclin D1 immunohistochemical analysis was used to evaluate 108 fixed, paraffin-embedded endometrial biopsy specimens and uterine resections obtained from 108 patients. Specimens included proliferative and secretory endometria, simple and complex hyperplastic lesions, and endometrioid adenocarcinoma. Normal and metaplastic surface epithelia were also evaluated independently of glandular morphologic features. RESULTS: Cyclin D1 was significantly overexpressed in glands with complex hyperplasia and endometrioid adenocarcinoma compared with proliferative or secretory endometrium and simple hyperplasia. Significant overexpression was also noted in papillary, syncytial, and squamous metaplasias compared with normal surface epithelium or epithelium with tubal metaplasia. CONCLUSION: Overexpression of cyclin D1 increases from normal endometrium to hyperplasia and carcinoma, suggesting that it may play a role in endometrial carcinogenesis. Overexpression of cyclin D1 in endometrial glands was independent from overexpression of cyclin D1 in surface metaplastic epithelium.     

β- And γ-catenin expression in endometrial carcinoma. Relationship with clinicopathological features and microsatellite instability

The activation of the adenomatous polyposis coli (APC)/beta-catenin/T-cell factor (Tcf) pathway due to beta-catenin gene mutation has been recently implicated in the development of some endometrial carcinomas. beta- and gamma-catenin are structurally and functionally related molecules that participate in cell adhesion and signal transduction. Nuclear accumulation of beta- and gamma-catenin have been related to the activation of the APC/beta-catenin/Tcf pathway. In this study, we investigate the immunohistochemical expression pattern (nuclear vs membranous) of beta- and gamma-catenin in 40 endometrial carcinomas and their correlation with clinicopathological features and microsatellite instability (MI) status. MI was detected at three or more loci in 12 tumors: 11 were endometrioid and one was non-endometrioid. Nuclear catenin expression was found in 13 carcinomas: ten carcinomas had nuclear beta-catenin expression and three carcinomas had nuclear gamma-catenin expression. The nuclear catenin expression pattern significantly correlated with the histological type, International Federation of Gynecology and Obstetrics (FIGO) grade, and the presence of a second neoplasm. Nuclear catenin expression was always observed in low-grade endometrioid carcinomas; it was also more frequently associated with a second carcinoma. No correlation was observed between the catenin expression pattern and the level of myometrial infiltration, stage, associated endometrial hyperplasia, the existence of a source of estrogenic stimulation, and MI. However, four of 13 endometrioid carcinomas in this series had both catenin nuclear expression and MI. These data suggest that at least two different neoplastic pathways can lead to endometrial carcinomas with an endometrioid phenotype. In one, MI would be a key event, while in the other, the APC/beta-catenin/Tcf signaling pathways could be activated. Probably, in some cases, both pathways could simultaneously occur.     

Mutation of ?-catenin gene in endometrial cancer but not in associated hyperplasia

Endometrial hyperplasia is a recognized effect of excessive or unopposed estrogen stimulation and is considered to be a precancerous condition of endometrial adenocarcinoma. We have previously shown that the subcellular localization of beta-catenin in the human endometrium is changed according to cell proliferation, suggesting a role of intercellular transduction in cell-growth control in human endometrium, not only in the physiological condition but also in the carcinogenic endometrium. In the present study, to clarify at which stage of endometrial carcinogenesis molecular alteration of the beta-catenin gene occurs, we analyzed the subcellular localization of beta-catenin by immunohistochemistry, and we analyzed exon 3 of the beta-catenin gene, in 25 patients - with endometrial hyperplasia and 20 patients with endometrial cancers associated with endometrial hyperplasia, digesting DNA from the cancer and hyperplasia parts, separately. Fourteen of the 25 (56.0%) endometrial hyperplasia samples, 12 (60.0%) endometrial cancers, and 11 (55.0%) associated hyperplasias of the 20 endometrial cancers associated with hyperplasia showed nuclear localization of beta-catenin. Mutation in exon 3 of the beta-catenin gene was found in 2 of the 20 endometrial cancer samples; however, it was not found in the 25 endometrial hyperplasias or the 20 associated hyperplasias. The results suggest that molecular alteration of the beta-catenin gene occurs in atypical hyperplasia or cancer, rather than in simple or complex hyperplasia without atypia, during endometrial carcinogenesis.     

