Priscilla Brebi

Epigenetic alterations in preneoplastic and neoplastic lesions of the cervix

Cervical cancer (CC) is one of the most malignant tumors and the second or third most common type of cancer in women worldwide. The association between human papillomavirus (HPV) and CC is widely known and accepted (99.7% of cases). At present, the pathogenesis mechanisms of CC are not entirely clear. It has been shown that inactivation of tumor suppressor genes and activation of oncogenes play a significant role in carcinogenesis, caused by the genetic and epigenetic alterations. In the past, it was generally thought that genetic mutation was a key event of tumor pathogenesis, especially somatic mutation of tumor suppressor genes. With deeper understanding of tumors in recent years, increasing evidence has shown that epigenetic silencing of those genes, as a result of aberrant hypermethylation of CpG islands in promoters and histone modification, is essential to carcinogenesis and metastasis. The term epigenetics refers to heritable changes in gene expression caused by regulation mechanisms, other than changes in DNA sequence. Specific epigenetic processes include DNA methylation, chromotin remodeling, histone modification, and microRNA regulations. These alterations, in combination or individually, make it possible to establish the methylation profiles, histone modification maps, and expression profiles characteristic of this pathology, which become useful tools for screening, early detection, or prognostic markers in cervical cancer. This paper reviews recent epigenetics research progress in the CC study, and tries to depict the relationships between CC and DNA methylation, histone modification, as well as microRNA regulations.

Immunohistochemical Expression of Phospho-mTOR Is Associated With Poor Prognosis in Patients With Gallbladder Adenocarcinoma

CONTEXT Advanced gallbladder carcinoma (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in cell growth and homeostasis. Its regulation is frequently altered in various tumors and is an attractive target for cancer therapy; however, its status in GBC remains unclear. OBJECTIVE To characterize immunohistochemical expression and prognostic significance of phospho-mTOR in advanced gallbladder carcinoma. DESIGN Phospho-mTOR expression was examined by immunohistochemistry in tissue microarrays containing 128 advanced GBCs and 99 cases of chronic cholecystitis, which were divided into 2 groups according to the presence or absence of metaplasia. To evaluate the association of the level of phospho-mTOR expression with clinical variables and patient survival, the advanced GBCs were classified as having low or high expression. Statistical analysis was performed by using a significance level of P < .05, and Kaplan-Meier curves were constructed for survival analysis. RESULTS Immunostaining for phospho-mTOR was positive in 82 of 128 tumors (64.1%) and in 24% of chronic cholecystitis cases (16% nonmetaplasia and 32% with metaplasia) (P < .001). Survival analysis indicated that a high phospho-mTOR immunohistochemical expression was associated with poorer prognosis in patients with advanced GBC (P = .02). CONCLUSIONS Metaplasia is a common finding in chronic cholecystitis and is considered a precursor lesion of dysplasia. Our results suggest that the activation of mTOR occurs very early during the development of GBC, contributing to the carcinogenesis process. Phospho-mTOR expression is correlated with poor survival, supporting the potential of mTOR for targeted therapy.

Emerging Role of miRNAs in the Drug Resistance of Gastric Cancer

Gastric cancer is the third leading cause of cancer mortality worldwide. Unfortunately, most gastric cancer cases are diagnosed in an advanced, non-curable stage and with a limited response to chemotherapy. Drug resistance is one of the most important causes of therapy failure in gastric cancer patients. Although the mechanisms of drug resistance have been broadly studied, the regulation of these mechanisms has not been completely understood. Accumulating evidence has recently highlighted the role of microRNAs in the development and maintenance of drug resistance due to their regulatory features in specific genes involved in the chemoresistant phenotype of malignancies, including gastric cancer. This review summarizes the current knowledge about the miRNAs’ characteristics, their regulation of the genes involved in chemoresistance and their potential as targeted therapies for personalized treatment in resistant gastric cancer.

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia, associated with HPV status and ethnicity in a Chilean population

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.

DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer.

