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

[Epigenetics in the pathogenesis and early detection of gastric cancer]. / 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.

[Human papillomavirus (HPV) genotypes in cervix uterine cancer patients in a public hospital and private clinic from Santiago, Chile]. / Genotipos de virus papiloma humano (VPH) en pacientes con cáncer cervico-uterino en un hospital público y una clínica privada de Santiago, Chile.

We compared HPV genotypes among squamous cervical cancer samples from a public hospital (n = 55) and a private clinic (n = 35 cases) of Santiago. Paraffin-embedded specimens were analyzed by PCR followed by an immunoenzimatic assay. Reverse line blotting was used for the identification of 36 HPV genotypes. We found HPVDNAm 94.4% of all cancers. Single mfections: HPV16: 40.0%, (clinic 37.1%, hospital 41.8%) VPH18:7.8% (clinic 2.9%, hospital 10.9%); single+multiple mfections: VPH16: 61.1% (clinic 53.1%, hospital 71.7%), VPH18: 34.4% (clinic 21.9%, hospital 45.2%). HPV16 orHPV18 occurredin 75.6% of cases, higher in the hospital than the clinic (87.3%-95% CI: 84.9-96.3 - and 57. l%-95% CI: 46.6-66 - respectively, p = 0.002). Other genotypes in single mfections: HPV 26, 31, 33, 45, 58, 67; in co-mfections: HPV 35,52,56,59 and 66. HPV16 but specially HPV 18 were significantly more frequent in the public hospital; 75.6% of squamous cervical cancer were associated to the vaccine preventable HPV16/18.

Genotipos de virus papiloma humano (VPH) en pacientes con cáncer cervico-uterino en un hospital público y una clínica privada de Santiago, Chile / Human papillomavirus (HPV) genotypes in cervix uterine cancer patients in a public hospital and private clinic from Santiago, Chile

We compared HPV genotypes among squamous cervical cancer samples from a public hospital (n = 55) and a private clinic (n = 35 cases) of Santiago. Paraffin-embedded specimens were analyzed by PCR followed by an immunoenzimatic assay. Reverse line blotting was used for the identification of 36 HPV genotypes. We found HPVDNAm 94.4 percent of all cancers. Single mfections: HPV16: 40.0 percent, (clinic 37.1 percent, hospital 41.8 percent) VPH18:7.8 percent (clinic 2.9 percent, hospital 10.9 percent); single+multiple mfections: VPH16: 61.1 percent (clinic 53.1 percent, hospital 71.7 percent), VPH18: 34.4 percent (clinic 21.9 percent, hospital 45.2 percent). HPV16 orHPV18 occurredin 75.6 percent of cases, higher inthe hospital than the clinic (87.3 percent-95 percent CI: 84.9-96.3 - and 57. l percent-95 percent CI: 46.6-66 - respectively, p = 0.002). Other genotypes in single mfections: HPV 26, 31, 33, 45, 58, 67; in co-mfections: HPV 35,52,56,59 and 66. HPV16 but specially HPV 18 were significantly more frequent in the public hospital; 75.6 percent of squamous cervical cancer were associated to the vaccine preventable HPV16/18.

Se comparan los genotipos de VPH en casos de cáncer cérvico-uterino escamocelular de una clínica privada (n: 35) y de un hospital público (n: 55) atendidos entre 1996 y 2006 en Santiago, Chile. Se analizaron por RPC y ensayo inmunoenzimático muestras tumorales en bloques de parafina, genotipificándose con reverse Une blotting para 36 genotipos de VPH. Se detectó VPH en 94,4 por ciento de los casos: infecciones únicas por: VPH 16: 40,0 por ciento>, (clínica 37,1 por ciento, hospital 41,8 por ciento) VPH 18: 7,8 por ciento (clínica 2,9 por ciento, hospital 10,9 por ciento); total de infecciones por VPH 16 61,1 por ciento (clínica 53,1 por ciento, hospital 71,7 por ciento), por VPH 18 34,4 por ciento (clínica 21,9 por ciento, hospital 45,2 por ciento). Co-infección: VPH 16/18 75,6 por ciento (clínica 57,1 por ciento; IC95 por ciento = 46,6-66,0 hospital 87,3 por ciento; IC95 por ciento = 84,9-96,3, p = 0,002). Se identificó otros 11 genotipos oncogénicos en infecciones únicas (VPH: 26, 31, 33, 45, 58, 67) o en co-infección con VPH 16/18 (VPH: 35, 52, 56, 59, 66). VPH 16 y VPH 18 fueron significativamente más frecuentes en el hospital público, particularmente VPH18; 75,6 por ciento> de los cánceres se asociaron a los genotipos VPH 16/18, tipos prevenibles por vacuna.

