RESUMEN
AIMS: No good predictive marker for the malignant transformation of potentially malignant oral lesions (PMOLs) is currently available. This study re-evaluated the value of p53 immunoexpression to predict malignant transformation of PMOLs after discounting possible confounding factors. METHODS: PMOLs from 18 patients who showed progression to carcinoma, 16 of the respective carcinomas, and PMOLs from 18 matched controls were evaluated by immunohistochemistry (IHC) for p53 expression. A mouse monoclonal antibody that detects wild-type and mutant forms of human p53 was used. The p53 immunostaining pattern was also correlated with the degree of dysplasia. RESULTS: Suprabasal p53 staining was significantly associated with high grades of dysplasia (p < 0.01). The specificity and positive predictive value (PPV) for malignant transformation of suprabasal p53 staining were superior to the assessment of dysplasia, but sensitivity was inferior. All carcinomas derived from PMOLs with suprabasal p53 showed strong p53 immunostaining. However, the absence of suprabasal p53 staining and/or dysplastic changes did not preclude malignant transformation in a considerable proportion of PMOLs. CONCLUSIONS: This study confirms and extends previous findings that suprabasal p53 immunoexpression has a high PPV for malignant transformation of PMOLs and can be used as a specific marker for lesions that are at high risk for malignant transformation. The absence of suprabasal p53 staining (that is, absence of, or basal, p53 staining) is non-informative for prognostic purposes. Because of its limited sensitivity, p53 IHC is not a substitute for the assessment of dysplasia in the evaluation of PMOLs. Instead, p53 IHC emerges as a clinically useful supplement of histopathological assessment in the prognosis of PMOLs.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Boca/metabolismo , Lesiones Precancerosas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Leucoplasia Bucal/metabolismo , Leucoplasia Bucal/patología , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/patología , Lesiones Precancerosas/patología , Valor Predictivo de las Pruebas , Pronóstico , Sensibilidad y EspecificidadRESUMEN
The role of a polymorphism at position 72 of the tumor suppressor gene TP53 in the development of cervical cancer is not well established. The arginine variant of the p53 protein could be more susceptible to degradation by human papillomavirus (HPV) E6 protein than the protein containing proline. Recent studies show controversial results. We investigated a possible association between TP53 polymorphism and cervical cancer in a Peruvian population with high prevalence of HPV infection. HPV status and TP53 polymorphism were determined for 119 cases of invasive cervical cancer and 127 control women from Peru. HPV infection was detected by PCR of cervical cells or tumor biopsies. For determination of TP53 polymorphism, exon 4 of the TP53 gene was amplified by PCR, and DNA was subsequently subjected to restriction enzyme digest. Associations between TP53 polymorphism, HPV infection, and cervical cancer were assessed using logistic regression. Women homozygotes for arginine had a 2.2-fold increased risk (95% confidence interval: 0.6-7.6) for cervical cancer. The odds ratio for women heterozygotes for Arg/Pro was 3.5 (95% confidence interval: 0.9-14). Similarly increased risks were found when restricting analysis to HPV-positive women only. The distribution of TP53 genotypes in this Peruvian population was comparable with that found in Caucasians. Our results cannot rule out an association between the TP53 polymorphism at codon 72, HPV infection, and the etiology of cervical cancer.