Gallbladder cancer is an infrequent neoplasia with noticeable geographical variations in its incidence around the world. In Chile, it is the main cause of death owing to cancer in women over 40 years old, with mortality rates up to 16.5 per 100,000 cases. The prognosis is poor with few therapeutic options; in advanced cases there is only a 10% survival at 5 years.Several studies mention the possible role of DNA methylation in gallbladder carcinogenesis. This epigenetic modification affects tumor suppressor genes involved in regulation pathways, cell cycle control, cell adhesion and extracellular matrix degradation, in a sequential and cumulative way. Determining DNA methylation patterns would allow them to be used as biomarkers for the early detection, diagnosis, prognosis and/or therapeutic selection in gallbladder cancer.

Cellular FLICE-like inhibitory protein long form (c-FLIPL) overexpression is related to cervical cancer progression.

Cervical cancer is a leading cause of cancer deaths in women worldwide and infection by high-risk human papillomavirus types is a precursor event. The cellular FLICE-like inhibitory protein (c-FLIP) has been found to be overexpressed in several types of cancers and could be associated with cervical cancer progression because of its ability to inhibit the apoptotic process. To detect c-FLIP expression in cervical cancer, an immunohistochemical staining was performed, using tissue microarrays, on a series of 536 archival biopsy samples, including normal cervical tissues, low-grade and high-grade squamous intraepithelial lesions, and squamous cervical carcinomas. The epithelium in the normal cervix and low-grade squamous intraepithelial lesions mainly stained negatively for c-FLIP, whereas high-grade intraepithelial lesions and cancer samples showed an elevated expression of c-FLIP. A direct association was observed between the increasing grade of the lesion and the intensity of c-FLIP staining, in which the frequency of intense c-FLIP expression increased from 12.5% in the normal tissue to 82.1% in the cervical cancer tissue. An increased expression of c-FLIP may be an important factor in the progression of cervical cancer. This finding could aid in identifying patients with preneoplastic lesions at greater risk of developing cervical cancer. c-FLIP expression in cervical tissue may be a potential cervical cancer progression marker.

Genotyping of human papillomavirus in cervical intraepithelial neoplasia in a high-risk population†

Infection with the human papillomavirus (HPV) is responsible for 99.7% of cervical cancers, the second most prevalent neoplasia in women worldwide and the fifth leading cause of death by cancer in this population. In Chile, the incidence rate is 14.4 cases per 100,000 women per year and it is considered a significant public health problem. The natural history of cervical cancer begins gradually from low‐grade and high‐grade squamous intraepithelial lesions to an invasive disease. In this study the frequency of HPV types was determined by HPV genotyping with reverse line blot hybridization in 200 cytobrushes of women with preneoplastic lesions in a high‐risk population. HPV DNA was found in 89% of the lesions (83.3% of low‐grade squamous intraepithelial lesions and 93.6% of high‐grade squamous intraepithelial lesions). Multiple HPV infections were found in 14.4% and 15.5% of low‐ and high‐grade lesions, respectively. HPV 16 was the most frequent genotype in single infections, followed by HPV 18. These results show that most of the preneoplastic lesions of the cervix (60%) were associated with HPV 16 and/or HPV 18, supporting the implementation of an HPV vaccination program in this high‐risk population. J. Med. Virol. 83:833–837, 2011.

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.

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G‐415 cells, and the effects on cell viability, anchorage‐independent growth and migration was assessed by comparing them to scrambled vector‐transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage‐independent growth (P < 0.05). An increased p27 expression was observed in G‐415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells.

Diagnóstico de la infección por virus papiloma humano en el hombre

El virus papiloma humano (VPH) corresponde a la infeccion de transmision sexual (ITS) mas frecuente en el mundo, afectando a hombres y mujeres por igual. En hombres, el VPH ha sido asociado principalmente a lesiones como verrugas ano-genitales y en los ultimos anos a neoplasias intraepiteliales de pene y ano. La prevalencia de VPH en el hombre es muy variada, dependiendo del tipo de muestra y las tecnicas de deteccion. Las muestras celulares del cuerpo del pene, glande, prepucio, surco coronal, orina y semen han sido las mas utilizadas y su deteccion se realiza habitualmente con tecnicas de reverse line blot (RLB) y captura hibrida (CH). Dado a que las mayores tasas de infeccion se encuentran en Africa y Latinoamerica, esta revision tiene como objetivo describir la patogenia de VPH y sus principales tecnicas de deteccion en el hombre.