Alejandro Corvalán R

Rol patogénico del gen supresor de tumores PTEN en cáncer ovárico asociado a endometriosis

Background: Endometrioid carcinoma and clear cell carcinoma of the ovary are associated to endometriosis. Somatic mutations of PTEN (10q23.3) are present in endometrial endometrioid carcinoma. Therefore, these mutations could be also present in ovarian tumors. Molecular studies show that solitary endometriotic cysts are monoclonal, have aneuploid DNA, have a loss of 9p,11q and 22q heterozygosity (LOH) and a higher cellular proliferation index of the epithelial component. Aim: To determine the cellular proliferation index using Ki 67, the immunohistochemical expression of PTEN and LOH in patients with ovarian endometriosis without atypia (EN), ovarian endometriosis with atypia (EA) and endometriosis with adjacent ovarian carcinoma (ET). Material and methods: Paraffin embedded samples of 37 endometrioid and clear cell carcinomas of the ovary (CC/CE), 15 solitary ovarian EN and 15 ovarian EA, were studied. Expression of Ki 67 and PTEN was measured by immunohistochemistry. LOH of 10q23.3 locus was measured by polymerase chain reaction. Results: Ki 67 was 5.5 and 2.3% in EA and EN, respectively (p <0.005). There was a histological correlation between EA and a higher cellular proliferation index. PTEN was negative in 5 of 15 EN, 9 of 15 EA and 30 of 37 CE/CC. There was a correlation between LOH and loss of PTEN protein in EN, EA and ET (60%). Conclusions: Negative expression on PTEN in EN; EA; ET and CE/CC is a manifestation of the inactivation of this gene. The mechanisms that cause this inactivation, must be elucidated (Rev Med Chile 2006; 134: 271-8).

Identificación de asociaciones clínico-patológicas e hipermetilación de genes supresores de tumores en cáncer gástrico difuso a través de análisis de Hierarchical clustering

Eighty three patients with diffuse gastric cancer with information about survival andinfection with Epstein Barr virus, were studied. DNA was extracted from pathological slides and themethylation status of genes p14, p15, p16, APC, p73, FHIT, E-caderin, SEMA3B, BRCA-1, MINT-2 yMGMT, was studied using sodium bisulphite modification and polymerase chain reaction. Resultswere grouped according to the methylation index or Hierarchical clustering (TIGR MultiExperimentViewer).

Características clínico-moleculares del cáncer gástrico cardial asociado al virus Epstein Barr

BACKGROUND Mortality caused by cardial gastric cancer in Chile, is increasing. Previously we demonstrated an association between Epstein Barr virus and this specific location of gastric cancer. AIM To perform a clinical and molecular characterization of cardial gastric cancer associated to Epstein Barr virus. MATERIAL AND METHODS Epstein Barr virus was identified in 93 cardial gastric tumors, by in situ hybridization. Clinical and pathological features, survival and expression of p53 and c-erbB2 were compared between tumors with or without the presence of the virus. RESULTS Twenty two (23.6%) tumors expressed Epstein Barr virus. No difference in sex or age of patients with tumors positive or negative for the virus was observed. Epstein Barr positive tumors had a tendency to have a higher frequency of Bormann III endoscopic appearance and a lower frequency of p53 accumulation (p=0.06). Five years survival was 67% and 42% of tumors positive and negative for the presence of the virus, respectively (p=0.57). CONCLUSIONS Our results, although not significant, show a tendency towards unique characteristics of cardial gastric tumors associated to Epstein Barr.

Bases epigenéticas del cáncer gástrico: oportunidades para la búsqueda de nuevos biomarcadores

Gastric cancer is the first cause of death for cancer in Chile. The recently identified genetic alterations in these tumors have not yielded new biomarkers for the disease. Epigenetics or the study of reversible genomic changes that do not affect protein codifying DNA sequences but cause phenotypic disturbances, is identifying new cancer biomarkers. Specifically, the loss of expression caused by the covalent link of a methyl group to carbon 5 of cytosine (DNA hypermethylation) is extensively evaluated. Performing an epigenetic evaluation of 24 genes, we have identified eight genes associated to the aggressive signet ring cell type gastric cancer, the association between APC hypermethylation and worse prognosis and BRCA1 hypermethylation association with early onset of gastric cancer. The most interesting findings are the hypermethylation of Reprimo gene in plasma as a population biomarker and the tissue over expression of p73 gene (as a consequence of hypomethylation) as a high risk indicator of progression to gastric cancer. All these findings are indicating an important role of epigenetics in the pathogenesis and early detection of gastric cancer.Gastric cancer is the first cause of death for cancer in Chile. The recently identified genetic alterations in these tumors have not yielded new biomarkers for the disease. Epigenetics or the study of reversible genomic changes that do not affect protein codifying DNA sequences but cause phenotypic disturbances, is identifying new cancer biomarkers. Specifically, the loss of expression caused by the covalent link of a methyl group to carbon 5 of cytosine (DNA hypermethylation) is extensively evaluated. Performing an epigenetic evaluation of 24 genes, we have identified eight genes associated to the aggressive signet ring cell type gastric cancer, the association between APC hypermethylation and worse prognosis and BRCA1 hypermethylation association with early onset of gastric cancer. The most interesting findings are the hypermethylation of Reprimo gene in plasma as a population biomarker and the tissue over expression of p73 gene (as a consequence of hypomethylation) as a high risk indicator of progression to gastric cancer. All these findings are indicating an important role of epigenetics in the pathogenesis and early detection of gastric cancer.