Asunto(s)
Genes p53 , Infecciones por Papillomavirus/genética , Infecciones Tumorales por Virus/genética , Neoplasias del Cuello Uterino/virología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Modelos Logísticos , Persona de Mediana Edad , Oportunidad Relativa , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Perú , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Factores de Riesgo , Encuestas y Cuestionarios , Neoplasias del Cuello Uterino/genéticaRESUMEN
BACKGROUND: High-risk human papillomavirus (HPV) types play a major role in the development of cervical cancer in vivo and can induce immortalization of primary human keratinocytes in vitro. Activation of the telomere-lengthening enzyme telomerase constitutes a key event in both processes. Because losses of alleles from chromosome 6 and increased telomerase activity have been observed in high-grade premalignant cervical lesions, we analyzed whether human chromosome 6 harbors a putative telomerase repressor locus that may be involved in HPV-mediated immortalization. METHODS: Microcell-mediated chromosome transfer was used to introduce chromosomes 6 and 11 to the in vitro generated HPV type 16 (HPV16)-immortalized keratinocyte cell line FK16A and to the in vivo derived HPV16-containing cervical cancer cell line SIHA: Hybrid clones were analyzed for growth characteristics, telomerase activity, human telomerase reverse transcriptase (hTERT) and HPV16 E6 expression, and telomere length. FK16A hybrid clones were also transduced with an hTERT-containing retrovirus to examine the effect of ectopic hTERT expression on growth. Statistical tests were two-sided. RESULTS: Introduction of human chromosome 6 but not of chromosome 11 to both cell lines yielded hybrid cells that demonstrated crisis-like features (i.e., enlarged and flattened morphology, vacuolation, and multinucleation) and underwent growth arrest after a marked lag period. In the chromosome 6 hybrid clones analyzed, telomerase activity and hTERT messenger RNA (mRNA) expression were statistically significantly reduced compared with those in the chromosome 11 hybrid clones (for telomerase activity, P =.004 for the FK16A hybrids and P =.039 for the SiHa hybrids; for hTERT mRNA expression, P =.003 for the FK16A hybrids). The observed growth arrest was associated with telomeric shortening. Ectopic expression of hTERT in FK16A cells could prevent the telomeric shortening-based growth arrest induced by chromosome 6. CONCLUSIONS: Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.
Asunto(s)
Cromosomas Humanos Par 6 , Papillomaviridae/genética , ARN , Telomerasa/metabolismo , Neoplasias del Cuello Uterino/genética , División Celular , Línea Celular Transformada , Cromosomas Humanos Par 11 , Proteínas de Unión al ADN , Femenino , Genes Reporteros , Humanos , Células Híbridas , Queratinocitos , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/antagonistas & inhibidores , Telómero/genética , Telómero/ultraestructura , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/genéticaRESUMEN
BACKGROUND: Although extensive information has been gathered about the prevalence and determinants of human papillomavirus infection among women, little is known about the prevalence and natural history of this infection among males. GOAL: To investigate the potential usefulness of urine specimens to assess the presence of genital human papillomavirus DNA infection. STUDY DESIGN: The authors conducted a study of 120 healthy men from Cuernavaca, Mexico. A urine specimen and urethral and coronal sulcus swab samples were collected and tested for human papillomavirus using the GP5+/6+ polymerase chain reaction enzyme immunoassay method. RESULTS: In 95% of the urethral-coronal sulcus samples, the beta-globin gene was detectable, indicating adequacy of the specimen for DNA amplification; however, only 14% of the urine specimens had detectable beta-globin. Removal of inhibitors by DNA purification in a sample of subjects produced beta-globin amplification, but no increase in human papillomavirus DNA positivity was detected. Human papillomavirus DNA was not detectable in penile-urethral swab samples in any of the subjects who reported not having engaged in sexual activity but was present in 43% of men who reported sexual activity, a strong indication of the sexual transmission of human papillomavirus. CONCLUSIONS: Human papillomavirus is a common sexually transmitted infection among Mexican males, and urine sample specimens cannot adequately detect the presence of this infection in males.
Asunto(s)
Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Infecciones Tumorales por Virus/epidemiología , Adolescente , Adulto , Secuencia de Bases , Cartilla de ADN , ADN Viral/análisis , ADN Viral/orina , Globinas/genética , Globinas/orina , Humanos , Masculino , México/epidemiología , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/orina , Pene/virología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Sensibilidad y Especificidad , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/orina , Uretra/virologíaRESUMEN
Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8(+) T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.
