David Guerrero

Hypermethylation of the thrombospondin-1 gene is associated with poor prognosis in penile squamous cell carcinoma

To evaluate the presence of human papillomavirus (HPV) infection, the methylation status in the promoter region of thrombospondin‐1 (TSP‐1), RAS association domain family 1A (RASSF1‐A) and p16 genes, and the expression of TSP‐1, CD31, p16 and p53 proteins in patients diagnosed with penile cancer, and the possible associations between these variables and clinical and pathological features.

Unmasking of epigenetically silenced candidate tumor suppressor genes by removal of methyl-CpG-binding domain proteins

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5′-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein γ-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.

Differential hypermethylation of genes in vulvar cancer and lichen sclerosus coexisting or not with vulvar cancer

Squamous cell carcinoma (SCC) of the vulva is a heterogeneous disease, associated or not with vulvar lichen sclerosus (LS). The precursor role of LS in vulvar cancer is unclear. We studied the epigenetic alterations of RASSF1A, RASSF2A, p16, TSP‐1 and MGMT genes in vulvar SCCs, LS associated with SCC, isolated LS and normal vulvar skin. Gene hypermethylation and human papillomavirus presence were evaluated by methylation‐specific PCR and PCR/reverse line blot, respectively. High‐risk human papillomavirus types were present in 16.7% of the patients with vulvar SCC. There were increasing percentages of hypermethylation of genes from isolated LS to LS associated with vulvar SCC and vulvar SCC. The genes were hypermethylated more frequently in vulvar SCC associated with LS than in those not associated with LS, MGMT and RASSF2A being unmethylated in LS not associated with vulvar SCC. TSP‐1 hypermethylation was related to recurrence in patients with vulvar cancer. Conclusions are as follows: (i) the epigenetic inactivation of genes is a common event in vulvar SCC and is also present in adjacent lesions, implying a possible precursor role for these alterations; (ii) MGMT and RASSF2A hypermethylation are present exclusively in vulvar SCC and LS associated with SCC, and absent from isolated LS; and (iii) TSP‐1 hypermethylation is a bad prognosis factor in vulvar SCC.

Bcl‐2 and CD10 expression in the differential diagnosis of trichoblastoma, basal cell carcinoma, and basal cell carcinoma with follicular differentiation

Background  Both trichoblastoma and basal cell carcinoma (BCC) of the skin are characterized morphologically by the proliferation of basaloid cells; however, BCCs are clinically associated with a more aggressive behavior. An accurate diagnosis of these lesions is essential for effective, timely treatment and appropriate therapeutic decisions.

Differences and molecular immunohistochemical parameters in the subtypes of infiltrating ductal breast cancer.

Breast cancer is a heterogeneous disease, and patients are categorized into subtypes according to gene expression. We studied the associations among molecular, immunohistochemical, and clinicopathologic features and their distribution according to the subtypes luminal, HER2, basal, and normal-like in 60 patients with invasive ductal breast carcinoma without distant metastasis at the time of diagnosis (M0). We evaluated the hypermethylation of the CDH-1, RASSF1A, SIAH-1 and TSLC-1 genes by methylation-specific polymerase chain reaction and the expression of p53, bcl-2, cyclin D1, E-cadherin, and beta-catenin proteins in tissue microarrays by immunohistochemical analysis. Expression of bcl-2 was associated with the luminal subtype (P=.003), and CDH-1 hypermethylation was present preferentially in HER2 tumors (P=.038). The basal subtype was characterized by the expression of beta-catenin (P=.003). The hypermethylation of CDH-1 and the expression of bcl-2, cyclin D1, and beta-catenin proteins differ among breast cancer subtypes.

Experimental model of non–heart-beating donors: oxidative stress metabolism in kidney after cardiac arrest (30 minutes of warm ischemia) and reimplantation 24 hours later

THE procurement of organs from non–heart-beating donors (NHBD) is a new way to increase the number of the grafts available for transplantation. Because of increasing organ demand, it is important to establish a protocol to select optimal donors. Postischemic renal failure is an important problem, especially if the kidney has been harvested from a NHBD, presumably because of the combined injury from warm and cold ischemia. The organs obtained from NHBDs go through potential periods of injury for ischemia reperfusion: warm ischemia (NHBD with cardiopulmonary resuscitation; hyperoxygenated reperfusion (cardiopulmonary resuscitation); cold ischemia (preservation and storage); and reperfusion (graft revasculation). Free radicals generated from oxygen by activated xanthine oxidase at reperfusion have been shown to be a major mediator of renal injury following either warm or cold ischemia. The process in human has not been reproduced in animals totally. The goals of the present study are to develop a NHBD process that reproduces the human one in animals; to study ischemia–reperfusion lesion repercution, determining ATP, reduced glutathione (GSH), malondialdehyde (MDA), and myeloperoxidase (MPO); to evaluate warm ischemia as an indicator of graft viability with 30 minutes of renal ischemia; and to determine the quality and quantity of damage in each phase. MATERIALS AND METHODS

Asociación entre la inestabilidad de microsatélites y las características clínicas y anatomopatológicas en pacientes con cáncer de colon esporádico

Universidad de Navarra. Pamplona. Navarra. Espana. Fundamento y objetivo: La inestabilidad de microsatelites derivada del fallo en la reparacion de los falsos emparejamientos del ADN es la alteracion caracteristica de los tumores de la via mutadora o inestables (MSI). Tales casos parecen presentar diferencias desde el punto de vista clinicopatologico con los tumores de la via supresora o estables (MSS). Los tumores con alto grado de inestabilidad (MSI-H) parecen constituir una nueva entidad de tumores con diferencias en determinadas caracteristicas anatomopatologicas y clinicas con respecto a los tumores estables (MSS) e inestables de bajo grado (MSI-L). En el presente estudio se valora la posible asociacion entre el alto grado de inestabilidad de microsatelites con la localizacion, contenido mucinoso, grado de diferenciacion, estadio, asi como el intervalo libre de enfermedad y supervivencia. Pacientes y metodo: Se clasifica a 117 pacientes con cancer de colon esporadico en las poblaciones MSS/MSI-L y MSI-H (siguiendo las recomendaciones del National Cancer Institute) mediante reaccion en cadena de la polimerasa y electroforesis de 7 microsatelites. Resultados: Los tumores MSI-H tendieron a localizarse en el colon derecho (p = 0,022) y a presentar contenido mucinoso (p = 0,04). El conjunto de pacientes MSI-H de estadios II y III no presento intervalos libres de enfermedad ni periodos de supervivencia mas prolongados (p = 0,54, p = 0,37, respectivamente). Los tumores MSI-H de estadio II presentaron periodos de supervivencia mas prolongados que los tumores MSS/MSI-L (p = 0,027). No observamos diferencias en la respuesta a quimioterapia con 5-fluorouracilo y leucovorin entre los grupos MSS/MSI-L y MSI-H (p = 0,38). Conclusiones: El alto grado de inestabilidad de microsatelites se asocia con determinadas caracteristicas patologicas, asi como con periodos de supervivencia mas prolongados para los tumores de estadios II.