Influence of nonalcoholic fatty liver disease severity on carotid adventitial vasa vasorum.

Introduction: Nonalcoholic fatty liver disease (NAFLD) affects a quarter of the world's population and encompasses a spectrum of liver conditions, from non-alcoholic steatohepatitis (NASH) to inflammation and fibrosis. In addition, NAFLD also links to extrahepatic conditions like diabetes or obesity. However, it remains unclear if NAFLD independently correlates with the onset and progression of atherosclerosis. Material and methods: This cross-sectional study aimed to explore the relationship between NAFLD severity, assessed via liver biopsy, and early atherosclerosis using adventitial vasa vasorum (VV) density. It included 44 patients with obesity (33 with steatosis, 11 with NASH) undergoing bariatric surgery. Results: Results revealed no significant differences in adventitial VV density between steatosis and NASH groups, neither in the mean values [0.759 ± 0.104 vs. 0.780 ± 0.043, P=0.702] nor left-right sides. Similarly, carotid intima-media thickness (cIMT) did not vary between these groups. Additionally, no linear correlation existed between VV density and cIMT. Only gender showed an association with VV density. Conclusion: These findings suggest that NASH severity doesn't independently drive early atherosclerosis or affects cIMT. Gender might play a role in early atherosclerotic disease in NAFLD, impacting VV density and cIMT. This highlights the need to consider other risk factors when evaluating cardiovascular risk in NAFLD patients.

The Role of Morbid Obesity in the Promotion of Metabolic Disruptions and Non-Alcoholic Steatohepatitis by Helicobacter Pylori.

BACKGROUND: Helicobacter pylory (HP) infection has been associated to an increased rate of type 2 diabetes (T2D) and liver disease through its effect on insulin resistance and systemic inflammation. However, results are inconstant and no studies exist in morbidly obese patients, in which both insulin resistance and inflammation coexist. MATERIAL AND METHODS: Cross-sectional study to evaluate the relationship between HP infection and alterations in carbohydrate metabolism, lipid profile, inflammation markers, and liver disease in patients awaiting for bariatric surgery. HP infection was histologically assessed in gastric antrum biopsy from 416 subjects. Liver biopsy was also available in 93 subjects. RESULTS: Both impaired fasting glucose and T2D were similar when comparing subjects with and without HP infection (24.2% vs. 22%, p = 0.290 and 29.4% vs. 29.1%, p = 0.916, respectively), with no differences between groups in the HOMA-IR, lipid profile neither inflammatory parameters. However, HP infection was higher among subjects with a BMI ≥ 40.0 kg/m2 in comparison with lower degrees of obesity (71.7% vs. 60.0%, p = 0.041). In addition, subjects without HP infection showed higher degrees of steatosis (44.1±26.4% vs. 32.0±20.7%, p = 0.038), as well as a lower prevalence of non-alcoholic steatohepatitis (9.3% vs. 30.7%, p = 0.023). CONCLUSIONS: In patients with morbid obesity, HP infection does not seem to be associated with abnormal carbohydrate metabolism. In addition, less advanced degrees of non-alcoholic fatty disease were observed. We suggest that low-grade inflammation that accompanies obesity mitigates the diabetogenic effect of HP, so the presence of obesity should be considered in studies that evaluate the HP metabolic effects.

Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer.

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.

Clinicopathological characteristics and outcome of nested carcinoma of the urinary bladder.

We present the clinicopathological features of 56 cases of the nested variant of urothelial bladder carcinoma. This is an uncommon variant of bladder cancer, recognized by the current WHO classification of urologic tumors. The nested component represented 100 % of the tumor in 24 cases. The architectural pattern of the tumor varied from solid expansile to infiltrative nests characterized by deceptively bland histologic features resembling von Brunn nests. Typical features of high-grade conventional urothelial carcinoma were present in 32 cases. Most neoplastic cells had nuclei of low to intermediate nuclear grade with occasional nuclear enlargement, most frequently seen in deep areas of tumor. The nested component expressed cytokeratins 7, 20, CAM5.2, and high molecular weight (34ßE12), p63, Ki67, p53, p27, and GATA3. Tumor extension was T1 (n = 9), minimally T2 (n = 10), T2a (n = 1), T2b (n = 4), T3a (n = 8), T3b (n = 13), and T4a (n = 11). On follow-up, 36 of patients died of or were alive with disease from 2 to 80 months (mean 21 months). Four patients died of other causes. Eleven other patients remained disease free. Univariate survival analysis showed no differences for nested carcinoma compared with conventional urothelial carcinoma. As in conventional urothelial carcinoma, in nested carcinoma of the bladder pT category defined different survival groups. In summary, nested variant of urothelial bladder carcinoma is typically associated with advanced stage. In samples of limited volume, it may be misdiagnosed as proliferation of von Brunn nests or other nested-like bladder lesions, delaying definitive therapy.