Asunto(s)
Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada/enzimología , Línea Celular Transformada/inmunología , Activación de Linfocitos/genética , ARN , Telomerasa/biosíntesis , Telomerasa/genética , Antígenos/fisiología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Clonales/enzimología , Células Clonales/inmunología , Citocinas/fisiología , Proteínas de Unión al ADN , Activación Enzimática/genética , Activación Enzimática/inmunología , Estabilidad de Enzimas/genética , Estabilidad de Enzimas/inmunología , Epítopos de Linfocito T/análisis , Epítopos de Linfocito T/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Inmunofenotipificación , Interleucina-7/biosíntesis , Interleucina-7/genética , Monofenol Monooxigenasa/inmunología , Ingeniería de Proteínas/métodos , ARN Mensajero/biosíntesis , Telómero/enzimología , Telómero/genética , Transducción Genética , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: To improve the accuracy of conventional cytology in cervical cancer screening, high risk human papillomavirus (HPV) testing and neural network based screening have been developed. This study assessed the power of both techniques to detect women at risk of developing incident CIN III; that is, CIN III detected during the follow up of women with normal cytology and borderline nuclear changes. METHODS: A cohort of 2250 women, 34-54 years of age, who attended population based cervical cancer screening from 1988 to 1991 and had normal smears or borderline nuclear changes was followed. All smears were tested for high risk HPV and the smears were rescreened using neural network based screening. The value of neural network based screening for predicting incident CIN III during a mean follow up period of 6.4 years was compared with that of high risk HPV testing. In addition, morphological markers presumed to be related to HPV were correlated with HPV status. RESULTS: Thirteen (0.6%) women had incident CIN III. Both high risk HPV positivity and abnormal cytology were associated with an increased risk for incident CIN III (odds ratio, 240 and 22, respectively) and high risk HPV positivity was associated with abnormal cytology. The sensitivity of high risk HPV testing for predicting incident CIN III was much higher than that of neural network based screening (92% and 46%, respectively). None of the morphological markers assessed, including koilocytosis, was correlated with high risk HPV status. CONCLUSION: High risk HPV testing is superior to neural network based screening in identifying women at risk of developing CIN III. For women with normal cytology and borderline changes and a negative high risk HPV test, the screening interval can be considerably prolonged.
Asunto(s)
Tamizaje Masivo/métodos , Redes Neurales de la Computación , Papillomaviridae/aislamiento & purificación , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Factores de Riesgo , Neoplasias del Cuello Uterino/virología , Frotis Vaginal , Displasia del Cuello del Útero/virologíaRESUMEN
BACKGROUND: The prevalence of the human papillomavirus (HPV) has been found to decrease with age, but whether this applies to all populations is not clear. Knowledge about the generalizability of this age dependency is important to understand the natural history of the infection, but may also have implications in relation to the use of HPV testing in cervical cancer screening. GOAL: To study the prevalence and risk factors for HPV infection in a selected population of female sex workers and to compare the results with persons with different sexual habits from two other populations in the same geographic area. STUDY DESIGN: A case-control study among 188 female sex workers from Copenhagen. Data were obtained by personal interviews. Cervical material was collected by a self-administered lavage kit, and HPV testing was done by means of GP5+/6+ primers based on HPV, polymerase chain reaction, enzyme immunoassay. RESULTS: Among sex workers, age was the most important risk factor for HPV infection. Number of private sex partners in the last 4 months was also a significant risk factor, and a protective effect of condom use was indicated. CONCLUSION: The HPV prevalence is declining in sex workers with age despite continuously high sexual activity, most likely indicating that an immune response is acquired over time.
Asunto(s)
Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Trabajo Sexual , Enfermedades Virales de Transmisión Sexual/epidemiología , Infecciones Tumorales por Virus/epidemiología , Adulto , Factores de Edad , Estudios de Casos y Controles , Anticoncepción , Dinamarca/epidemiología , Femenino , Humanos , Entrevistas como Asunto , Papillomaviridae/inmunología , Infecciones por Papillomavirus/prevención & control , Prevalencia , Factores de Riesgo , Conducta Sexual , Enfermedades Virales de Transmisión Sexual/prevención & control , Factores de Tiempo , Infecciones Tumorales por Virus/prevención & control , Frotis VaginalRESUMEN
Infection with high risk human papillomavirus (hrHPV) plays a central aetiological role in cervical cancer. Still, cervical carcinogenesis is a multistep process which requires other events in addition to hrHPV infection. Recent data have resulted in the following concept of cervical carcinogenesis: hrHPV infects normal squamous epithelium. In most cases this will not lead to a lesion or at worst give rise to a regressing low grade cervical intraepithelial neoplasia (CIN). Both phenomena involve viral clearance. Only persistent hrHPV infections will lead to a high grade CIN lesion, a subset of which may undergo malignant transformation. At the transition of CIN 2 to CIN 3 deregulated expression of the viral oncogenes E6 and E7 takes place, resulting in genetic instability. Subsequently, activation of the telomere-lengthening enzyme, telomerase occurs, at the result of which cells obtain an infinite replication capacity. Ultimately, successive allele losses occur at different chromosomal locations which, followed by a clonal outgrowth result in an invasive carcinoma.
