Promoter hypermethylation contributes to TIMP3 down-regulation in high stage endometrioid endometrial carcinomas.

AIMS: Expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) has been found to be decreased in several types of cancer by promoter gene hypermethylation. However, little is known regarding the silencing effect of TIMP3 promoter hypermethylation on gene and protein expression in endometrial carcinomas and its prognostic significance. METHODS AND RESULTS: TIMP3 promoter hypermethylation and gene copy number variations were evaluated using a methylation-specific multiplex ligation-dependent probe amplification approach in 60 cases of endometrioid endometrial carcinomas. TIMP3 expression was also evaluated at the transcript and protein levels. Loss of TIMP-3 protein expression was found in 44 (73%) of 60 carcinomas. Promoter hypermethylation was identified in 25% (15 of 60); was more frequent in stages II-IV (55%, six of 11) than in stage I (18%, nine of 49; P = 0.021); and was found more commonly in tumours with deep myometrial invasion. MLH1 and TIMP3 promoters were hypermethylated simultaneously in the same group of tumours (P < 0.001). A correlation between TIMP3 methylation and microsatellite instability (MSI) was found (P = 0.005). TIMP3 copy number changes were frequently a loss (35%), whereas a gain was detected in only 5%. CONCLUSIONS: TIMP3 promoter hypermethylation was associated with high stage endometrioid endometrial tumours with extrauterine spread. Nevertheless, promoter hypermethylation and loss of heterozygosity are not the only mechanisms for TIMP3 inactivation.

Genetic up-regulation and overexpression of PLEKHA7 differentiates invasive lobular carcinomas from invasive ductal carcinomas.

Molecular differentiation between invasive lobular carcinomas (ILCs) and invasive ductal carcinomas (IDCs) of the breast has not been well defined. We investigated gene expression differences between ILCs and IDCs and their correlation with variations in invasiveness and tumor growth. Total RNA was isolated from 30 frozen tumor samples: 10 from ILCs and 20 from IDCs. Gene expression was investigated using the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Data and validation were performed by reverse transcriptase polymerase chain reaction and immunohistochemistry. Gene expression differences between ILCs and IDCs were found in 140 genes. Overall, ILCs showed up-regulation of genes related with cell migration, lipid and fatty acid metabolism, and some transcription factors and showed down-regulation of cell adhesion, actin cytoskeleton, cell proliferation, and energetic metabolism of the tumor cells. Our reverse transcriptase polymerase chain reaction results showed that PLEKHA and TMSB10 expression discriminated ILCs from luminal A IDCs, whereas PLEKHA7, TMSB10, PRDX4, and SERPINB5 discriminated ILCs from luminal B IDCs. At the protein level, Plekha7 was overexpressed in ILCs but not in normal tissue or low-grade IDCs. Moreover, Plekha7 overexpression had an inverse relation with E-cadherin expression. The gene expression profile in ILCs and IDCs differs in several signaling pathways. Our findings suggest that overexpression of PLEKHA7 is common in ILCs and could be a molecular marker to differentiate ILCs from IDCs.

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.

Low-density lipoprotein receptor-related protein 1 is associated with proliferation and invasiveness in Her-2/neu and triple-negative breast carcinomas.

Low-density lipoprotein receptor-related protein 1, a member of the low-density lipoprotein cholesterol receptor family, has been implicated in the progression of certain tumors; but it remains unclear whether it plays a role in infiltrating ductal breast carcinomas. We studied a series of 81 ductal breast tumors to determine the correlation of low-density lipoprotein receptor-related protein 1 overexpression with clinicopathologic and immunohistochemical characteristics associated with prognosis. Low-density lipoprotein receptor-related protein 1 overexpression was identified in 14% (11/81) of tumors and was correlated with a high nuclear grade (P = .043), high mitotic index (P = .006), and Ki-67 greater than 20% (P = .047). Furthermore, low-density lipoprotein receptor-related protein 1 expression was associated with aggressive carcinomas (triple-negative tumors [21%, 7/33] and Her-2/neu tumors [17%, 4/24]) but not with hormone-dependent carcinomas (0%, 0/24) (P = .040). There was no correlation between low-density lipoprotein receptor-related protein 1 expression and survival, but a trend was found between low-density lipoprotein receptor-related protein 1 overexpression and tumor recurrence. Low-density lipoprotein receptor-related protein 1 overexpression was related to proliferation and invasiveness in Her-2/neu and triple-negative breast carcinoma. Moreover, patients with low-density lipoprotein receptor-related protein 1-positive tumors had higher cholesterol levels (62.5%, 5/8) than those with low-density lipoprotein receptor-related protein 1-negative tumors (40%, 19/47). Nevertheless, the correlation between low-density lipoprotein receptor-related protein 1 and hypercholesterolemia was not statistically significant; but cholesterol levels were higher in patients with triple-negative breast carcinoma (60%, 15/25) and Her-2/neu carcinomas (40%, 6/15) than in luminal-A carcinomas (20%, 3/15) (P = .046). These findings suggest a relationship between hypercholesterolemia and aggressiveness of ductal breast carcinomas.

