RESUMO
The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.
Assuntos
Ácidos Nucleicos Livres , DNA Viral , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Reação em Cadeia da Polimerase , Humanos , DNA Viral/sangue , DNA Viral/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/sangue , Infecções por Papillomavirus/virologia , Ácidos Nucleicos Livres/sangue , Reação em Cadeia da Polimerase/métodos , Neoplasias Orofaríngeas/virologia , Neoplasias Orofaríngeas/sangue , Neoplasias Orofaríngeas/diagnóstico , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Feminino , Sensibilidade e Especificidade , Masculino , Pessoa de Meia-Idade , Idoso , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus HumanoRESUMO
BACKGROUND: HPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and â¼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. RESULTS: The prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. CONCLUSIONS: Our findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.
Assuntos
Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Humanos , Papillomavirus Humano 16/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Brasil/epidemiologia , Neoplasias Bucais/complicações , Biomarcadores , DNA Viral/análise , RNA , Neoplasias de Cabeça e Pescoço/complicaçõesRESUMO
Increasing evidence pinpoints metronomic chemotherapy, a frequent and low dose drug administration with no prolonged drug-free intervals, as a potential tool to fight certain types of cancers. The primary identified targets of metronomic chemotherapy were the tumor endothelial cells involved in angiogenesis. After this, metronomic chemotherapy has been shown to efficiently target the heterogeneous population of tumor cells and, more importantly, elicit the innate and adaptive immune system reverting the "cold" to "hot" tumor immunologic phenotype. Although metronomic chemotherapy is primarily used in the context of a palliative setting, with the development of new immunotherapeutic drugs, a synergistic therapeutic role of the combined metronomic chemotherapy and immune checkpoint inhibitors has emerged at both the preclinical and clinical levels. However, some aspects, such as the dose and the most effective scheduling, still remain unknown and need further investigation. Here, we summarize what is currently known of the underlying anti-tumor effects of the metronomic chemotherapy, the importance of the optimal therapeutic dose and time-exposure, and the potential therapeutic effect of the combined administration of metronomic chemotherapy with checkpoint inhibitors in preclinical and clinical settings.
RESUMO
INTRODUCTION: This population-based study aimed to fill the knowledge gap on Human Papillomavirus (HPV) prevalence and associated sociodemographic risk factors of the general population in the North Tongu District, Ghana. These results are needed to guide cervical cancer prevention efforts, as the leading type of female cancers. METHODS: A cross-sectional study including 2002 women in the North Tongu District, Ghana investigated HPV prevalence and associated sociodemographic risk factors. Women were recruited by geographical distribution through the local community-based health system and samples collected using a self-sampling device. For HPV genotyping BSGP5+/6+-PCR with Luminex-MPG readout was used. Multivariate logistic regression analyzed sociodemographic risk factors for HPV positivity. RESULTS: Of 2002 self-collected samples, 1943 were eligible, contained sufficient DNA and provided valid HPV genotyping results. Prevalence of single high risk HPV types was 32.3% and of multiple high risk types 9.7%. The five most common detected HPV types were HPV16 (7.4%; 95%CI: 6.3-8.7), HPV52 (7.2%; 95%CI: 6.1-8.5), HPV35 (4.8%; 95%CI: 3.9-5.8), HPV59 (4.7%; 95%CI: 3.8-5.8), HPV56 (3.9%; 95%CI: 3.1-4.8). Highest prevalence was observed among women aged 18-24 years, while age 25-54 years was inversely associated with high risk HPV positivity in multivariate analysis. Sociodemographic risk factors identified were i) having any sexual partner, ii) more partners increased the odds for high risk HPV positivity, iii) independently from this marital status, in particular not being married. DISCUSSION & CONCLUSION: Most importantly, the high risk HPV prevalence detected from this study is higher than estimates reported for Western Africa. This needs be considered, when deciding on the cervical cancer screening algorithms introduced on a wider scale. Follow-up and triage, depending on the methods chosen, can easily overburden the health system. Self-sampling worked well and provided adequate samples for HPV-based screening. Women with increasing number of sexual partners and not being married were found to have higher odds of being high risk HPV positive, therefore could be a higher prioritized screening target group.