Asunto(s)
Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/complicaciones , Infecciones Tumorales por Virus/complicaciones , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Transformación Celular Neoplásica , Transformación Celular Viral , Femenino , Humanos , Modelos Biológicos , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Factores de Riesgo , Infecciones Tumorales por Virus/virologíaRESUMEN
More than 99% of all cervical cancers contain high risk HPV. Only a persistent infection with high risk HPV of the cervical epithelium results in cervical cancer. Because the risk of cervical cancer is identical for all different HPV types, tests which detect all 14 high risk HPV types at one time are sufficient for clinical management. Testing for hr-HPV is mandatory for women with mild dyskaryosis and for the follow-up of women treated for CIN lesions. Based on efficiency to detect CIN3 and cervical cancer and preliminary cost benefit analysis, the combination of a high risk HPV test in conjunction with a cervical smear appears to be the best way of cervical cancer screening. A definite point of view on using high risk HPV testing for primary screening for cervical cancer will be obtained after the completion of a randomized trial of 44,000 women, in which the efficiency to detect CIN3 and cervical cancer by high risk HPV testing in conjunction with a cytomorphological smear is compared with screening by classical cytology.
Asunto(s)
Sondas de ADN de HPV , Tamizaje Masivo/métodos , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Infecciones Tumorales por Virus/complicaciones , Displasia del Cuello del Útero/prevención & control , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/prevención & control , Frotis Vaginal , Adulto , ADN Viral , Femenino , Humanos , Incidencia , Persona de Mediana Edad , Países Bajos/epidemiología , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Infecciones Tumorales por Virus/epidemiología , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/epidemiología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/clasificación , Displasia del Cuello del Útero/epidemiologíaRESUMEN
Infection with oncogenic human papillomavirus (HPV) types is associated with the development of cervical neoplasia (CIN). The E6 and E7 oncoproteins are constitutively expressed in these lesions and are therefore putative targets for the immune response against HPV. The relation between HPV 16-specific memory cytotoxic T-cell precursor (mCTLp) activity to both oncoproteins and the natural course of cervical dysplasia was analyzed in 38 patients participating in a nonintervention cohort study of women with CIN and 11 HPV 16-positive cervical carcinoma patients. In a cross-sectional study at the end of follow-up prior to biopsy, 8 of 20 patients with a persistent HPV 16 infection had specific mCTLp against at least one of the two oncoproteins. By contrast, no specific mCTLp activity was detected in 11 HPV-negative patients or in 7 patients who had cleared an HPV 16 infection at the end of follow-up. However, 5 of 11 cervical carcinoma patients showed mCTLp activity against the E7 protein only. This study demonstrates that HPV 16 oncogene-specific mCTLp are present in women with HPV 16-positive CIN prior to any intervention. Since HPV-specific mCTLp were detected predominantly in women with high-grade lesions or invasive cervical carcinoma and not in women who cleared the virus, the role of naturally occurring mCTLp in the protection against HPV-associated cervical neoplasia remains to be established.
Asunto(s)
Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Proteínas Represoras , Linfocitos T Citotóxicos/inmunología , Infecciones Tumorales por Virus/inmunología , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Estudios de Cohortes , Estudios Transversales , Femenino , Humanos , Memoria Inmunológica/inmunología , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Linfocitos T Colaboradores-Inductores/inmunología , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/inmunología , Displasia del Cuello del Útero/inmunologíaRESUMEN
Before guidelines can be set for the use of high-risk human papillomavirus (HR HPV) testing in cervical cancer screening and vaccine preparation, age-related prevalence of HR HPV types in cytologically normal smears has to be known. Therefore, in a cross-sectional study the prevalence of 37 different HPV genotypes and putatively unidentified HPV types was determined in 3,305 cytologically normal cervical smears from the general female population (15-69 years of age) using an HPV general primer GP5+/bioGP6+ mediated PCR assay. Subsequently, HPV-positive cervical smears were typed for 19 HR and 18 low-risk (LR) HPVs with an enzyme immunoassay using HPV type-specific oligoprobes in cocktails and individually, respectively. Overall, -HR and -LR HPV prevalences appeared to be of 4.6%, 3.3%, and 1.0%, respectively. Twenty-six different HPV types were detected in the 152 HPV-positive samples, the most prevalent types being HPV 16, 31, and 18. With regard to age, a peak prevalence of 19.6% for all HPVs was found in women 25-29 years of age, which declined to a mean of 4.3% in women over 30 years. With regard cytologically normal cervical smears (n = 3, 011) of women participating in the population-based screening program in the Netherlands (30 to 60 years), all HR HPVs showed decreased occurrence with increasing age, whereas the prevalence of LR HPV types remained constant. We suggest that screening for abnormal cytology implies screening for HR HPV infections and the subsequent treatment results in a decline of HR HPV prevalence in contrast to LR HPV prevalence during the years of screening.
