RESUMEN
HPV73 is classified as possibly oncogenic. It is neither routinely evaluated in HPV screening, nor covered by any of the prophylactic vaccines. We sought to investigate the carcinogenic characteristics of HPV73. Molecular studies were performed on eight cervix cancer biopsy specimens containing HPV73 from a cross-sectional cancer cohort of 590 women referred to the National Cancer Institute in Rio de Janeiro, Brazil. Transcriptional activity of HPV73 was evaluated by detection of spliced transcripts of E6/E6* and E1^E4 in cDNA created from RNA isolated from fresh tissue. Disruption of viral E1 and E2 genes in the tumor DNA was assessed by overlapping PCR amplification. Evaluation of viral integration was performed using a customized capture panel and next-generation sequencing, and an in-house bioinformatic pipeline. HPV73 E6/E6* transcripts were found in 7/7 specimens with available RNA, and three also had HPV73 E1^E4 transcripts. Disruption of E1 and E2 genes was observed in 4/8 specimens. Integration of HPV73 sequences into the cancer cell genomes was identified in all cervix cancer tissues. These results provide evidence that HPV73 is an oncogenic virus that can cause invasive cervix cancer. With current molecular screening and HPV vaccination, not all cervix cancers will be prevented.
Asunto(s)
Infecciones por Papillomavirus/complicaciones , Neoplasias del Cuello Uterino/etiología , Neoplasias del Cuello Uterino/virología , Adulto , Anciano , Brasil , Estudios Transversales , ADN de Neoplasias/genética , Femenino , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Integración Viral/genéticaRESUMEN
INTRODUCTION: Protein p16 has been extensively studied as a potential biomarker for precursor lesions to distinguish cervical Intraepithelial neoplasia (CIN) from their mimics. However, the use of p16 as prognostic biomarker for diagnosis of cervical cancer and precancer is controversial. This study focuses on the assessment of peer-reviewed scientific data related to the use of p16 to predict disease severity and its controversies. METHODS: We reviewed publications in MEDLINE/PubMed assessing the clinical, diagnostic and prognostic significance of p16 in CIN and cervical cancer; we included publications from 2009 to June 2017. RESULTS: The use of p16 as a prognostic marker is still unreliable, although it could be a useful tool for diagnosis of Cervical Intraepithelial Neoplasia lesions with undetermined morphology. Moreover, p16 appears to be a specific marker of high-risk oncogenic HPV infection. CONCLUSION: This review shows the potential utility and drawbacks of p16 for clinical practice and the diagnosis of cervical cancer. Further studies are required to substantiate the role of p16 in conjunction with other more sensitive and specific biomarkers for diagnosing CIN and predicting its progression.
INTRODUÇÃO: A proteína p16 tem sido estudada como um biomarcador potencialmente específico de lesões cervicais precursoras e como uma forma de diferenciar as lesões parecidas com Neoplasia intra-epitelial cervical (NIC). Contudo existem várias controvérsias sobre a utilização de p16 como um biomarcador prognóstico e como uma ferramenta para o diagnóstico de câncer cervical e de lesões pré-câncer. O objetivo deste estudo foi a revisão de dados científicos por pares de bases, relacionados com a utilização da p16 e suas controvérsias. MÉTODOS: O estudo foi projetado como uma revisão da literatura das publicações do Medline/PubMed que avaliam o significado clínico, diagnóstico ou prognóstico do p16 em lesões de NIC e no câncer cervical no período de janeiro de 2009 a junho de 2017. RESULTADOS: o uso do p16 como um marcador prognóstico ainda não é confiável, apesar de que a p16 poderia ser uma ferramenta útil para o diagnóstico em lesões de NIC com morfologia indeterminada. Além disso, a p16 parece ser um marcador específico de infecção por HPV de alto risco oncogênico. CONCLUSÃO: A presente revisão mostra a potencial utilidade da proteína p16, bem como os inconvenientes para uso clínico-patológico e diagnóstico no câncer cervical. Contudo são necessários mais estudos para fundamentar o papel da p16 em conjunto com os outros biomarcadores mais sensíveis e específicos para diagnosticar NIC e prever a sua progressão.
Asunto(s)
Humanos , Biomarcadores de Tumor , Neoplasias del Cuello Uterino/diagnóstico , Displasia del Cuello del Útero , Infecciones por PapillomavirusRESUMEN
UNLABELLED: Cell cycle protein expression plays an important role in the pathophysiology of cervical cancer. However, few studies have attempted to correlate the use of these biomarkers with the clinical progression of the tumor. OBJECTIVES: 1) To analyze the expression of Ki-67, p53 and p16(INK4a) in cervical cancer, 2) to correlate the relative expression of these proteins as well as clinical parameters with the stage of disease, and 3) to determine the HPV DNA prevalence and subtype distribution. METHODS: Tissue Micro-Arrays (TMA) from patients with invasive cervical cancer (ICC) and controls were analyzed. HPV DNA detection was done by PCR and in situ hybridization. Ki-67, p53 and p16(INK4a) were analyzed by immunohistochemistry; clinical data was derived from the chart review. RESULTS: Advanced tumor stage (III and IV) was strongly associated (p<0.005) with advanced age (>55 years old), with more than four pregnancies and with the lack of formal education. HPV DNA was found in 94.3% of cases with the most prevalent types being HPV16 (67.5%), followed by HPV33 (12.0%) and HPV35 (3.6%). High expression of Ki-67 and p16 was more common in the advanced FIGO stages (pâ=â0.023). Women with HPV16 tended to be younger (50.9 years; SE 1.9) compared to women with other types (59.9 years; SE 2.8). CONCLUSION: We found that Ki-67 and p16 expression were independently associated with the tumor stage. We also noted that about 1/3 of the cervical cancers in this Brazilian cohort were not associated with HPV types directly targeted by the current HPV vaccines.