Assuntos
Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia , Adolescente , Adulto , Idoso , Estudos Transversais , Feminino , Genótipo , Gana/epidemiologia , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade , Papillomaviridae/classificação , Papillomaviridae/genética , Prevalência , Fatores de Risco , Fatores Socioeconômicos , Adulto JovemRESUMO
Colorectal cancer is a leading cause of morbidity and mortality worldwide in both men and women. The gut microbiome is increasingly recognized as having an important role in human health and disease. Fusobacterium has been identified in former studies as a leading gut bacterium associated with colorectal cancer, but it is still not clear if it plays an oncogenic role. In the current study, fecal samples were collected prior to bowel preparation from participants of screening colonoscopy in the German BliTz study. Using 16S rRNA gene analysis, we examined the presence and relative abundance of Fusobacterium in fecal samples from 500 participants, including 46, 113, 110 and 231 individuals with colorectal cancer, advanced adenomas, non-advanced adenomas and without any neoplasms, respectively. We found that the abundance of Fusobacterium in feces was strongly associated with the presence of colorectal cancer (P-value < 0.0001). This was confirmed by PCR at the species level for Fusobacterium nucleatum. However, no association was seen with the presence of advanced adenomas (P-value = 0.80) or non-advanced adenomas (P-value = 0.80), nor were there any associations observed with dietary or lifestyle habits. Although a causal role cannot be ruled out, our observations, based on fecal microbiome, support the hypothesis that Fusobacterium is a passenger that multiplies in the more favorable conditions caused by the malignant tumor rather than a causal factor in colorectal cancer development.
Assuntos
Neoplasias Colorretais/microbiologia , Fusobacterium/isolamento & purificação , Microbioma Gastrointestinal/genética , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Fezes/microbiologia , Feminino , Fusobacterium/genética , Fusobacterium/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genéticaRESUMO
To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh-frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV-driven in the presence of HPV16 DNA and HPV16 transformation-specific RNA transcript patterns (E6*I, E1⧠E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16-driven. HPV16 E6 seropositivity was the best predictor of HPV16-driven OPSCC (diagnostic accuracy 97% [95%CI 92-99%], Cohen's kappa 0.93 [95%CI 0.8-1.0]). Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non-HPV-driven OPSCC, resulting in a sensitivity of 96% (95%CI 88-98) and a specificity of 98% (95%CI 90-100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16-driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19-81), but was highly specific (100%, 95%CI 96-100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16-driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16-driven HNSCC outside the oropharynx.
Assuntos
Anticorpos Antivirais/imunologia , Carcinoma de Células Escamosas/imunologia , Papillomavirus Humano 16/imunologia , Proteínas Oncogênicas Virais/imunologia , Neoplasias Orofaríngeas/imunologia , Infecções por Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/virologia , Feminino , Interações Hospedeiro-Patógeno/imunologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Neoplasias Orofaríngeas/diagnóstico , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Sensibilidade e EspecificidadeRESUMO
The carcinogenic role of high-risk human papillomavirus (HR-HPV) types in the increasing subset of vulvar intraepithelial neoplasia and vulvar cancer in young women has been established. However, the actual number of vulvar cancer cases attributed to HPV is still imprecisely defined. In an attempt to provide a more precise definition of HPV-driven vulvar cancer, we performed HPV-type-specific E6*I mRNA analyses available for 20 HR-/possible HR (pHR)-HPV types, on tissue samples from 447 cases of vulvar cancer. HPV DNA genotyping was performed using SPF10-LiPA25 assay due to its high sensitivity in formalin-fixed paraffin-embedded tissues. Data on p16INK4a expression was available for comparative analysis via kappa statistics. The use of highly sensitive assays covering the detection of HPV mRNA in a broad spectrum of mucosal HPV types resulted in the detection of viral transcripts in 87% of HPV DNA+ vulvar cancers. Overall concordance between HPV mRNA+ and p16INK4a upregulation (strong, diffuse immunostaining in >25% of tumor cells) was 92% (K=0.625, 95% confidence interval (CI)=0.531-0.719). Among these cases, 83% were concordant pairs of HPV mRNA+ and p16INK4a+ and 9% were concordant pairs of HPV mRNA- and p16INK4a-. Our data confirm the biological role of HR-/pHR-HPV types in the great majority of HPV DNA+ vulvar cancers, resulting in an HPV-attributable fraction of at least 21% worldwide. Most HPV DNA+ vulvar cancers were associated with HPV16 (85%), but a causative role for other, less frequently occurring mucosal HPV types (HPV26, 66, 67, 68, 70 and 73) was also confirmed at the mRNA level for the first time. These findings should be taken into consideration for future screening options as HPV-associated vulvar preneoplastic lesions have increased in incidence in younger women and require different treatment than vulvar lesions that develop from rare autoimmune-related mechanisms in older women.