Asunto(s)
Papillomaviridae/clasificación , Papillomaviridae/genética , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Factores de Edad , Anciano , Cuello del Útero/metabolismo , Cuello del Útero/virología , Femenino , Genotipo , Humanos , Técnicas para Inmunoenzimas , Tamizaje Masivo , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Prevalencia , Valores de Referencia , Factores de Riesgo , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/metabolismo , Frotis VaginalRESUMEN
p53 is a tumour suppressor gene encoding a protein whose function is impaired in a very large proportion of human cancers. The objectives of this study were to determine the natural history of p53 alterations during stages of oral carcinogenesis, by comparing p53 immunoexpression in oral squamous cell carcinomas (OSCCs), their non-malignant adjacent mucosa, and respective metastases; and to define the potential practical consequences for clinical management of p53 staining in the non-malignant adjacent mucosa. Forty-two samples of non-malignant mucosa adjacent to OSCCs, the respective carcinomas, and six lymph node metastases derived from six of the OSCCs were investigated for p53 protein expression by immunohistochemistry. Seven out of 42 (17%) non-malignant mucosal samples immediately adjacent to OSCC showed suprabasal p53 staining and this was significantly associated with moderate/severe dysplasia (p=0.02). In six of these cases (86%), the respective carcinoma showed p53 immunoexpression in more than 50% of the neoplastic cells and in the remaining case, p53 immunoexpression was found in more than 25% of the neoplastic cells. In all p53-negative carcinomas that showed p53 immunoexpression in the non-malignant adjacent mucosa, p53 staining was never detected above the basal cell layer. Lymph node metastases showed the same patterns of p53 immunoexpression as the carcinomas from which they were derived. When suprabasal p53 staining is present in non-malignant mucosa immediately adjacent to OSCCs, this suggests stable p53 alterations which are maintained upon progression to overt malignancy. The immunostaining in non-malignant mucosa of the resection margins of OSCCs might be a valuable predictor for local recurrences and may therefore have implications for the management of patients who have received surgical treatment for OSCC.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico , Mucosa Bucal/metabolismo , Neoplasias de la Boca/diagnóstico , Lesiones Precancerosas/diagnóstico , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Valor Predictivo de las Pruebas , Coloración y EtiquetadoRESUMEN
OBJECTIVE: To study the significance of the presence of high risk human papillomavirus (HPV) in women with initially normal cervical cytology for the development of abnormal cytology and an abnormal colposcopic impression. DESIGN: Prospective, observational study PARTICIPANTS AND METHODS: Sixty-eight women with cytomorphologically normal smears and at least one positive HPV test result were evaluated every six months by cytology, colposcopy and HPV testing. The endpoint of the study was abnormal cervical cytology. RESULTS: The median time of follow up from the first positive HPV test was 34 months. A total of 17 women developed abnormal cytology, of whom 16 (94%) had persistence of a high risk HPV infection. Women with persistent high risk HPV were more likely to develop abnormal cervical cytology than women without high risk HPV (hazard ratio 28.2, 95% CI 3.72-215.2); they also had an increased risk of developing an abnormal colposcopic impression (hazard ratio 4.4, 95% CI 1.69-11.7). Among the 17 women with abnormal cytology, high grade dysplasia was histopathologically demonstrated in eight women. CONCLUSION: Persistent presence of high risk HPV in normal cervical smears is associated with a significantly increased risk of developing abnormal cytology and to a lesser degree with developing an abnormal colposcopic impression.