Biología molecular en Infectología: Parte I: Desarrollo y metodologías

RESUMENEl origen de la biologia molecular se puederastrear hasta fines del siglo XIX; sin embargo,el descubrimiento de la estructura del ADN seconsidera como el inicio de esta disciplina. Losavances producidos por la biologia molecularen la decada del ´60 nos permiten contar hoycon herramientas para el estudio de microorga-nismos a nivel molecular. En particular, el des-cubrimiento de la ADN polimerasa y las pro-piedades de hibridacion del ADN son algunosde los descubrimientos que aplicados hoy dia enla reaccion de polimerasa en cadena, la amplifi-cacion isotermica y la hibridacion con ADNramificado, se han transformado en herramien-tas utiles en el diagnostico y cuantificacion deagentes infecciosos. Finalmente la secuenciacionde genomas bacterianos completos permitira eldesarrollo de nuevos metodos para el diagnosti-co y tratamiento de enfermedades infecciosas.BIBLIOGRAFIA 1.- WATSON J D, CRICK F H C. A structure for Deo-xiribose Nucleic Acid. Nature 1953: 171; 737-8. 2.- CORVALAN, A. Biologia Molecular Fundamentosy aplicaciones diagnosticas. Rev Med Clinica LasCondes 1997; 8: 4-9. 3.- FREIFELDER D M. The DNA molecule Structureand Properties. WH Freeman, 1978 pp: 1-7. 4.- AVERY O, MACLEOD C, MCCARTY M. Studieson the chemical nature of the substance inducingtransformation of pneumococcal types. J Exp Med1944; 79: 137-57. 5.- CHARGAFF E. Chemical specificity of nucleic acidsand mechanism of their enzymatic degradation.Experentia 1950; 6: 201-9. 6.- FIERRO A. Breve historia del descubrimiento de laestructura del ADN. Rev Med Clinica Las Condes2001; 20: 71-75. 7.- ALBERTS B, BRAY D, LEWIS J, RAFF M,ROBERTS K, WATSON J D. Recombinant DNAtechnology in:Molecular Biology of the Cell 3thEdition Gardland 1994 pp: 291-334. 8.- DOTY P, MARMUR J, EIGNER J, SCHILDKRAUTC. Strand separtation and specific recombination indeoxyribonucleic acids: physical chemical studies.Proc Natl Acad Sci USA 1960; 46: 461-76. 9.- WATSON J D, TOOZE J. The DNA story: A docu-mentary history of gene cloning. New York: WHFreeman, 198110. NATHANS D, SMITH H J O. Restricction endo-nucleases in the analysis and restructuring of DNAmolecules. Ann Rev Biochem 1975: 44; 449-67.11.- GILBERT W, VILLA-KOMAROFF L. Usefulproteins from recombinant bacteria. Sci Am 1980;242: 74-94.12.- SOUTHERN E M. Detection of specific sequencesamong DNA fragments separated by gelelectrophoresis. J Mol Biol 1975; 98: 503-17.13.- SANGER F, NICKLEN S, COULSON A R. DNAsequencing with chain-terminating inhibitors. ProcNatl Acad Sci USA 1977; 74: 5463-7.14.- MULLIS K B. The unusual origin of the polymerasechain reaction. Sci Am 1990; 262: 56-61, 64-5.15. INNIS M A, MYAMBO K B, GELFAND D H,BROW M A. DNA sequencing with Thermusaquaticus DNA polymerase and direct sequencingof polymerase chain reaction-amplified DNA. ProcNatl Acad Sci USA 1988; 85: 9436-40.16.- BEN-EZRA J, JOHNSON DA, ROSSI J, COOK N,WU A. Effect of fixation on the amplification ofnucleic acids from paraffin-embedded material bythe polymerase chain reaction. J HistochemCytochem 1991; 39: 351-4.17.- GIBSON U E, HEID C A, WILLIAMS P M. A novelmethod for real time quantitative RT-PCR. GenomeRes 1996; 6: 995-1001.18.- HEID C A, STEVENS J, LIVAK K J, WILLIAMS PM. Real time quantitative PCR. Genome Res 1996;6: 986-94.19.- LIVAK K J, FLOOD S J, MARMARO J, GIUSTIW, DEETZ K. Oligonucleotides with fluorescentdyes at opposite ends provide a quenched probesystem useful for detecting PCR product and nucleicacid hybridization. PCR Methods Appl. 1995; 4:357-62.20.- JUNG R, SOONDRUM K, NEUMAIER M. Quanti-tative PCR. Clin Chem Lab Med. 2000; 38: 833-6.21.- KIEVITS T, VAN GEMEN B, VAN STRIJP D,SCHUKKINK R et al. NASBA isothermal enzymaticin vitro nucleic acid amplification optimized for thediagnosis of HIV-1 infection. J Virol Methods 1991;35: 273-86.22.- ROMANO J W, VAN GEMEN B, KIEVITS T.NASBA: a novel, isothermal detection technologyfor qualitative and quantitative HIV-1 RNAmeasurements. Clin Lab Med 1996; 16: 89-103.23.- MAHONY J B, SONG X, CHONG S, FAUGHT M,SALONGA T, KAPALA J. Evaluation of the Nucli-sens Basic Kit for detection of