Assuntos
Papillomaviridae , Infecções por Papillomavirus/virologia , Neoplasias Vulvares/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/patologia , Neoplasias Vulvares/patologiaRESUMO
Although the prevalence of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is increasing in developed countries and becoming a relevant health issue, the natural history of oral HPV infection is still unclear. Estimating the infection's prevalence in specific populations and identifying risk factors can widen our understanding of its natural history and help to delineate appropriate prevention strategies. This study sought to (i) determine oral HPV prevalence and genotype distribution in a large series of young Italian adults, (ii) validate an oral rinse sampling/storage protocol, and (iii) pinpoint factors associated with oral HPV infection. Five hundred students, nurses, and technicians (19-35 years-old) studying and working at/for the University of Padua were recruited. Each participant was provided with an oral rinse sampling kit and instructions for use. They were also asked to complete an anonymous questionnaire concerning their demographic characteristics and behaviors. The questionnaires and oral rinse containers were labeled with the same identification code number. The oral rinse samples were tested using a bead-based multiplex BSGP5+/6+-MPG genotyping assay which amplifies the L1 region of 51 mucosal HPV types. The prevalence of oral HPV infection was 4.0% (95% confidence interval (CI), 2.5%-6.1%); those of 14 high-risk HPV types and of HPV-type 16 (HPV16) infection were 2.2% (95% CI, 1.1%-3.9%) and 1.6% (95% CI, 0.6%-3.1%), respectively. HPV16 was the most frequent genotype (40.0% of oral HPV infections). No association was found between oral infection and the co-variables studied (gender, tobacco, alcohol and illegal drug use, number of sex and oral sex partners, HPV vaccination status, history of HPV and sexually transmitted infections, abnormal pap smears, recurrent tonsillitis and tonsillectomy). The oral rinse sampling protocol outlined here proved to be simple, efficient and well tolerated, and the prevalence rate can be considered reliable and thus useful to guide future research. Determinants of oral HPV infection are still unclear and further studies are certainly warranted.
Assuntos
Infecções por Papillomavirus/epidemiologia , Adulto , Papillomavirus Humano 16/genética , Humanos , Itália/epidemiologia , Masculino , Doenças da Boca/epidemiologia , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/genética , Infecções por Papillomavirus/etiologia , Prevalência , Fatores de Risco , Inquéritos e Questionários , Adulto JovemRESUMO
HIV-positive men who have sex with men (MSM) have an increased risk for anal human papillomavirus (HPV) infection, anal high-grade intraepithelial lesions (HSIL), and anal cancer. Smoking is associated with abnormal anal cytology and with an increased risk for anal cancer. We collected 3736 intraanal swabs from 803 HIV-positive MSM who participated in an anal cancer screening program between October 2003 and August 2014. HPV prevalence, anal cytology and HPV DNA load of high-risk (HR) HPV-types 16, 18, 31 and 33 of non-smokers and smokers were compared. HPV-typing was performed by alpha-HPV genus-specific PCR and hybridization with 38 type-specific probes using a multiplex genotyping assay. In samples positive for HPV16, 18, 31, or 33, HPV DNA loads were determined by type-specific real-time PCRs and expressed as HPV DNA copies per betaglobin gene copy. At baseline, HR-HPV DNA (80.5 vs. 89.0%, p=0.001), HPV16 DNA (41.6 vs. 52.3%, p=0.003), HPV18 DNA (15.5 vs. 26.0%, p<0.001), anal dysplasia (LSIL+HSIL; 51.5 vs. 58.4%, p=0.045) and HSIL (17.2 vs. 22.7%, p=0.048) were detected more frequently in smokers compared to non-smokers. Throughout the study period 32.7% of non-smokers and 39.9% of smokers developed HSIL (p=0.011), and three smokers developed anal cancer. Considering swabs from the entire study period (median HPV load value per patient per cytology grade), smokers with normal anal cytology had significantly higher HPV16 loads (median 0.29 vs. 0.87, n=201, p=0.007) and cumulative high-risk-HPV loads (median 0.53 vs. 1.08, n=297, p=0.004) than non-smokers. Since elevated HR-HPV DNA loads are associated with an increased risk for HPV-induced anogenital cancers, HPV-infected HIV-positive MSM should be counseled to refrain from smoking. Additionally, for smokers, shorter anal cancer screening intervals than for non-smokers may be appropriate.
