Methylation of p16 ink4a promoter is independent of human papillomavirus DNA physical state: a comparison between cervical pre-neoplastic and neoplastic samples

BACKGROUND Epigenetic modifications in host cells, like p16 ink4a methylation, have been considered as putative complementary mechanisms for cancer development. Because only a small proportion of infected women develop cervical cancer, other factors might be involved in carcinogenesis, either independently or in association with high-risk human papillomavirus (HR-HPV) infections, including epigenetic factors. OBJECTIVES We hypothesised that p16 ink4a methylation might have a role in cancer development driven by HPV16, mainly in the presence of intact E1/E2 genes. Thus, our objectives were to assess the status of p16 ink4a methylation and the HPV16 E1/E2 integrity in samples in different stages of cervical diseases. METHODS Presence of HPV16 was determined by E6 type-specific polymerase chain reaction (PCR). Methylation status of the p16 ink4a promoter was assessed by methylation-specific PCR in 87 cervical specimens comprising 29 low-grade (LSIL), 41 high-grade (HSIL) lesions, and 17 cervical cancers (CC). Characterisation of E1 and E2 disruption (as an indirect indicator of the presence of episomal viral DNA) was performed by PCR amplifications. FINDINGS We observed a significantly increased trend (nptrend = 0.0320) in the proportion of methylated p16 ink4a in cervical samples during cancer development. Concomitant E1 and E2 disruptions were the most frequent pattern found in all groups: CC (76%), HSIL (54%), and LSIL (73%). No statistically significant differences between p16 ink4a methylation and E1/E2 integrity, in histological groups, was observed. MAIN CONCLUSIONS There was an increase in methylation of the p16 ink4a promoter from pre-neoplastic lesions to cancer. Additionally, a high frequency of E1/E2 disruptions in LSIL/HSIL suggested that viral DNA integration was an early event in cervical disease. Moreover, the methylation status was apparently independent of HPV16 integrity.

Tipificación viral en el seguimiento de conización: rol pronóstico de la persistencia del HPV post cono con asa / Viral typing in conization monitoring: prognostic role of HPV persistence post cone with loop

RESUMEN Fundamento: La persistencia del virus papiloma posterior a la conización del cuello uterino, se ha considerado un factor de riesgo para la persistencia de lesiones intra epiteliales (LIE) causadas por virus papiloma. Para determinar la asociación entre persistencia de lesión cervical y la presencia del virus papiloma posterior a la conización del cuello uterino, se realizó un estudio observacional prospectivo en un grupo de 123 pacientes portadoras de lesiones intraepiteliales de alto grado (LIEAG) tratadas con conización. Material y métodos: Se siguieron a 123 pacientes portadoras de LIEAG, ingresadas a la Unidad de Patología Cervical entre Abril de 2013 y Abril de 2014, las que fueron seguidas por 2 años hasta Abril de 2016. Se realizó genotipificación antes, y entre 4 a 6 meses posterior a la conización. Los datos se tabularon considerando la edad, paridad, tipo de virus, persistencia de LIE, reconización o requerimiento de histerectomía posterior. Resultados: La mediana de la edad fue de 37 años, el 91% fueron multíparas, y solo el 9% fueron nulíparas. El 56% ingresó por NIE III y el 44% por NIE II. Los virus más frecuentes fueron el 16, 31,58, 52 y 56. La persistencia de virus papiloma se constató en el 37% de las pacientes conizadas. La persistencia de LIE se observó en el 27% de las pacientes que fueron positivas para virus papiloma posterior a la conización, en comparación a sólo el 5% en las que fueron negativas. Del total de pacientes positivas para virus papiloma posterior a la conización, 12 de ellas presentaron persistencia de lesión confirmadas histológicamente por biopsia cervical, 8 pacientes requirieron recono por LIE de alto grado, 2 pacientes fueron a histerectomía y en 2 casos se realizó seguimiento estricto por NIE I. Cuando la tipificación post cono fue negativa solamente 3 pacientes requirieron conización y en sólo una se realizó seguimiento estricto por NIE I. Conclusión: La persistencia del virus papiloma posterior a la conización se asocia a mayor persistencia de LIEAG, mayor frecuencia de reconización o histerectomía posterior.