Biología molecular en Infectología: Parte II: Diagnóstico molecular de agentes infecciosos

Resumen Las aplicaciones diagnosticas de la biologiamolecular para enfermedades infecciosas sonextremadamente variadas y aplicables a cualquierproblema diagnostico. En agentes virales de lafamilia Herpesviridae , los mas usados se basanen la amplificacion del gen de la enzima ADNpolimerasa que permite la deteccion de virusherpes simplex (VHS) 1 y 2, virus varicela-zoster(VVZ), citomegalovirus (CMV), virus de Epstein-Barr (VEB) y herpesvirus humano (HVH) 6 enforma simultanea. Esta metodologia ha detectadola coinfeccion por VHS 1 y VZV en muestras deliquido cefalorraquideo. En CMV son utilizadosen el monitoreo de la reactivacion de CMV enpacientes inmunosuprimidos siendo capaz de de-tectar reactivacion viral con una semana de anti-cipacion a la aparicion de los sintomas. Losmetodos moleculares han permitido la identifica-cion del VEB en una proporcion de 8 a 20% decasos de cancer gastrico los cuales poseen unacepa unica a pesar de la presencia de multiplescepas en la poblacion sana. Estas asociacionesentre virus y cancer tambien se han descrito parael virus papiloma humano y cancer pulmonar. Enagentes bacterianos, la deteccion y cuantificacionde

Identificación de Virus Papiloma Humano 16 (vph-16) en carcinoma queratinizante de pulmón

El cancer pulmonar constituye la primera causa de muerte por cancer en el mundo y la cuarta causa de muerte por cancer en Chile. El carcinoma escamoso de pulmon representa entre el 35% a 50% de los casos de cancer pulmonar. Existe fuerte evidencia, aunque aun controversial, respecto de la asociacion entre esta forma histologica y la infeccion por Virus Papiloma Humano (VPH), siendo los genotipos VPH 16 y 18 los que se han asociado a lesiones malignas y premalignas de diversos tejidos epiteliales. Analizamos casos de carcinoma escamoso de pulmon del tipo queratinizante para evaluar la presencia de genotipos de VPH 16 y 18 en Chile. Quince casos con diagnostico histologico de carcinoma escamoso moderada y altamente diferenciados en tejido incluido en parafina, fueron tratados con xilol y etanol y resuspendidos en proteinasa K durante 48 horas a 56o C para la extraccion de ADN. Este se amplifico mediante la reaccion de polimerasa en cadena (PCR) usando partidores especificos para VPH generico, VPH 16, VPH 18 y betaglobina humana como control positivo interno. Los amplificados fueron revelados en geles de polacrilamida y tincion con nitrato de plata. Identificamos la presencia de VPH generico en 6 (42,2%) de 13 casos amplificables. De estos casos todos correspondieron al genotipo VPH 16 y ninguno correspondio al genotipo VPH 18. La presencia de VPH 16 en la serie analizada indicaria que VPH puede tener algun rol en cancer pulmonar del tipo escamoso - queratinizante. Es interesante la ausencia de VPH 18 en la serie analizada lo cual podria indicar caracteristicas epidemiologicas propias en nuestra poblacion. En esta serie analizada, una muestra mostro no corresponder a los genotipos estudiados. Es necesario realizar un estudio mas amplio con otros genotipos de VPH y un universo mayor de casos para confirmar estos resultados