Alejandra Andana

Evaluation of ZAR1 and SFRP4 methylation status as potentials biomarkers for diagnosis in cervical cancer: exploratory study phase I

Abstract Context: Aberrant hypermethylation of promoter region of tumor suppressor genes could be used as cancer biomarkers. Objective: To test methylation status of ZAR1 and SFRP4 promoter regions as potentials biomarkers for diagnosis of preneoplastic and neoplastic lesions of cervix. Materials and methods: Cytobrush samples were evaluated by Methylation specific PCR (MSP) and quantitative MSP (qMSP). Results: ZAR1 and SFRP4 methylation frequency increased as the grade of lesion increased and the differences between normal and cervical cancer (CC) are statistically significant (p < 0.0001). qMSP showed higher ZAR1 and SFRP4 methylation levels in cancer than normal epithelia (p < 0.001) and preneoplastics lesions (p < 0.01). Discussion: qMSP quantify methylation levels and have high sensitivity and specificity. Conclusion: ZAR1 and SFRP4 qMSP could be used as potential biomarker for CC diagnosis.

Infecciones de Transmisión Sexual en Semen: El Hombre como Vector de Transmisión

En los ultimos anos el estudio de las infecciones de transmision sexual ha cobrado gran importancia debido principalmente al incremento de estas en parejas heterosexuales y hombres que tienen sexo con hombres. En mujeres existe mucha informacion de epidemiologia y patogenesis de estas infecciones, sin embargo, en hombres la informacion es muy escasa debido a que la mayoria no presenta sintomatologia. En los ultimos anos se ha evidenciado un creciente interes en el estudio del semen como via de transmision, debido principalmente a la afinidad de algunos patogenos con los espermatozoides. Dentro de los principales microorganismos infectantes en semen se encuentran Chlamydia trachomatis, Neisseria gonorrhoeae, Mollicutes, Virus de la Inmunodeficiencia Humana tipos 1 y 2, Virus Herpes Simplex 1 y 2, Virus Papiloma Humano, Virus de la Hepatitis B y C, Citomegalovirus, Virus Epstein-Barr y Trichomonas vaginalis.

Genotipificación del virus papiloma humano en mujeres bajo 25 años de edad participantes del Programa Nacional del Cáncer Cérvico-uterino en la Región de la Araucanía, Chile

Resumen Introduccion : En Chile, el cancer cervico-uterino (CCU) es la segunda causa de muerte por neoplasias malignas en la mujer. El principal agente causal es el virus papiloma humano (VPH), descrito como la infec-cion de transmision sexual mas frecuente entre jovenes sexualmente activas. El comienzo precoz de la vida sexual incrementa las posibilidades de infeccion con VPH; esto puede implicar un eventual desarrollo prematuro de neoplasia intraepitelial cervical y CCU, creando un importante problema de salud publica. Objetivo: Presentar la frecuencia del VPH en mujeres bajo 25 anos de edad, participantes del programa de CCU y su seguimiento post-lesion. Material y Metodos: Se genotipificaron 173 muestras cervicales, mediante reaccion de polimerasa en cadena e hibridacion no radioactiva (reverse line blot). Resultados: La frecuencia global del VPH fue 84,8%. El genotipo mas frecuente fue VPH16. En 12,3% la lesion cervical persistio o evoluciono a una mayor. Se encontro 28,9% de mujeres con seguimiento post-lesion irregular; en este grupo, 88% fue VPH (+) y 52% no tuvo registro de Papanicolaou en los ultimos tres anos.BACKGROUND In Chile, cervical cancer (CC) is the second leading cause of death from malignancy in women. The main causal agent of cervical cancer is the human papillomavirus (HPV). This virus is the most common sexually transmitted infection among sexually active youth. An early onset of sexual life increases the chances of HPV infection; this may involve a possible early development of cervical intraepithelial neoplasia and CC, creating a major public health problem. OBJECTIVE To present HPV frequency in women under the age of 25, treated in the CC screening program and their follow-up after histopathological diagnosis. METHODS 173 cervical samples were genotyped by polymerase chain reaction and non-radioactive reverse hybridization (line blot). RESULTS The overall frequency of HPV was 84.8%. HPV16 was the most prevalent. In 12.1% of women the cervical lesion persisted or progressed. 28.9% of women had irregular follow-up; in this group, 88% were HPV(+) and 52% had no record of Pap smear in the past 3 years. DISCUSSION The results reaffirm the usefulness of complementing the Pap and HPV detection as a primary screening tool in sexually active women. They also suggest the possibility of extending the age coverage of the national screening program.