ABSTRACT Backgroud: The persistence of papilloma virus after conization of the cervix has been considered a risk factor for the persistence of cervical intra epithelial lesion (CIN) caused by papilloma virus. Aim: In order to determine the association between cervical lesion persistence and the presence of papilloma virus after conization, a prospective observational study was performed in a group of 123 patients with intraepithelial lesions treated with conization. Material and methods: We followed 123 patients with high grade CIN who were admitted to the Cervical Pathology Unit, between April 2013 and April 2014; they were followed for 2 years until April 2016. Viral genotyping was done before, and among the 4 to 6 months after the LEEP. Data were tabulated considering age, parity, type of virus, persistence of CIN, reconization or requirement of posterior hysterectomy. Results: The median age was 37 years, 91% were multiparous, and only 9% were nulliparous. 56% had NIE III and 44% NIE II. The most frequent viruses were 16, 31, 58, 52 and 56. The persistence of papillomavirus was present in 37% of patients. The persistence of CIN was observed in 27% of patients who were positive for papilloma virus after conization, compared to only 5% in those who were negative. Of the total number of patients positive for papilloma virus, in 12 of them had intra epitelial lesions were confirmed by cervical biopsy, 8 patients required recone for high grade CIN, 2 patients underwent hysterectomy, and 2 patients underwent follows up strictly by CIN I. When post cone typing was negative only 3 patients required conization and only one was followed strictly by CIN I.

Human papillomavirus and genome instability: from productive infection to cancer

Infection with high oncogenic risk human papillomavirus types is the etiological factor of cervical cancer and a major cause of other epithelial malignancies, including vulvar, vaginal, anal, penile and head and neck carcinomas. These agents affect epithelial homeostasis through the expression of specific proteins that deregulate important cellular signaling pathways to achieve efficient viral replication. Among the major targets of viral proteins are components of the DNA damage detection and repair machinery. The activation of many of these cellular factors is critical to process viral genome replication intermediates and, consequently, to sustain faithful viral progeny production. In addition to the important role of cellular DNA repair machinery in the infective human papillomavirus cycle, alterations in the expression and activity of many of its components are observed in human papillomavirus-related tumors. Several studies from different laboratories have reported the impact of the expression of human papillomavirus oncogenes, mainly E6 and E7, on proteins in almost all the main cellular DNA repair mechanisms. This has direct consequences on cellular transformation since it causes the accumulation of point mutations, insertions and deletions of short nucleotide stretches, as well as numerical and structural chromosomal alterations characteristic of tumor cells. On the other hand, it is clear that human papillomavirus-transformed cells depend on the preservation of a basal cellular DNA repair activity level to maintain tumor cell viability. In this review, we summarize the data concerning the effect of human papillomavirus infection on DNA repair mechanisms. In addition, we discuss the potential of exploiting human papillomavirus-transformed cell dependency on DNA repair pathways as effective antitumoral therapies.

Regulation of HPV transcription

Human papillomavirus infection is associated with the development of malignant and benign neoplasms. Approximately 40 viral types can infect the anogenital mucosa and are categorized into high- and low-risk oncogenic human papillomavirus, depending on their association with the development of cervical carcinoma. High-risk human papillomavirus 16 and 18 are detected in 55% and 15% of all invasive cervical squamous cell carcinomas worldwide, respectively. Low-risk human papillomavirus 6 and 11 are responsible for 90% of genital warts and are also associated with the development of recurrent respiratory papillomatosis. Human papillomavirus preferentially infects mitotic active cells of the basal layer from both mucosal and cutaneous epithelium through microabrasions. The viral life cycle synchronizes with the epithelial differentiation program, which may be due, in part, to the binding of differentially expressed cellular transcription factors to the long control region throughout the various epithelial layers. This review aimed to summarize the current knowledge regarding the mechanisms by which viral gene expression is regulated and the influence of human papillomavirus heterogeneity upon this phenomenon. A better understanding of the regulatory mechanisms may elucidate the particularities of human papillomavirus-associated pathogenesis and may provide new tools for antiviral therapy.

Anal canal squamous carcinoma / Carcinoma epidermóide do canal anal

ABSTRACT Background: Anal canal carcinoma is a rare neoplasm, representing 2% of the digestive tumors, and the most common is squamous cell carcinoma, with an increasing incidence. Objective: The study aims to elucidate the pathogenesis of an increasingly prevalent disease, as well as to update treatment and prognosis. Methods: A literature search in Pubmed database, including articles from 2005 to 2015 and cross-research articles with the initial research. Results: Several studies prove the role of HPV as a major risk factor in the development of squamous cell carcinoma of anal canal, as well as a greater prevalence of this neoplasia in HIV-positive people and in those who practice receptive anal intercourse. In the last two decades chemoradiotherapy remains the treatment of choice, and abdominoperineal resection is reserved for those cases of treatment failure or recurrence. Evidence advances in order to adapt the treatment to each patient, taking into account individual prognostic factors and biological tumor characteristics. Conclusions: Squamous cell carcinoma of the anal canal is a neoplasm associated with HPV; therefore, screening and vaccination programs of male individuals, by way of prevention, should be started. Many studies are needed in order to achieve development in the treatment as well as in the evaluation of the biological characteristics of the tumor.