Role of local bioactivation of vitamin D by CYP27A1 and CYP2R1 in the control of cell growth in normal endometrium and endometrial carcinoma.

Vitamin D (VD) deficiency has been suggested as a risk factor for cancer. One recognized mechanism is that the low-serum 25-hydroxyvitamin D (25(OH)D) of VD deficiency reduces intratumoral 25(OH)D conversion to 1α,25-dihydroxyvitamin D (1,25D, the hormonal form of VD), compromising 1,25D-VD receptor (VDR) antitumoral actions. Reduced tumoral VDR and increased CYP24A1, the enzyme that degrades 1,25D and 25(OH)D, further worsen cancer progression. Importantly, in cells expressing CYP27A1 and/or CYP2R1, which convert inert VD into 25(OH)D, low-serum VD may reduce intratumoral 25(OH)D synthesis thereby compromising VDR antitumoral actions because 25(OH)D can activate the VDR directly and enhance 1,25D-VDR action. Therefore, this study examined whether abnormal endometrial expression of CYP27A1 and/or CYP2R1 may impair VDR-antiproliferative properties in endometrial carcinoma (EC). Immunohistochemical analysis of tissue microarrays of normal human endometrium (NE; n=60) and EC (n=157) showed the expected lower VDR expression in EC (P=0.0002). Instead, CYP24A1 expression was lower in EC compared with NE, while CYP27A1 and CYP2R1 expressions were higher (P=0.0002; P=0.03). Furthermore, in NE and EC, CYP2R1 and CYP27A1 expression correlated directly with nuclear VDR levels, an indicator of ligand-induced VDR activation, and inversely with the proliferation marker Ki67. Accordingly, in the endometrioid carcinoma cell lines IK, RL95/2 and HEC1-A, which express VDR, CYP27A1, and CYP2R1, VD efficaciously reduced cell viability and colony number, with a time course that paralleled actual increases in both intracellular 25(OH)D and nuclear VDR levels. Thus, VD may protect from EC progression in part through increased intratumoral 25(OH)D production by CYP27A1 and CYP2R1 for autocrine/paracrine enhancement of 1,25D-VDR-antiproliferative actions.

Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues.

In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P < .05) and included a high target retrieval solution, 1:100 primary antibody dilution (2.925 mg/L), FLEX diluent, and EnVisionFLEX+ detection method, with a sensitivity and specificity of 72.33% and 78.57%, respectively. Staining specificity was confirmed in cell lines and animal models. Endometrial carcinomas with PTEN genetic abnormalities showed statistically lower staining than tumors without alterations (mean histoscores, 34.66 and 119.28, respectively; P = .01). Controlled preanalytical factors (delayed fixation and overfixation) did not show any statistically significant effect on staining with optimal protocol (P > .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01).

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias.

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ER(T) under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

FGFR2 alterations in endometrial carcinoma.

Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase receptor involved in many biological processes such as embryogenesis, adult tissue homeostasis and cell proliferation. Mutations in FGFR2 have been reported in up to 10-12% of endometrial carcinomas identical to those found in congenital craniofacial disorders. Inhibition of FGFR2 could be a new therapeutic target in endometrial carcinoma. FGFR2 immunostaining was assessed in three tissue microarrays: one constructed from paraffin-embedded blocks of 60 samples of normal endometrium in different phases of menstrual cycle, and two tissue microarrays containing endometrial carcinoma samples (95 and 62 cases). FGFR2 expression was correlated with stage, histological type and grade as well as with immunostaining of PTEN, RASSF1A, estrogen and progesterone receptors, KI67, Cyclin D1, STAT-3 and SPRY2. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 31 paraffin-embedded endometrial carcinoma samples. In normal endometrium, FGFR2 expression was higher in the secretory than in the proliferative phase (P=0.001), with an inverse correlation with Ki67 (P=0.00032), suggesting a tumor-suppressor role for FGFR2 in normal endometrium. Cytoplasmic expression of FGFR2 was higher in endometrial carcinoma when compared with the atrophic endometrium from the same patients (P=0.0283), but was lower in comparison with normal endometrium from women in the menstrual cycle. Interestingly, nuclear staining was observed in some cases, and it was less frequent in endometrial carcinoma when compared with the adjacent atrophic endometrium (P=0.0465). There were no statistical differences when comparing superficial and myoinvasive endometrial carcinoma samples. Endometrioid endometrial carcinomas showed higher expression of FGFR2 than nonendometrioid endometrial carcinomas (fold change 2.56; P=0.0015). Grade III endometrioid endometrial carcinomas showed decreased FGFR2 expression when compared with grade II endometrioid endometrial carcinomas (P=0.0055). No differences were found regarding pathological stage. Two missense mutations of FGFR2 gene were detected in exons 6 and 11 (S252W and N549K, respectively; 6.45%). Results support the hypothesis that FGFR2 has a dual role in the endometrium, by inhibiting cell proliferation in normal endometrium during the menstrual cycle, but acting as an oncogene in endometrial carcinoma.