[Hierarchical clustering analysis to detect associations between clinical and pathological features of gastric tumors and hypermethylation of suppressor genes]. / Identificación de asociaciones clínico-patológicas e hipermetilación de genes supresores de tumores en cáncer gástrico difuso a través de análisis de Hierarchical clustering.

BACKGROUND: Methylation is an inactivation mechanism for tumor suppressor genes, that can have important clinical implications. AIM: To analyze the methylation status of 11 tumor suppressor genes in pathological samples of diffuse gastric cancer. MATERIAL AND METHODS: Eighty three patients with diffuse gastric cancer with information about survival and infection with Epstein Barr virus, were studied. DNA was extracted from pathological slides and the methylation status of genes p14, p15, p16, APC, p73, FHIT, E-cadherin, SEMA3B, BRCA-1, MINT-2 y MGMT, was studied using sodium bisulphite modification and polymerase chain reaction. Results were grouped according to the methylation index or Hierarchical clustering (TIGR MultiExperiment Viewer). RESULTS: Three genes had a high frequency of methylation (FHIT, BRCA1, APC), four had an intermediate frequency (p15, MGMT, p14, MINT2) and four had a low frequency (p16, p73, E-cadherin, SEMA3B). The methylation index had no association with clinical or pathological features of tumors or patients survival. Hierarchical clustering generated two clusters. One grouped clinical and pathological features with FHIT, BRCA1, and APC and the other grouped the other eight genes and Epstein Barr virus infection. Two significant associations were found, between APC and survival and p16/p14 and Epstein Barr virus infection. CONCLUSIONS: Hierarchical clustering is a tool that identifies associations between clinical and pathological features of tumors and methylation of tumor suppressor genes.

Identificación de asociaciones clínico-patológicas e hipermetilación de genes supresores de tumores en cáncer gástrico difuso a través de análisis de Hierarchical clustering / Hierarchical clustering analysis to detect associations between clinical and pathological features of gastric tumors and hypermethylation of suppressor genes

Background:Methylation is an inactivation mechanism for tumor suppressor genes, that can have important clinical implications. Aim: To analyze the methylation status of 11 tumor suppressor genes in pathological samples of diffuse gastric cancer. Material and methods: Eighty three patients with diffuse gastric cancer with information about survival and infection with Epstein Barr virus, were studied. DNA was extracted from pathological slides and the methylation status of genes p14, p15, p16, APC, p73, FHIT, E-caderin, SEMA3B, BRCA-1, MINT-2 y MGMT, was studied using sodium bisulphite modification and polymerase chain reaction. Results were grouped according to the methylation index or Hierarchical clustering (TIGR MultiExperiment Viewer). Results: Three genes had a high frequency of methylation (FHIT, BRCA1, APC), four had an intermediate frequency (p15, MGMT, p14, MINT2) and four had a low frequency (p16, p73, E-cadherin, SEMA3B). The methylation index had no association with clinical or pathological features of tumors or patients survival. Hierarchical clustering generated two clusters. One grouped clinical and pathological features with FHIT, BRCA1, and APC and the other grouped the other eight genes and Epstein Barr virus infection. Two significant associations were found, between APC and survival and p16/p14 and Epstein Barr virus infection. Conclusions: Hierarchical clustering is a tool that identifies associations between clinical and pathological features of tumors and methylation of tumor suppressor genes.

[Pathogenic role of PTEN tumor suppressor gene in ovarian cancer associated to endometriosis]. / 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.

Rol patogénico del gen supresor de tumores PTEN en cáncer ovárico asociado a endometriosis / Pathogenic role of PTEN tumor suppressor gene in ovarian cancer associated to 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.

Características clínico-moleculares del cáncer gástrico cardial asociado al virus Epstein Barr / Clinical and molecular features of cardial gastric cancer associated to Epstein Barr virus

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 virus.

Biología molecular en infectología: parte II, diagnóstico molecular de agentes infecciosos / Molecular biology in infectius diseases: part II molecular diagnosis of infectious agents