RESUMO Introdução: O carcinoma do canal anal é uma neoplasia rara, representando 2% dos tumores digestivos, sendo o epidermóide o mais comum com uma incidência crescente. Objetivo: Este estudo pretende elucidar sobre a etiopatogenia desta patologia cada vez mais prevalente, assim como atualizar sobre o tratamento e prognóstico. Métodos: Pesquisa bibliográfica na base de dados Pubmed, incluindo artigos de 2005 a 2015, assim como artigos de pesquisa cruzada com os artigos iniciais. Resultados: Diversos estudos provam o papel do HPV como um fator de risco major no desenvolvimento de carcinoma epidermóide do canal, assim como uma maior prevalência desta neoplasia na população HIV positiva e nos que praticam sexo anal recetivo. O tratamento continua a ser desde há duas décadas a quimioradioterapia, reservando a resseção abdominoperineal para casos de falência do tratamento ou recorrência. A evidência avança no sentido de adequar o tratamento a cada doente, tendo em conta fatores prognósticos individuais e as características biológicas do tumor. Conclusões: O carcinoma epidermóide do canal anal é uma neoplasia associada ao HPV, logo deveria iniciar-se programas de rastreio e vacinar o sexo masculino como prevenção. Muitos estudos são necessários para evoluir no tratamento, assim como na avaliação das características biológicas do tumor.

Knowledge of HPV and Surgery among Women Who Underwent Cervical Conization: A Korean Multi-Center Study

PURPOSE: Human papillomavirus (HPV) infection is a well-known cause of cervical cancer, which, along with its precursors, can be diagnosed and treated with cervical conization (CC). This study aimed to assess HPV- and procedure-related knowledge among women who had undergone CC. MATERIALS AND METHODS: Between February and May 2014, consecutive women who had undergone CC at five different educational hospitals were recruited. All patients had undergone a loop electrosurgical excision procedure as the method of CC. A survey was conducted with a self-developed, 29-item questionnaire, measuring knowledge related to HPV and CC. We analyzed the responses of 160 patients who completed the questionnaire. RESULTS: Mean total knowledge scores (±standard deviation) for HPV and CC were 5.2±3.0 of a possible 13.0 and 8.3±4.2 of a possible 16.0, respectively. While 73% of the patients knew that HPV is the main cause of cervical cancer, only 44% knew that HPV is sexually transmitted. The purpose of CC was correctly identified by 71% of the patients. However, 35% failed to indicate the anatomical area resected at the time of CC in the schematic diagram. Women who were younger (p<0.001), had higher education level (p<0.001), and higher family income (p=0.008) had higher knowledge scores. In contrast, neither interval from CC to survey nor disease severity were associated with total knowledge score. CONCLUSION: The level of knowledge related to HPV and CC was unexpectedly low in women who had undergone CC. Intuitive educational resources may improve this knowledge, and further cohort studies are warranted.

Associação entre a infecção pelo papilomavírus humano (HPV) e outras infecções genitais femininas / Association between human papillomavirus (HPV) infection and other genital infections

Avaliação da associação entre a infecção pelo HPV e outras infecções genitais, sendo acompanhadas 80 pacientes, submetidas a exame ginecológico com colposcopia, biópsia de colo uterino e captura híbrida. Observou-se redução da carga viral do HPV após o tratamento cervical, seja nas lesões de baixo ou alto grau (p=0,02). Além disso, as infecções genitais (vaginose bacteriana e candidíase) foram mais comuns nas pacientes com HPV identificadas à captura híbrida.

To evaluate the association between HPV infection and other genital infections were studied 80 patients submitted to gynecological examination with colposcopy, biopsy of the cervix and hybrid capture. There wasreduction in viral load of HPV after treatment cervical, or in lesions of low or high grade (p = 0.02). Moreover, genital infections (bacterial vaginosis and candidiasis) were more common in patients with the hybrid captureHPV identified.

Characterization of newly established oral cancer cell lines derived from six squamous cell carcinoma and two mucoepidermoid carcinoma cells

Since genetic abnormalities of human cancer are greatly geographically dependent, cultural and environmental backgrounds are thought to be closely related to the carcinogenic process. In the present study, eight human cell lines were established by culture from untreated carcinomas of the oral cancer, of which five were from primary oral squamous cell carcinomas (OSC), one from a mucoepidermoid carcinoma (MEC) and one each originating from metastatic OSC and MEC. All the studied tumor lines grew as monolayers, and showed: i) an epithelial origin by the presence of cytokeratin, and ii) tumorigenic potential in nude mice. Western blot analysis revealed i) over expression of EGFR in six of the cell lines ii) decreased expression of E- cadherin in six cell lines compared to normal human oral mucosa. A mutational analysis showed: point mutations of p53 at exon 7, with transversion, and at exon 8, with transition. These well-characterized human YD cell lines should serve as useful tools in the study of the molecular pathogenesis and biological characteristics of head and neck cancer cells, and in the future testing of new therapeutic reagents for oral cancer.