Asunto(s)
Cuello del Útero/virología , Papillomaviridae/aislamiento & purificación , Adolescente , Adulto , Cuello del Útero/patología , Colposcopía , Femenino , Humanos , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , Frotis VaginalRESUMEN
The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the beta-globin gene appeared to be superior for the GTC-based assays. Using competitive beta-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 x 10(5) to 3 x 10(5) genome equivalents in smears containing 5 x 10(5) to 20 x 10(5) nucleated cells, indicating a mean efficiency of 26% (range of 15-44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3-6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 +/- 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears.
Asunto(s)
Cuello del Útero/patología , Cuello del Útero/virología , Prueba de Papanicolaou , Papillomaviridae/aislamiento & purificación , Neoplasias del Cuello Uterino/virología , Frotis Vaginal , Cuello del Útero/citología , ADN Viral/análisis , Desinfectantes , Femenino , Genoma Viral , Guanidinas , Humanos , Proteínas Asociadas a Pancreatitis , Papillomaviridae/genética , Reacción en Cadena de la Polimerasa/métodos , Estudios Retrospectivos , Manejo de Especímenes , Tiocianatos , Factores de Tiempo , Neoplasias del Cuello Uterino/patologíaAsunto(s)
Diagnóstico por Computador/tendencias , Neoplasias del Cuello Uterino/patología , Frotis Vaginal/tendencias , Femenino , Humanos , Papillomaviridae , Infecciones por Papillomavirus/diagnóstico , Infecciones Tumorales por Virus/diagnóstico , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Reports on the association of EBV with oral squamous-cell carcinomas (OSCCs) are scarce and inconclusive. To determine the potential role of EBV in oral carcinogenesis, we investigated 36 EBV DNA PCR-positive OSCCs for the expression of EBV transcripts and proteins. From these EBV DNA-positive OSCCs, 13 were analysed for the presence of EBV products, either at RNA and/or protein level. EBER transcripts were investigated by RNA in situ hybridisation. EBNA-1, EBNA-2, LMP-1, LMP-2, BHRF1 and BARF0 transcripts were investigated by RT-PCR and/or NASBA. EBNA-1, LMP-1 and ZEBRA protein expressions were investigated by immunohistochemistry. All 36 OSCCs were positive for EBV DNA, using the highly sensitive BamHI W PCR, and 18 of these (50%) were positive using the less-sensitive PCR, which targets BNLF-1. However, virtually all OSCCs tested failed to reveal EBV transcripts, including EBERs and EBNA-1 transcripts. No ZEBRA and LMP-1 proteins were found in the neoplastic or any other cells of the OSCCs investigated. Immunohistochemistry using a monoclonal antibody (MAb) raised against EBNA-1 (2B4) resulted in positive staining in some cases of OSCCs, but these results were non-specific, since EBV-negative epithelial tissues showed extensive non-specific staining and no EBNA-1-specific transcripts were detected by RT-PCR or NASBA. The absence of expression of EBV encoded transcripts and proteins indicate that, with the present knowledge on EBV, an active role in oral carcinogenesis for this virus is unlikely.