Las aplicaciones diagnósticas de la biología molecular para enfermedades infecciosas son extremadamente variadas y aplicables a cualquier problema diagnóstico. En agentes virales de la familia Hepersviridae, los más usados se basan en la amplificación del gen de la enzima ADN polimerasa que permite la detección de virus herpes simplex (VHS) 1 y 2, virus varicela-zoster (VVZ), citomegalovirus (CMV), virus de Epstein Barr (VEB) y herpesvirus humano (HVH) 6 en forma simultánea. Esta metodología ha detectado la coinfección por VHS 1 y VZV en muestras de líquido cefalorraquideo. En CMV son utilizados en el monitoreo de la reactivación de CMV en pacientes inmunosuprimidos siendo capaz de detectar reactivación viral con una semana de anticipación a la aparición de los síntomas. Los métodos moleculares han permitido la identificación del VEB en una proporción de 8 a 20 por ciento de casos de cáncer gástrico los cuales poseen una cepa única a pesar de la presencia de múltiples cepas en la población sana. Estas asociaciones entre virus y cáncer también se han descrito para el virus papiloma humano y cáncer pulmonar. En agentes bacterianos, la detección y cuantificación de Bordetella pertussis es otra aplicación relevante ya que podría convertirse en un método de diagnóstico rápido y predictivo de severidad de enfermedad en niños menores de 6 meses. La caracterización de cepas de Helicobacter pylori en relación con cáncer gástrico y enfermedadulcerosa péptica, y la caracterización de cepas nosocomiales de Staphylococcus aureus resistente a meticilina (SAMR), son ejemplos de las potencialidades de los métodos moleculares en la tipificación de microorganismos. En el diagnóstico de agentes causantes de patologías respiratorias como Mycobacterium tuberculosis, Pneumocystis carinil y agentes atipicos de infecciones respiratorias, estos métodos han permitidoel diagnóstico a partir de muestras de obtención no invasora. Finalmente, también han demostrado su aporto en el diagnóstico de infecciones micóticas (candidiasis y aspergilosis), n particular en pacientes inmunocomprometidos

Identificación de virus papiloma humano 16 en carcinoma queratinizante de pulmón / Identification of human papillomavirus 16 in keratinizing lung carcinoma

El cáncer pulmonar constituye la primera causa de muerte por cáncer en el mundo y la cuarta causa de muerte por cáncer en Chile. El carcinoma escamoso de pulmón representa entre 35 por ciento a 50 por ciento de los casos de cáncer pulmonar. Existe fuerte evidencia, aunque aun controversial, respecto de la asociación entre esta forma histológica y la infección 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 carcinomas escamosos de pulmón del tipo queratinizante para evaluar la presencia de genotipos de VPH 16 y 18 en Chile. Quince casos con diagnóstico histológico 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 56 C para la extracción de ADN. Este se amplificó mediante la reacción de polimerasa en cadenas (PCR) usando partidores específicos para VPH genérico, VPH 16, VPH 18 y betaglobina humana como control positivo interno. Los amplificados fueron revelados en geles de polacrilamida y tinción con nitrato de plata. Identificamos la presencia de VPH genérico en 6 (42,2 por ciento) de 13 casos amplificables. De estos casos todos correspondieron al genotipo VPH 16 y ninguno correspondió al genotipo VPH 18. La presencia de VPH 16 en la serie analizada indicaría que VPH puede tener algún rol en cáncer pulmonar del tipo escamoso - queratinizante. Es interesante la ausencia de VPH 18 en la serie analizada lo cual podría indicar características epidemiológicas propias en nuestra población. En esta serie analizada, una muestra mostró no corresponder a los genotipos estudiados. Es necesario realizar un estudio más amplio con otros genotipos de VPH y un universo mayor de casos para confirmar estos resultados

Reordenamiento de inmunoglobulinas en el diagnóstico diferencial del linfoma gástrico primario / Immunoglobulin rearrangement in the differential diagnosis of primary gastric lymphoma

Background: The traditional methods to distinguish Chronic Follicular Gastritis and Primary Gastric Lymphoma do not allow an adequate definitive diagnosis in a significant number of cases. The Molecular Biology diagnostic methods are based on the rearrangement of immunoglobulin genes. The polymerase chain reaction (PCR) specifically amplifies this rearrangement and allows molecular analysis of minimal tissue samples obtained with endoscopical biopsies. Aim: To test the usefulness of this PCR method in the differential diagnosis between Chronic Follicular Gastritis and Primary Gastric Lymphoma. Material and methods: We analyzed the endoscopical biopsies of six Chronic Follicular Gastritis cases and eight surgically treated Primary Gastric Lymphoma cases, six with the correct diagnosis in the endoscopical biopsies and two with a diagnosis of Chronic Follicular Gastritis. Results: A policlonal immunoglobulin rearrangement was found in the six cases with Chronic Follicular Gastritis. A monoclonal arrangement was found in 5 of 6 biopsies with the diagnosis of Primary Gastric Lymphoma. The same monoclonal rearrangement was observed in the two biopsies incorrectly diagnosed as Chronic Follicular Gastritis. Conclusions: PCR analysis of immunoglobulin rearrangement is a useful method in the differential diagnosis between Chronic Follicular Gastritis and Primary Gastric Lymphoma