Gene expression analysis identifies two groups of ovarian high-grade serous carcinomas with different prognosis.

Gene expression profiling is an important tool to evaluate genetic heterogeneity in carcinomas and is useful to develop expression-based classifications for many types of cancer, as well as markers of disease outcome. In this study, we have investigated the expression profile of 22 genes involved in the PI3K-AKT pathway in 26 high-grade ovarian carcinomas (19 serous and 7 clear cell carcinomas). Unsupervised hierarchical clustering divided high-grade ovarian carcinomas into three groups. Although all clear cell carcinomas clustered in one group, high-grade serous carcinomas were segregated into two separate groups with different prognosis (P=0.05). High expression of CASP3, XIAP (X-linked inhibitor of apoptosis) , NFKB1, FAS, and GSK3B mRNAs identified high-grade serous carcinomas with better prognosis. In multivariate analysis, these cluster groups were of prognostic significance independent of age, tumor size, and tumor stage (P=0.008). To validate the mRNA expression data, we studied the immunohistochemical expression of caspase-3 and XIAP on a tissue microarray. Immunoreaction for caspase-3 was concordant with the results obtained by mRNA expression analysis (Spearman r=0.762, P=0.000). Caspase-3 was exclusively expressed by the macrophages. Furthermore, co-expression of caspase-3 and XIAP identified high-grade serous carcinomas with different prognosis (P=0.03). Our results suggest that there are different biological subtypes of high-grade serous carcinomas.

Prognostic significance of the Fas-receptor/Fas-ligand system in cervical squamous cell carcinoma.

We studied whether Fas-receptor (Fas-R; CD95) expression, single-nucleotide polymorphisms (SNPs) in the Fas promoter region, and/or Fas-ligand (Fas-L) production could determine individual susceptibility to cervical cancer progression. The clinicopathologic features of 38 patients with cervical squamous carcinomas (22 stage I, 8 stage II, and 8 stage III+) were reviewed and related with: (a) Fas-R expression by immunohistochemistry; (b) Fas-R SNPs at -670 and -1377 locations by restriction fragment length polymorphism and DNA sequencing; and (c) Fas-L expression by immunohistochemistry. Overall and disease-free survival curves showed significant differences in relation to stage (p < 0.001). Fas-R was identified in 20 of 38 (52.6%) tumors without statistical differences in survival, stage, or Fas-L overproduction. Fas-R GG genotype was more common than expected in advanced tumors (p = 0.065). The Fas-R-1377A allele and AA genotype were unrelated with survival, stage, or Fas-R expression. Fas-L overproduction was detected in 20 of 38 (52.6%) tumors; it was more frequent in advanced-stage tumors and was inversely related to survival (p = 0.03) and decrease in host inflammatory response (p = 0.01). Fas-R expression by tumor cells seems unrelated to stage or lymphoid infiltrate. Tumor production of Fas-L may represent an attempt to destroy the host's lymphocytic reaction.

Molecular pathology of atypical polypoid adenomyoma of the uterus.