Expression of nm23 in normal, hyperplastic and neoplastic endometrial tissues

This study evaluated the expression of nm23 in curettage specimens from 63 cases of normal, hyperplastic and neoplastic endometrial tissues by immunohistochemistry. The histological diagnoses were as follows: normal proliferative (N = 5) or secretory (N = 5), simple hyperplasia (N = 11), complex hyperplasia (N = 9), atypical hyperplasia (N = 8) and adenocarcinoma (N = 25), consisting of endometrioid adenocarcinoma (N = 15), clear cell (N = 7) and serous papillary adenocarcinoma (N = 3). There was no immunostaining for nm23 protein in the 10 cases of normal endometria and in the 28 cases of endometrial hyperplasia. In contrast, 52% of the adenocarcinomas displayed a cytoplasmic staining pattern which was moderate to strong. This difference was statistically significant (p < 0.0001, chi-square test). nm23 expression in curettage specimens had no predictive value for determining the FIGO stage in the hysterectomy specimens (p = 0.2709, chi-square test). No significant difference for nm23 immunoreactivity was found between the histologic subtypes of endometrial adenocarcinoma (endometrioid versus serous papillary and clear cell, p = 0.1413, chi-square test). In this study, there was no immunostaining of normal endometria or of endometrial hyperplasia (including atypical endometrial hyperplasia) to support the hypothesis that expression of the nm23 gene product is related to the development of endometrial adenocarcinoma. In contrast, nm23 expression was upregulated in many cases of endometrial adenocarcinomas irrespective of the histologic subtype.     

β-catenin、Glut-1和PTEN蛋白在子宫内膜样腺癌发生过程中的表达及意义

目的 通过分析子宫内膜上皮内瘤变(EIN)诊断、分类与子宫内膜增生WHO(2003)分类的关系,探讨β-catenin、Glut-1和PTEN蛋白在子宫内膜样腺癌发生过程中的表达及其意义.方法 根据EIN诊断及分类标准,对83例子宫内膜增生病例进行再分类.采用免疫组织化学SP法,对10例增殖期子宫内膜、83例子宫内膜增生及24例子宫内膜样腺癌组织中β-catenin、Glut-1及PTEN蛋白的表达进行检测.结果 (1)83例子宫内膜增生病例中共检出24例EIN病例,总检出率为28.9%(24/83).24例EIN病例中,来自复杂型不典型性增生16例(66.7%,16/24),但EIN的检出率与不典型增生的分级无明显关系(P＞0.05).(2)β-catenin蛋白在增殖期子宫内膜中呈正常表达,良性子宫内膜增生的异常表达率为10.2%(6/59),而EIN和子宫内膜样腺癌的异常表达率(50%,12/24;66.7%,16/24)分别明显高于良性子宫内膜增生(P＜0.01),但二者间的异常表达率差异无统计学意义(P＞0.05).(3)Glut-1蛋白在增殖期子宫内膜、良性子宫内膜增生组织中均呈低表达,而在EIN和子宫内膜样腺癌中的高表达率分别为58.3%(14/24)和70.8%(17/24),均显著高于增殖期子宫内膜及良性子宫内膜增生(P＜0.01),但二者高表达率间差异无统计学意义(P＞0.05).(4)PTEN蛋白在EIN病例中的失表达率(37.5%,9/24)与子宫内膜样腺癌(62.5%,15/24)、增殖期子宫内膜(2/10)及良性子宫内膜增生(28.8%,17/59)比较差异均无统计学意义(P＞0.05),但子宫内膜样腺癌的失表达率则明显高于增殖期子宫内膜及良性子宫内膜增生病例,其差异均具有统计学意义(P＜0.05).结论 β-catenin蛋白的异常表达和Glut-1蛋白高表达是子宫内膜样腺癌发生过程中的早期事件,二者在区别良性子宫内膜增生、EIN和子宫内膜样腺癌中可能是有用的免疫标志物.PTEN蛋白失表达是子宫内膜样腺癌发生过程中的极早期事件,但将其作为EIN病变的诊断性标志物并不恰当.     

Markers of proliferative activity are predictors of patient outcome for low-grade endometrioid adenocarcinoma but not papillary serous carcinoma of endometrium.