Frecuencia de Virus Papiloma Humano en Tumores no Ginecológicos de la Región de la Araucanía, Chile

El Virus Papiloma Humano (HPV por sus siglas en ingles) es una de las infecciones de transmision sexual mas frecuentes del mundo y se encuentra presente en la mayoria de los canceres de cuello uterino. Se ha descrito su presencia en otros tipos de cancer no ginecologicos como lo son esofago y prostata. Sin embargo, las frecuencias de HPV descritas hasta el momento para estos tipos de cancer son muy variables, y no hay articulos donde se muestren la presencia de HPV en estas neoplasias en Chile. El objetivo de este estudio fue determinar la frecuencia de HPV en muestras de biopsias de tumores no ginecologicos y tejido inflamatorio de pacientes de la region de La Araucania. Se extrajo DNA desde un total de 47 biopsias de pacientes con esofagitis, 25 con carcinoma escamoso esofagico, 20 con hiperplasia nodular de la prostata y 39 con adenocarcinoma prostatico. Estas fueron analizadas por PCR de la region L1 del virus y posterior genotipificacion por reverse line blot. Se detecto HPV en el 53,2% de las muestras de esofagitis, 48% en muestras de carcinoma escamoso esofagico, 15% en hiperplasia nodular de la prostata y un 15,4% en los casos de adenocarcinoma prostatico. Siendo los mas frecuentes los genotipos de HPV 16 y 18, ya sea en infecciones simples o junto con otros genotipos, en lesiones preneoplasicas y neoplasicas de los tejidos estudiados. Existe una alta frecuencia de infeccion por HPV en biopsias de esofagitis y tejido inflamatorio esofagico de pacientes de la region de la Araucania. En los casos de adenocarcinoma prostatico e hiperplasia nodular de la prostata se observa una baja frecuencia de HPV.

Abstract 647: Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC New biomarkers are needed to improve cervical cancer screening technologies, which are mostly based on cytological examination and HPV detection. However, PAP-smear has a low-sensitivity to detect low grade squamous intraepithelial lesions and not all HPV infected women will develop preneoplastic or neoplastic lesions. Previous results of our group showed that genes Gen Z (patent pending), CDH1 and MEGF9 could be hypermethylated in cervical cancer and not in normal epithelia. The aim of this study was to determinate if promoter methylation status of genes (Gen Z, CDH1 and MEGF9) are related with progression or diagnosis of cervical carcinogenesis. For this study, 107 citobrush, urine and blood samples were collected from women who attended to gynecological care in a public health center in Temuco, Chile. Citobrush DNA from 50 normal, 40 low grade squamous intraepithelial lesion (LSIL), 40 high grade squamous intraepithelial lesion (HSIL) and 17 squamous cervical cancer were bisulfite converted for methylation specific PCR (MSP). Bisulfite conversion was confirmed by amplification of a 133-bp fragment of the β-actin. MSP primers were specifically design for CpG island of promotor region of each gene. Gen Z was found 100% methylated in SCC samples, 65% in HSIL, 43% in LSIL and in normal samples only a 26%. MEGF9 and CDH1 genes were found methylated in 36% and 48% of normal samples, 45% and 55% of LSIL, 70% and 77% of HSIL and 47% and 71% of SCC, respectively. All promoter regions studied showed a higher methylation frequency in LSIL, HSIL and SCC than normal samples. Significant statistical differences in Gen Z and CDH1 Methylation frequencies between normal and SCC samples were found (P<0.05). Methylation of Gen Z increased in a sequential and cumulative way as the lesion progress. Our results suggest that the Gen Z could be useful tool for identifying women with a higher risk of progression to cervical cancer. Examination of these biomarkers in a larger, independent cohort is warranted. Grant Support: This investigation was financed by Proyect CORFO-INNOVA N°07CN13PBT-222 and Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141 Citation Format: Priscilla Brebi, Alejandra Andana, Rene Hoffstetter, Carmen Ili, Tamara Viscarra, Ramon Silva, Patricia Garcia, Pamela Leal, Helga Weber, Juan C. Roa. Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2013-647

Abstract 4789: Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix.

Human papillomavirus (HPV) is the most frequent microorganism sexually transmitted and is the causal agent of cervical carcinoma were it could be found in 99.7% of cases. The detection of HPV en women with or without lesions of cervix is generally performed in biopsies and cytobrush, which are invasive samples; therefore many patients do not attend to health care to get HPV test. The aim of this study was to develop a technique capable of detect and genotype HPV in urine samples of women with squamous intraepithelial lesions of cervix. For this study, 87 urine samples from patients with normal epithelia of cervix (25), low grade squamous intraepithelial lesion (LSIL) (24), high grade squamous intraepithelial lesion (HSIL) (38) were collected from women who attended to gynecological care in a public health center in Temuco, Chile. DNA extraction was performed with a manual method. For protein precipitation, Phenol-Chloroform and ammonium acetate were used. DNA precipitation was performed with absolute ethanol. HPV detection and genotyping was performed with the kit HPV Typing (patent pending) which is capable of detect 18 more frequent HPV types and beta globin gen as control of DNA integrity. 20% of urines from normal epithelia women were positive for HPV. Three genotypes were found in normal samples: HPV52, HPV42 and HPV53. LSIL there was a 92% of HPV positivity and the most frequent genotypes were HPV16 and HPV18. In HSIL, 95% of urine sample were positive for HPV. HPV16, HPV18 and HPV31 were the most frequent genotypes in HSIL. Genotypes found in urine samples were similar to world reports. Using the DNA extraction described before and HPV easy typing kit it is possible to detect and genotype HPV in urine samples. These results suggest that is possible a HPV primary screening test in non-invasive sample, complementing PAP smear in order to determinate which women have a higher risk of developing preneoplastic and neoplastics lesions. Grant Support: Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141. Citation Format: Tamara Viscarra, Alejandra Andana, Priscilla Brebi, Doris Menzel, Carmen G. Ili, Ramon Silva, Juan C. Roa, Raul Sanchez. Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2013-4789