Atypical polypoid adenomyoma (APA) is an uncommon and benign tumor of the uterus. In some patients, however, APA has been found to coexist with or to precede the development of an endometrioid adenocarcinoma similarly to complex endometrial hyperplasia. The molecular changes underlying the progression from APA to adenocarcinoma are unknown. DNA from paraffin-embedded tissue of 6 APAs was evaluated for microsatellite instability (MI), MLH-1 promoter hypermethylation, and CTNNB-1 mutations. Tissue sections were also subjected to MLH-1, MSH-2, and beta-catenin immunostaining. MI was not detected in any case. Two tumors exhibited MLH-1 promoter hypermethylation and showed focal negative MHL-1 immunostaining; 1 of these showed marked architectural complexity and cellular pleomorphism. Five cases presented beta-catenin nuclear immunoreactivity, but none of them had CTNNB-1 mutations. The results of this study suggest that APA and complex endometrial hyperplasia may share some molecular alterations. Some APAs exhibit MLH-1 promoter hypermethylation with focal lack of MLH-1 immunostaining, a molecular abnormality involved in the transition from complex atypical hyperplasia to endometrioid adenocarcinoma.

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). METHODS: Cutaneous (eight sebaceous adenomas, one sebaceous carcinoma and one keratoacanthoma) and internal tumors (four colonic adenocarcinomas, two endometrial carcinomas, two transitional cell carcinomas of renal pelvis and ureter, one adenocarcinoma of the small bowel, one ovarian carcinoma and one colonic tubular adenoma) were obtained from six patients with MTS and were subjected to microsatellite instability (MI) analysis, and to immunostaining for MLH-1 and MSH-2. MI was assessed by evaluating three (CA)n dinucleotide repeats (D2S123, D5S346, D17S250) and the mononucleotide tracts BAT 26 and BAT 25. RESULTS: All cutaneous and internal tumors exhibited MI. An immunohistochemical concordance between all tumors within each single patient was obtained in five cases. In these five patients all tumors exhibited a lack of MSH-2 staining, consistent with a germline abnormality in this gene. In the one remaining case, the immunohistochemical staining in the sebaceous adenoma was negative for MLH-1 and positive for MSH-2, consistent with a germline alteration in MLH-1. However, the colonic adenocarcinoma in that patient showed positivity for MSH-2 and an equivocal positivity for MLH-1. CONCLUSIONS: The results confirm that tumors from patients with MTS exhibit MI. Moreover, immunostaining for MLH-1 and MSH-2 may be useful to identify the most probable gene responsible for the disease in each family.

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.

Depresión en la adolescencia. Su relación con actividad deportiva y consumo de drogas / Depression in the adolescent. Its relationship with sports activities and drug consumption

Objetivo: determinar la relación entre depresión, actividad deportiva y consumo de drogas en adolescentes.Material y métodos: de la Escuela Nacional Preparatoria 9, Universidad Nacional Autónoma de México, durante el periodo comprendido entre marzo y abril de 1997, se seleccionaron 324 estudiantes a quienes se les aplicó el Inventario para depresión (de Beck), formándose dos grupos con práctica o no de actividad deportiva: uno sin depresión y otro con diagnóstico de dicha condición. El diseño fue transversal, comparativo y observacional.Resultados: las edades oscilaron entre 15 y 18 años; predominó ligeramente el género femenino (54.3 por ciento); 142 alumnos (43.8 por ciento) cursaban el cuarto grado de bachillerato, 114 (35.2 por ciento) el quinto y 68 (21 por ciento) el sexto; 135 (41.7 por ciento) asistían al turno matutino y 189 (58.3 por ciento) al vespertino. La prevalencia de depresión fue de 28.4 por ciento (92 alumnos), siendo más frecuente la leve (72.8 por ciento). De 199 alumnos que no realizaban deporte 64 tuvieron depresión; de 125 que practicaban deporte, 28 manifestaron depresión. La diferencia entre un grupo y otro no fue estadísticamente significativa (p = 0.05825). En cambio, se encontró asociación estadísticamente significativa entre consumo de alcohol y tabaco con depresión (p < 0.05), así como consumo de tabaco con falta de actividad físico-deportiva (p = 0.01).Conclusiones: la depresión es un padecimiento frecuente en la población adolescente, asociada en ocasiones con el consumo de sustancias. Es necesario buscar opciones de prevención y tratamiento. Aunque en este estudio no es significativa la diferencia entre el grupo de deprimidos con actividad física y sin ella, tendría que realizarse un diseño diferente para dar seguimiento al mismo tema.