On the basis of pathogenesis, two types of endometrial cancer can be recognized. Type 1 endometrial carcinomas are relatively indolent tumors that develop after prolonged estrogen stimulation, on a background of endometrial hyperplasia. Type 2 endometrial carcinomas are aggressive tumors that are not associated with hyperplasia or estrogen excess. The aim of this study is to evaluate the prognostic significance of tumor proliferative activity in early-stage endometrial cancer by using mitotic index and immunostaining, comparing Type 1 (endometrioid) and Type 2 (papillary serous carcinoma) tumors. The mitotic index, MIB-1, and p53 immunostaining in 39 tumors from patients with low-grade Stage Ia or Ib endometrioid adenocarcinoma; as well as 23 tumors from patients with Stage I papillary serous carcinoma. In low-grade endometrioid adenocarcinoma, mitotic and MIB-1 indices were statistically significant independent prognostic indicators (P =.004 and P =.018, respectively), and both were strongly correlated with p53 expression (P =.01 and P =.006, respectively). The mean mitotic index was 5 mitoses/10 high-power fields, and mean MIB-1 index was 27.5%. There was no significant correlation between mitotic or MIB-1 indices and patient outcome or p53 expression in papillary serous carcinoma. The mean mitotic index was 31 mitoses/10 high-power fields, and mean MIB-1 index was 30.5% in these tumors. p53 expression and proliferative indices are strongly correlated in low-grade endometrioid adenocarcinoma. MIB-1 and mitotic indices are independent prognostic indicators in these tumors. Papillary serous carcinoma of endometrium is rapidly proliferative in tumors even at an early stage, and quantification of proliferative activity in these tumors does not allow prediction of patient outcome.     

Theories of endometrial carcinogenesis: a multidisciplinary approach.

Historical observations have suggested that endometrial carcinomas vary in histopathologic appearance and clinical features. More recent, systematic studies have provided epidemiologic, clinicopathologic, and molecular support for these observations. Specifically, studies suggest that the most common type of endometrial carcinoma, endometrioid adenocarcinoma, develops from endometrial hyperplasia in the setting of excess estrogen exposure and usually pursues an indolent clinical course. In contrast, a minority of endometrial carcinomas, best represented by serous carcinoma, do not seem to be related to estrogenic risk factors or elevated serum hormone levels, and these tumors seem to develop from atrophic rather than hyperplastic epithelium. We have proposed that serous carcinomas develop from "endometrial intraepithelial carcinoma," a lesion representing malignant transformation of the endometrial surface epithelium. Whereas endometrioid carcinoma and endometrial hyperplasia are associated with microsatellite instability and ras and PTEN mutations, serous carcinoma and endometrial intraepithelial carcinoma are associated with p53 mutations and abnormal accumulation of p53 protein. Based on these data regarding the pathogenesis of endometrioid and serous carcinoma, we have proposed a dualistic model of endometrial carcinogenesis incorporating a "classic" estrogen-driven pathway and an "alternative" pathway seemingly unrelated to hormones. It is hoped that further studies may permit the extension and modification of this model and that these advances will lead to improved diagnosis, management, and prevention.     

MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a cost-effectiveness study

The analytical algorithm of Lynch syndrome (LS) is increasingly complex. BRAF V600E mutation and MLH1 promoter hypermethylation have been proposed as a screening tool for the identification of LS. The aim of this study was to assess the clinical usefulness and cost-effectiveness of both somatic alterations to improve the yield of the diagnostic algorithm of LS. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed microsatellite instability and/or loss of mismatch repair (MMR) protein expression were studied. MMR germline mutations were detected in 57 cases (40 MLH1, 15 MSH2 and 2 MSH6). BRAF V600E mutation was assessed by single-nucleotide primer extension. MLH1 promoter hypermethylation was assessed by methylation-specific multiplex ligation-dependent probe amplification in a subset of 71 cases with loss of MLH1 protein. A decision model was developed to estimate the incremental costs of alternative case-finding methods for detecting MLH1 mutation carriers. One-way sensitivity analysis was performed to assess robustness of estimations. Sensitivity of the absence of BRAF mutations for depiction of LS patients was 96% (23/24) and specificity was 28% (13/47). Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared with BRAF study and germinal MLH1 mutation study. Somatic hypermethylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.     

Beta-catenin and E-cadherin expression patterns in high-grade endometrial carcinoma are associated with histological subtype.