Abstract 2956: c-FLIP (L) dual function in cervical cancer.

Cellular FLICE-like inhibitory protein (c-FLIP) is a caspasa-8/10 homolog catalytically inactive that interferes with the efficient formation of DISC. There are three isoforms of this protein: c-FLIPL (long) of 55kDa, c-FLIPS (short) of 26kDa y c-FLIPR (Raji) of 24kDa. c-FLIPS and c-FLIPR functions have been well established: both can block extrinsic pathway of apoptosis by inhibiting procaspasa-8 activation in DISC. However, c-FLIPL function still remains unclear. When c-FLIPL is overexpressed, as in some cancers, has an anti-apoptotic function very similar to c-FLIPS, but also can be a proapoptotic molecule at low concentrations. The aim of this study is to characterize c-FLIP functions in cervical uterine carcinogenesis. Three cervical cancer cell lines were used in this study: SiHa, C-4I and C-33A. c-FLIP expression in cell lines was determined by real time PCR and western blottting. c-FLIP expression was transiently downregulated by siRNA and the silencing effects on cell viability, proliferation and apoptosis were analysed, by comparing with a control negative siRNA-transfected cells. Using MTS analysis, SiHa and C4I c-FLIP transfected cells showed a increment of viability compared with scramble, since 24 hours after transfection in C 4I and 72 hours in SiHa (P Our results suggest that c-FLIPL could be having a dual function in cervical cancer cell lines, both inhibiting proliferation and apoptosis. Grant Support: This investigation was financed by Proyect CORFO-INNOVA N°07CN13PBT-222 and Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141 Citation Format: Carmen G. Ili, Priscilla Brebi, Alejandra Andana, Patricia Garcia, Pamela Leal, Oscar Tapia, Tamara Viscarra, Helga Weber, Juan Roa. c-FLIP (L) dual function in cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2013-2956 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4037: Downregulation of ZNF516 and INTS1 is related with promoter methylation in cervical uterine cancer cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: One of the most common neoplasia in developing countries is cervical uterine carcinoma (CC). HPV infection is recognized as the major risk factor for carcinogenesis of cervix. However, not all infected women will develop a cervical cancer. Several studies have proposed DNA methylation as early biomarker in CC. A higher promoter methylation is associated with lower gene expression. Aim: To evaluate the association between DNA methylation and gene expression of ZNF516 and INTS1 in cervical cancer cell lines. Materials and Methods: DNA isolated from 12 normal and 7 cervical cancer tissue samples was enriched by Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K CpG Islands plus Promoter arrays (MeDIP-chip). Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions by Methylation Specific PCR in a cohort of 221 normal, premalignant, and cervical cancer samples. Relative expression analysis was performed in a normal cervical epithelium cell line (ECT1 E6/E7) and three cervical cancer cell lines (C-4I, SiHa and C-33A) using real time PCR. MSP was also used to examine promoter methylation in normal cervical and cervical cancer cells lines. Results: ZNF516 and INTS1 were differential methylated in the promoter region of normal and cervical cancer samples in the MeDIP-chip discovery project. The methylation frequency was 100% in cervical cancer cell lines. Relative expression of ZNF516 was significantly lower in C-4I and SiHa (p <0.05) in relation with the normal cell line. C-33A showed elevated promoter methylation and a low expression of ZNF516, however, this result was not significant. INTS1 expression was significantly lower in C-33A and SiHa (p <0.05) in relation with the normal cell line. C-4I showed a high promoter methylation and a low expression of INTS1, however, this result was not significant. Conclusions: We have identified ZNF516 and INTS1 as potentials biomarkers for cervical cancer. These genes may be a useful tool for diagnosis and clinical management of cervical cancer. These results need to be confirmed in clinical samples, from an independent laboratory before the gene panel is examined in a Phase II Biomarker Development Trial. Work sponsored by CORFO Project 09CN14-5960 and Postdoctoral Fondecyt Research Project 3120141. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4037. doi:1538-7445.AM2012-4037