Assuntos
Doenças do Ânus/virologia , DNA Viral/isolamento & purificação , Infecções por HIV/complicações , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Fumar/efeitos adversos , Carga Viral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças do Ânus/patologia , DNA Viral/genética , Feminino , Técnicas de Genotipagem , Homossexualidade Masculina , Humanos , Masculino , Pessoa de Meia-Idade , Papillomaviridae/classificação , Papillomaviridae/genética , Medição de Risco , Lesões Intraepiteliais Escamosas Cervicais/patologia , Adulto JovemRESUMO
OBJECTIVE: Cervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women. METHODS: We developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C. RESULTS: HPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5-24 months later had progressed to ≥CIN2. CONCLUSIONS: We successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression.
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Papillomavirus Humano 16/genética , RNA Viral/genética , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , DNA Viral/genética , Detecção Precoce de Câncer/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias do Colo do Útero/patologia , Adulto Jovem , Displasia do Colo do Útero/patologiaRESUMO
Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1ß) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1ß production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1ß processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1ß regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1ß was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1ß that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1ß is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1ß levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1ß regulation which ultimately inhibits the secretion of IL-1ß in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1ß towards cervical cancer could be discerned. Hence, attenuation of IL-1ß by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.
Assuntos
Transformação Celular Viral/imunologia , Papillomavirus Humano 16/imunologia , Imunidade Inata , Interleucina-1beta/imunologia , Proteínas Oncogênicas Virais/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Proteólise , Proteínas Repressoras/imunologia , Evasão Tumoral/imunologia , Proteína Supressora de Tumor p53/imunologia , Ubiquitina-Proteína Ligases/imunologia , Linhagem Celular Transformada , Transformação Celular Viral/genética , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Evasão Tumoral/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.
Assuntos
Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/fisiologia , Neoplasias Orofaríngeas/genética , Infecções por Papillomavirus/genética , Família Aldeído Desidrogenase 1 , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Ilhas de CpG , Metilação de DNA , Intervalo Livre de Doença , Fator de Transcrição GATA4/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Proteínas de Homeodomínio/genética , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Humanos , Estimativa de Kaplan-Meier , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Orofaríngeas/mortalidade , Neoplasias Orofaríngeas/virologia , Infecções por Papillomavirus/mortalidade , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Receptores de AMPA/genética , Retinal Desidrogenase/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , TranscriptomaRESUMO
Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.
Assuntos
Detecção Precoce de Câncer/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Papillomavirus Humano 18/isolamento & purificação , Proteínas E7 de Papillomavirus/análise , Infecções por Papillomavirus/diagnóstico , Vagina/virologia , Virologia/métodos , Anticorpos Monoclonais , Estudos de Viabilidade , Feminino , Papillomavirus Humano 18/imunologia , Humanos , Proteínas E7 de Papillomavirus/imunologia , Esfregaço VaginalRESUMO
BACKGROUND: While the association of the Merkel cell polyomavirus (MCV) with the neuroendocrine Merkel cell carcinomas (MCC) has been shown recently, it is unknown whether other human polyomaviruses (HPyV) may be associated with neuroendocrine tumours (NETs) of distinct entities. METHODS: Using novel, highly sensitive polyomavirus genotyping assays, we evaluated the prevalence of eight distinct HPyVs in a selection of 51 NETs from different entities. In addition, we analyzed these NETs for the presence of DNA from 12 adeno-associated virus (AAV) genotypes, adeno virus-5, 27 mucosal human papillomavirus (HPV) genotypes, hepatitis B (HBV), 8 human herpes viruses (HHV), and xenotropic murine leukemia virus-related virus (XMRV). RESULTS: 43 of the 50 (86%) NETs were positive for the DNA integrity control. Of these, 2 of 3 MCCs (67%) were positive for MCV. NETs from other entities, however, were negative for all HPyVs. Only a small subset of lung and appendix NETs were positive for EBV, HHV-6, and -7. CONCLUSION: While the association of MCV with MCC was confirmed, other human viruses could not be identified as potentially causative agents of other NETs. IMPACT: Our findings suggest that the human viruses tested for in this study do not play a comparable role in NETs like the polyomavirus MCV in MCC.