Both beta-catenin and E-cadherin are epithelial cell adhesion molecules. In addition, beta-catenin is an important element of the Wnt signal transduction pathway, which has been implicated in embryogenesis and carcinogenesis, including the development of endometrial and ovarian endometrioid carcinomas. We hypothesized that the expression pattern of these two adhesion molecules may depend upon the histological subtype of endometrial carcinomas. Therefore, we compared the immunohistochemical expression of beta-catenin and E-cadherin in a set of uterine adenocarcinomas matched for high histologic grade, that is, poorly differentiated (International Federation of Gynecology and Obstetrics [FIGO] Grade III) uterine endometrioid carcinomas and uterine serous carcinomas. Seventeen FIGO Grade III endometrioid adenocarcinomas and 17 serous carcinomas were evaluated histologically and immunohistochemically with commercially available monoclonal antibodies against beta-catenin and E-cadherin. Nuclear expression of beta-catenin was observed in 8 of 17 (47%) endometrioid adenocarcinomas but in none of the serous carcinomas (P = .003). Moderate or strong E-cadherin expression was identified in 7 of 17 (41%) serous carcinomas as opposed to in only 1 of 17 (6%) endometrioid adenocarcinomas (P = .02). The majority of endometrioid adenocarcinomas showed strong beta-catenin expression coupled with weak E-cadherin expression; serous carcinomas did not exhibit a comparable trend. Our results indicate that the expression of beta-catenin and E-cadherin in high-grade endometrial cancers is strongly associated with histological subtype. These data provide further support for the distinct molecular profiles of endometrioid adenocarcinoma and serous carcinoma. Notably, differences in cell adhesion molecule expression could account for variations in patterns of tumor dissemination. The immunohistochemical staining pattern may also be useful for diagnostic purposes.     

Clinicopathological and molecular characteristics of endometrial neuroendocrine carcinomas reveal preexisting endometrial carcinoma origin

Endometrial neuroendocrine carcinoma is a rare disease with unknown clinicopathological and molecular characteristics. Therefore, we conducted the present study to elucidate the clinicopathological and molecular characteristics of endometrial neuroendocrine carcinoma, as compared to conventional endometrial carcinoma, and to determine the origin of the former. We analyzed 22 endometrial neuroendocrine carcinomas and 22 conventional endometrial neoplasia cases with respect to clinical, histological and genetic features. Of these, 21/22 neuroendocrine carcinoma cases were admixed carcinomas, with 15 admixed with endometrioid adenocarcinoma. Genetic analysis of hotspot mutations in 50 cancer-related genes revealed that the neuroendocrine carcinoma group carried mutations in PIK3CA (12/22 cases; 54%) and PTEN (8/22 cases; 36%), commonly encountered in endometrioid adenocarcinoma. Comparative statistical analysis of neuroendocrine carcinoma and conventional endometrial neoplasia cases showed a significant trend only in PIK3CA mutation. Moreover, in six mixed-type neuroendocrine carcinoma cases, macrodissection was used to separate the neuroendocrine carcinoma and endometrioid adenocarcinoma components for next-generation sequencing, which revealed several mutations common among the two. These findings suggest that endometrial neuroendocrine carcinoma could originate from conventional endometrial neoplasia, especially endometrioid adenocarcinoma.     

Abnormalities of E- and P-cadherin and catenin (beta-, gamma-catenin,and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia

Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumours. This study aimed to evaluate the immunoreactivity of E- and P-cadherin, beta- and gamma-catenin, and p120ctn in premalignant and malignant endometrial lesions and to correlate their membranous expression with clinicopathological features. In addition, we examined whether or not LOH and promoter hypermethylation of the CDH1 gene were associated with E-cadherin expression and clinicopathological variables. Finally, we studied the frequency of beta-catenin mutations in premalignant endometrial lesions. Immunohistochemical staining was performed in 21 atypical endometrial hyperplasias (AEHs), 95 endometrioid carcinomas (EECs), and 33 non-endometrioid carcinomas (NEECs). Reduced E-cadherin expression was observed in 57.8% of the cases, being more frequent in NEECs (87.1%, p = 0.001) and carcinomas of more advanced stage (85.7% of stage III-IV carcinomas, p = 0.01). LOH of CDH1 gene was found in 57.1% of NEECs but only in 22.5% of EECs (p = 0.011) and showed a trend towards association with reduced E-cadherin expression (p = 0.089). CDH1 promoter hypermethylation was found in 21.2% of endometrial carcinomas but was not associated with clinicopathological or immunohistochemical variables. Reduced expression of beta- and gamma-catenin and p120ctn was found in 76.1%, 94.3%, and 63.6% of the cases, respectively, being more frequent in lesions with reduced E-cadherin expression. In addition, beta-catenin, but not gamma-catenin or p120ctn expression, was associated with the histology of the lesion, since it was reduced in 35% of AEHs, 80.3% of EECs, and 96.9% of NEECs (p = 0.000). Mutations in exon 3 of the beta-catenin gene, associated with beta-catenin nuclear expression, were detected in 3 (14.0%) AEH, a frequency similar to that previously reported in this series of ECs. Finally, upregulation of P-cadherin was observed in 28.6% of cases. This alteration was associated with the histology of the lesion, since it was found in 9.5% of AEHs, 27.7% of EECs, and 46.2% of NEECs (p = 0.021).     