KSR1 is overexpressed in endometrial carcinoma and regulates proliferation and TRAIL-induced apoptosis by modulating FLIP levels.

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

Promoter hypermethylation and expression of sprouty 2 in endometrial carcinoma.

Sprouty 2 is a key antagonist regulator of receptor tyrosine kinases, and downstream signaling pathways, like fibroblastic growth factor (FGF) and Ras-mitogen-activated protein kinase (RAS-MAPK). By controlling these pathways, sprouty 2 is involved in regulation of cell proliferation, differentiation, and angiogenesis. Alterations in fibroblastic growth factor receptor (FGFR) and members of the RAS-MAPK pathway are frequent in endometrial carcinoma. The expression of sprouty 2 has been found to be decreased in several types of human cancer, by mechanisms of promoter methylation. In the present study, we have assessed the expression of sprouty 2 in endometrial carcinoma, in correlation with sprouty 2 promoter methylation. Sprouty 2 immunohistochemical expression was assessed using 3 different tissue microarrays: one constructed from paraffin blocks of 80 samples of normal endometrium and 2 tissue microarrays containing samples of 157 endometrial carcinoma (1 tissue microarray constructed with 95 endometrial carcinomas previously studied for microsatellite instability and alterations in phosphatase and tensin homolog (PTEN), k-ras, and b-catenin, and 1 tissue microarray containing 62 endometrial carcinoma, which were also subjected to sprouty 2 promoter methylation analysis). The immunohistochemical expression of sprouty 2 was correlated with cellular proliferation (Ki67) and clinicopathologic data. Sprouty 2 promoter methylation was assessed by methylation-specific polymerase chain reaction, with DNA obtained from fresh-frozen samples of endometrial carcinoma and corresponding normal tissues, and correlated with promoter methylation of RAS association domain family-1A (RASSF1A). A highly significant decrease in sprouty 2 immunoexpression was seen in the proliferative phase of normal endometrium (P < .001). Differences were detected between types I and II endometrial carcinoma, but they were not statistically significant. Reduced immunoexpression of sprouty 2 was seen in 19.85% of endometrial carcinoma and was strongly and inversely associated with increased cell proliferation (Ki67; r = -0.367; P = .001). Sprouty 2 promoter methylation was detected in 31 (53.4%) of 58 endometrial carcinomas. Results from our study show that alterations in sprouty 2 may be involved in endometrial carcinogenesis by controlling cell proliferation.

A review of the applications of tissue microarray technology in understanding the molecular features of endometrial carcinoma.

OBJECTIVE: To review the literature regarding the use of tissue microarray (TMA) technology in understanding the biology, diagnosis and prognosis of endometrial carcinoma (EC). STUDY DESIGN: This review of TMA technology in EC was based on a large number of published articles. We focused on the use of TMA technology as a tool to gain insight in endometrial carcinogenesis and to validate data obtained from DNA microarrays, proteomics and cellular models. RESULTS: We summarized the technical aspects of the 37 articles that were reviewed. The number of EC cases in each series varied from 32-485 (median, 128). The number of cores ranged from 1-4 (median, 2), and the size of the cores ranged from 0.6-2 mm (median, 0.6 mm). Only 3 studies applied fluorescence in situ hybridization technology, while the remaining 34 studies used immunohistochemistry. CONCLUSION: TMA can help to establish new prognostic markers and to define protein biomarkers that help in differential diagnosis.

CK2beta is expressed in endometrial carcinoma and has a role in apoptosis resistance and cell proliferation.

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

Targeted therapies in gynecologic cancers and melanoma.