Asunto(s)
Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Adulto , Anciano , Biomarcadores de Tumor , Brasil/epidemiología , Estudios de Casos y Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Estudios de Cohortes , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Prevalencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Adulto JovenRESUMEN
Although several studies have evaluated the role of p16(INK4a) as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16(INK4a) in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16(INK4a) as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16(INK4a) was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16(INK4a) expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16(INK4a) in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16(INK4a) under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16(INK4a) expression in CIN 2-3.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Estudios de Casos y Controles , Femenino , Infecciones por VIH/complicaciones , VIH-1 , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Curva ROC , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/complicaciones , Neoplasias del Cuello Uterino/metabolismo , Displasia del Cuello del Útero/complicaciones , Displasia del Cuello del Útero/metabolismoRESUMEN
Although several studies have evaluated the role of p16INK4a as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16INK4a in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16INK4a as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16INK4a was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16INK4a expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16INK4a in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16INK4a under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16INK4a expression in CIN 2-3.
Asunto(s)
Adulto , Femenino , Humanos , Persona de Mediana Edad , Displasia del Cuello del Útero/diagnóstico , /metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias del Cuello Uterino/diagnóstico , Estudios de Casos y Controles , Displasia del Cuello del Útero/complicaciones , Displasia del Cuello del Útero/metabolismo , Infecciones por VIH/complicaciones , VIH-1 , Inmunohistoquímica , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Curva ROC , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/complicaciones , Neoplasias del Cuello Uterino/metabolismoRESUMEN
BACKGROUND: Minichromosome maintenance proteins (MCM) are highly expressed in actively replicating cells. The need for biological markers for cervical carcinoma and its precursor lesions is emerging. Our main aim was to determine the immunohistochemical expression of MCM-2 in HIV-positive and -negative dysplastic cervical specimens. METHODS: Immunohistochemical analysis of MCM-2 was performed in a total of 352 cervical TMA specimens of normal control, low-grade CIN, high-grade CIN and invasive tumor. 38 specimens were from HIV-positive women. A receiver operating characteristic (ROC) curve was constructed to determine the best cutoff to diagnose high-grade CIN and invasive cervical cancer. RESULTS: In the progression from normal epithelium to high-grade CIN and invasive tumor we found significant differences in the MCM-2 expression (p<0.05). Based on the ROC curve of 80% with an area under the curve (AUC) of 0.78, expression of MCM-2 to diagnose high-grade CIN and invasive tumor resulted in sensitivity of 81%, specificity of 66%, a positive predictive value (PPV) of 86% and a negative predictive value (NPV) of 57%. HIV-positive cervices revealed a decreasing expression of MCM-2 in both LGCIN and HGCIN compared with HIV-negative specimens (p<0.0001). CONCLUSIONS: The present study suggests that immunohistochemical MCM-2 may not be a promising biomarker for diagnosing high-grade CIN and invasive cancer.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Proteínas de Ciclo Celular/biosíntesis , Proteínas Nucleares/biosíntesis , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Cuello del Útero/metabolismo , Cuello del Útero/patología , Cuello del Útero/virología , Coinfección , Epitelio/metabolismo , Epitelio/patología , Epitelio/virología , Femenino , Expresión Génica , VIH/fisiología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Inmunohistoquímica , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Clasificación del Tumor , Proteínas Nucleares/genética , Papillomaviridae/fisiología , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Valor Predictivo de las Pruebas , Curva ROC , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virologíaRESUMEN
This manuscript focuses on the detection of viral nucleic acids by in situ based methodologies. The optimal protocol depends on the virus. We will describe protocols for viral RNA detection by reverse transcriptase (RT) in situ PCR. We will also directly compare this method to the detection of viral RNA using standard in situ hybridization with locked nucleic acid (LNA) probes. Most DNA viruses are associated with high viral copy number and, thus, can be detected by standard in situ hybridization. Retroviral provirus is an exception as the single integrated DNA is best detected by PCR in situ hybridization. We will also describe protocols for the co-localization of viral DNA and RNA with host cytokines. Our protocol typically has the protein immunohistochemistry as the second step, with the key features being pretreatment step, antibody concentration, co-labeling analyses with a computer-based system, and co-analyzes with serial sections.