Promoter hypermethylation and reduced expression of RASSF1A are frequent molecular alterations of endometrial carcinoma.

Alterations in the regulation of the RAS-MAPK pathway are frequent in endometrial carcinoma. RASSF1A is a tumor-suppressor gene that can regulate this pathway negatively. RASSF1A has been found to be inactivated by promoter methylation in some human tumors. The aim of the study was to assess the immunohistochemical expression of RASSF1A in normal endometrium and endometrial carcinoma, and to correlate its expression with K-RAS mutations, presence of microsatellite instability, RASSF1A promoter methylation, and clinicopathological data. RASSF1A immunostaining was evaluated in one tissue microarray constructed from 80 paraffin-embedded samples of normal endometrium, and two tissue microarrays constructed with a total of 157 endometrial carcinomas (one constructed with 95 endometrial carcinomas previously evaluated for K-RAS mutations, and microsatellite instability, and another one containing 62 endometrial carcinomas that were also subjected to RASSF1A promoter methylation analysis). RASSF1A immunostaining was correlated with cell proliferation (Ki67), Cyclin D1 expression and clinicopathological data. Promoter methylation of RASSF1A was assessed by methylation-specific PCR. RASSF1A immunostaining was variable during the menstrual cycle in normal endometrium. RASSF1A expression was significantly reduced in 48% of endometrial carcinomas, particularly in tumors exhibiting microsatellite instability. RASSF1A-promoter methylation was very frequent in endometrial carcinoma (74%), and was frequently associated with reduced expression of RASSF1A. RASSF1A-promoter hypermethylation was common in advanced-stage endometrial carcinoma. The results suggest that reduced expression of RASSF1A may play a role in endometrial carcinogenesis by controlling cell proliferation and apoptosis through the MAPK-signaling pathway.     

PIK3CA mutations and amplification in endometrioid endometrial carcinomas: relation to other genetic defects and clinicopathologic status of the tumors

Alterations of the PIK3CA gene occur in endometrial carcinomas, but their role in the carcinogenesis of those malignancies is still poorly understood. In this study, PIK3CA mutations and amplification in 196 endometrioid endometrial carcinomas and 20 endometrial hyperplasias were assessed by single-strand conformation polymorphism (PCR-SSCP), sequencing, and quantitative polymerase chain reaction. Results were correlated with mutations in the PTEN, KRAS, and CTNNB1 genes and with the clinicopathologic parameters of the tumors. PIK3CA mutations were found in 39 (20%) carcinomas. Six new mutations were identified. No PIK3CA mutations were found in endometrial hyperplasias. PIK3CA amplification was observed in 24 (12.2%) carcinomas and in 2 (10%) hyperplasias. The PIK3CA mutations and amplifications (with the exception of 6 cases) occurred independently. PIK3CA mutations were significantly associated with PTEN mutations (P = .0414) and tended to be associated with CTNNB1 (P = .0833), but not with KRAS mutations. Conversely, the PIK3CA amplifications significantly negatively correlated with PTEN mutations (P = .0038) and did not coexist with CTNNB1 and KRAS mutations. The PIK3CA mutations were significantly associated with poorly differentiated tumors (P = .0423). Interestingly, PIK3CA amplifications, but not mutations, were strongly associated with older age (≥63 years, P = .0018). Our data show that mutations and amplification of PIK3CA are significant genetic alterations in endometrioid endometrial carcinomas associated with adverse clinicopathologic parameters (grade and stage). These data also demonstrate that PIK3CA mutations cooperate with PTEN mutations, suggesting an additive effect to PTEN, whereas PIK3CA amplification can, as an isolated event, enable the development of those tumors. Moreover, for the first time, a possible role of PIK3CA amplification in initiation and progression of endometrial carcinomas in older women is suggested, but this preliminary suggestion requires further research.     

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.