The article reviews the main molecular pathology alterations of endometrial and ovarian carcinomas and melanoma. Several promising drugs targeting the genes most frequently altered in these tumors are under consideration. The most promising signaling pathways to be targeted for therapies in these tumors are the tyrosine kinase receptor (EGFR, HER2, c-KIT), the RAS/B-RAF/MAPK, the PI3K-mTOR, and apoptosis signaling pathways.

Loss of heterozygosity in endometrial carcinoma.

Inactivation of a tumor suppressor gene typically occurs in two steps, thus fulfilling Knudson hypothesis. One "hit" is frequently a point mutation or a small deletion. The other alteration is usually a large genomic loss of part of a gene, or even part of a chromosome, or the whole chromosome. However, it is not clear which of these two events occurs first. Loss of heterozygosity (LOH) analysis allows the identification of one of the 2 hits. Although microsatellite polymerase chain reaction is the technique most frequently used to assess LOH, other different approaches can also be used. The LOH can also be assessed by restriction fragment length polymorphism analysis, single strand conformation polymorphism analysis, oligonucleotide microarrays capable to simultaneously determine the genotype of thousands of single-nucleotide polymorphism (single-nucleotide polymorphism arrays), comparative genomic hybridization, multiplex amplification and probe hybridization, and multiplex ligation-dependent probe amplification. In this article, the authors review the results obtained with molecular analysis of LOH in the understanding of development and progression of endometrial carcinoma. Particular attention is given to: (1) the presence of widespread LOH in nonendometrioid carcinoma, probably reflecting the existence of chromosomal instability; and (2) specific LOH patterns associated with some clinicopathologic features.

Nuclear factor-kappaB activation is associated with somatic and germ line RET mutations in medullary thyroid carcinoma.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. NF-kappaB has been shown to be activated through several signaling pathways that involve growth factor receptors. The aim of the study was to assess the immunohistochemical expression of members of the NF-kappaB family and the putative targets of NF-kappaB in a series of medullary thyroid carcinomas (MTCs), in correlation with RET mutational status. A tissue microarray was constructed from paraffin-embedded blocks of 48 MTCs (13 familial, 35 sporadic) previously evaluated for germ line and somatic RET mutations. Immunohistochemical evaluation included members of the NF-kappaB (p50, p65, p52, c-Rel, RelB) family, as well as putative targets of NF-kappaB such as Flip, Bcl-xL, and cyclin D1. Nuclear immunostaining for members of NF-kappaB was frequent in MTCs (p50, 19%; p65, 68%; p52, 86.6%; c-Rel, 75%; RelB, 36%). MTCs with germ line or somatic RET mutations (29 cases) showed NF-kappaB nuclear translocation (particularly of p65, P = .035) more frequently than MTCs without RET mutations (19 cases). Immunostaining for putative targets of NF-kappaB showed a significant statistical association between p65 and Bcl-xL (P = .024). In addition, Bcl-xL expression was statistically higher in the tumors with exon 16 RET mutation in comparison with those with exon 10 and 11 RET mutations or wild-type RET (P = .002). Moreover, the significance of RETsignaling in NF-kappaB activation was evaluated in the RET-mutated TT cell line. TT cells were infected with lentiviruses carrying short hairpin RNA to knock down RET expression, and NF-kappaB activity was assessed by luciferase reporter assays. Silencing of RET in the TT cell line produced a significant decrease in NF-kappaB activation and reduction in ERK1/2. The results suggest that the NF-kappaB is frequently activated in MTCs. The results also support the hypothesis that RET activation by somatic or germ line mutations may be responsible for NF-kappaB activation in MTCs.

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.

Metastatic small cell carcinoma to the thyroid gland: a pathologic and molecular study demonstrating the origin in the urinary bladder.

Small cell carcinomas may occur in the thyroid gland. Infrequently, they are primary tumors, and have been interpreted as variants of medullary thyroid carcinoma. However, the vast majority of small cell carcinomas involving the thyroid gland are metastatic tumors. In some cases, demonstration of the primary tumor is not easy. An example of a small cell carcinoma metastatic to the thyroid is presented in this report. The primary tumor was a small cell carcinoma that occurred as a minor component in a transitional carcinoma of the urinary bladder. The microscopical and immunohistochemical features of both tumors, in the thyroid and the bladder, were identical. Moreover, both tumors exhibited an identical mutation in p53, as well as similar loss of heterozygosity at 10q23 and RASSF1A promoter hypermethylation, clearly indicating that the bladder tumor was the site for the primary tumor of the patient.

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.