Mecanismos de oncogénesis viral / Mechanisms of viral oncogenesis

Actualmente se sabe que el 20 por ciento de los cánceres humanos están asociados con virus oncogénicos. El virus papiloma humano con cáncer anogenital, los virus de la hepatitis B y C con carcinoma hepatocelular, el virus Epstein Barr con carcinomas nasofaríngeos y linfomas, el virus de la leucemia-linfoma T con leucemias en el adulto. Un rasgo común en todos los tumores asociados con infección viral es el largo período de latencia entre la infección y la aparición de la neoplasia y la baja proporción de individuos infectados que desarrollan un tumor maligno. Estas observaciones indican que los virus oncogénicos son necesarios pero no suficientes para inducir cáncer, otros factores podrían estar involucrados. Esta actualización resume informaciones recientes acerca de los mecanismos de carcinogénesis viral, en particular, la interacción de oncoproteínas virales y proteínas supresoras tumorales. La inactivación de estas proteínas supresoras podría representar una estrategia común a través de la cual los virus tumorales pueden contribuir a la transformación maligna de la célula

Prevalencia de los genotipos del virus del papiloma humano en neoplasia intraepitelial cervical y cáncer cervical en Bogotá

Se decribe la prevalencia del Virus del Papiloma Humano(VPH) mediante Hibridación in Situ(HIS), genotipos 6-11-16-18 y 31-33-35, en una muestra de 266 biopsias cervicales de mujeres residentes en Bogotá, seleccionadas según el criterio de infección por VPH (atipia coilocítica), presencia de Neoplasia Intracervical o Cáncer. 27 biopsias(10.1 por ciento) fueron desechadas. La HIS fue positiva en 81/239 casos (33.9 por ciento). VPH 6-11 se detectó e 6/81 casos(7.4 por ciento), los tipos 16-18 en 47/81 (58 por ciento), y los tipos 31-33-35 en 28/81(34.6 por ciento). 3/12 casos (25 por ciento) de Cáncer Infiltrante fueron positivos a la HIS, todos para 16-18. Teniendo en cuenta la baja prevalencia de 6-11 y la alta de 16-18 y 31-35 demostradas en el presente estudio en relación a las informadas en la literatura internacional, se confirma una variación geográfica de la prevalencia de los diferentes tipos de VPM. En esta muestra de Bogotá las displasias, tanto de Bajo como de Alto Grado, tienen una alta prevalencia de los genotipos de VPH de riesgo oncogénico elevado. Este hallazgo es consistente con los estudios epidemiológicos que demuestran que en esta zona geográfica se encuentran los índices más altos del mundo de Carcinoma de Cérvix

El Papel del papiloma virus humano en las displasias del cuello uterino / Role of human papillomavirus in uterine cervix dysplasia

Partiendo del concepto de que el cáncer cérvico uterino es curable cuando se trata al inicio de su evolución, se ha insistido en la necesidad de efectuar un diagnóstico temprano, y hasta hoy la práctica de la prueba de Papanicolau -como método de rastreo- no ha sido superada y, mas bien, por el contrario el que una mujer no tenga los controles citológicos anuales constituye el principal factor de riesgo de desarrollar, primero las lesiones displasias y luego, el cáncer en todas sus etapas. Actualmente la responsabilidad del papiloma virus humano (HIP) esta siendo objeto de múltiples estudios, a fin de determinar su responsabilidad en la génesis de esta neoplasia.

Multistep carcinogenesis and genital papillomavirus infection: implications for diagnosis and vaccines

Activated cellular oncogenes (myc and ras, for example) and inactivated anti-oncogenes (p53 or Rb) participate in multistep carcinogenesis. In addition, some high risk human papillomaviruses (HPV) are also involved in uterine cervix carcinomas. Typification of HPV is important for clinical diagnosis. Unravelling the complexities of the immune system and understanding the biochemistry and molecular genetics of cellular oncogenes and tumor viruses have opened up new possibilities for vaccination

Interacción del Virus del Papiloma Humano con genes supresores de Tumors / Interaction of the CPH with Tumor Suppressor genes

Los virus del papiloma humano de tipo 16 y 18 está asociados al desarrollo de cáncer ano-genital. La actividad transformante de estos virus depende de la expresión de los oncogenes virales E6 y E7. Estos dos genes son necesarios y suficientes para la transformación de ketatinocitos humanos. Esta actividad transformante involucra la interacción de estos oncogenes con genes supresores de tumores. El oncogen E7 se une al gen supresor de tumors retinoblastoma (Rb) impidiendo que actúe en forma normal. Por otro lado; el oncogen E6 se asocia al gen supresores de tumores p53 estimulando su degradación. De esta forma los oncogens virales al inactivar funcionalmente genes que controlan negativamente proliferación celular conducen a la transformación.