Asunto(s)
Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/virología , ADN Viral/análisis , Herpesvirus Humano 4/aislamiento & purificación , Neoplasias de la Boca/etiología , Neoplasias de la Boca/virología , ARN Viral/análisis , Proteínas Virales/análisis , Herpesvirus Humano 4/genética , Humanos , Inmunohistoquímica , Reacción en Cadena de la PolimerasaRESUMEN
HPV types 16 and 18 have been categorized as human carcinogens based on their strong associations with cervical cancer in previous case-control studies. Recent IARC studies in the Philippines, Thailand and Morocco show strong associations between invasive cervical cancer and less common HPV types, including HPV 31, 33, 45, 51, 52 and 58. We present results of a further IARC case-control study conducted in Asunción, Paraguay, to examine the association between specific HPV types and invasive cervical cancer as well as risk factors other than HPV. One-hundred thirteen incident histologically confirmed invasive cervical cancer cases and 91 age-matched hospital controls were recruited. A standardized questionnaire was administered to investigate known and suspected risk factors for cervical cancer. For HPV status determination, cervical biopsy specimens from case subjects and exfoliated cervical cells from control subjects were obtained. HPV DNA was ascertained using a GP5+/6+ PCR-based assay capable of detecting more than 33 HPV types. Overall HPV prevalence was 97% in the cervical cancer cases and 20% in the control subjects. As a single infection, HPV 16 was the predominant type with a prevalence of 48% among case subjects and 5.5% among control subjects. Significant associations with the risk of cervical cancer were detected as follows: any HPV type (OR = 114; 95% CI: 36-361); HPV 16 (OR = 910); HPV 18 (infinite OR); HPV 31 (OR = 110); HPV 33 (OR = 261); HPV 45 (OR = 129); and HPV 58 (OR = 36). In the multivariate model, risk factors other than HPV significantly associated with cervical cancer risk were a higher number of lifetime sexual partners, lower educational status and never having had a Pap smear. Strong associations were found between invasive cervical cancer and specific HPV types 16, 18, 31, 33, 45 and 58.
Asunto(s)
Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/patología , Infecciones Tumorales por Virus/patología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Cuello del Útero/citología , Cuello del Útero/patología , Cuello del Útero/virología , ADN Viral/aislamiento & purificación , Escolaridad , Femenino , Humanos , Persona de Mediana Edad , Análisis Multivariante , Invasividad Neoplásica , Papillomaviridae/clasificación , Infecciones por Papillomavirus/epidemiología , Paraguay/epidemiología , Factores de Riesgo , Conducta Sexual , Infecciones Tumorales por Virus/epidemiología , Neoplasias del Cuello Uterino/epidemiologíaRESUMEN
This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.
Asunto(s)
Genes p53 , Papillomaviridae/genética , Papillomaviridae/patogenicidad , Proteínas Represoras , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Secuencia de Bases , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/genética , Codón/genética , Cartilla de ADN/genética , ADN Viral/genética , Femenino , Variación Genética , Genotipo , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Polimorfismo Genético , Factores de Riesgo , Homología de Secuencia de Ácido Nucleico , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Neoplasias del Cuello Uterino/etiología , Displasia del Cuello del Útero/etiologíaRESUMEN
BACKGROUND: The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. AIMS: To determine the intermethod agreement between different GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. METHODS: For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. RESULTS: In the intermethod comparison, pairwise agreement of the different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% (kappa values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). CONCLUSIONS: The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used.
Asunto(s)
Cuello del Útero/virología , ADN Viral/análisis , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Infecciones Tumorales por Virus/diagnóstico , Estudios de Evaluación como Asunto , Femenino , Humanos , Técnicas para Inmunoenzimas , Distribución Aleatoria , Reproducibilidad de los Resultados , Frotis VaginalRESUMEN
Serum samples from 194 cases and 217 controls participating in a case-control study of invasive cervical cancer in Brazil were examined for antibodies to human papillomavirus (HPV) 16 virus-like particles (VLPs) by ELISA. The prevalence of antibody in cases and controls was 47.4 versus 24.4% (P < 0.001). The prevalence was higher in women who had HPV-16 DNA in the genital tract (54.2%) than in those with other HPVs (36.8%) or no HPVs (44.8%), but the differences were not statistically significant. Among cases and controls, HPV-16 VLP antibodies were associated with a greater number of lifetime sexual partners (chi2 for trend, P < 0.001). Among controls, age was inversely associated with HPV-16 VLP seroreactivity (chi2 for trend, P = 0.019). The sera were previously tested for antibodies to HPV-16 E6 and E7 oncoproteins; there was no correlation between antibody titers to HPV-16 E6 or E7 and VLPs. The HPV-16 serological assays were compared as screening tests for invasive cervical cancer. The sensitivity and specificity estimates were 47.4 and 75.6% for HPV-16 VLP serology, 63.4 and 89.9% for either HPV-16 E6 or E7 serology, and 53.6 and 93.6% for high titers of either HPV-16 E6 or E7 or VLP antibodies. The utility of HPV-16 VLP ELISA as a screening test for invasive cervical cancer is limited by a high seroprevalence in women with probable prior exposure to HVP 16 but without disease.