p16INK4A immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16 INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.

Telomerase activity in the vaginal margins of radical hysterectomy in patients with carcinoma of the cervix: Correlation with histology and human papillomavirus

This study was undertaken to evaluate the telomerase activity both in the tumor and in the vaginal margins of radical hysterectomy in patients with squamous cell carcinoma (SCC) of the cervix. Thirty-three patients with SCC of the cervix (study group) and 13 patients with uterine myoma (control group) were prospectively studied. Tissue samples were taken from the tumor or cervix, anterior vaginal margin (AVM), and posterior vaginal margin (PVM). The specimens were analyzed by histopathology, by a telomerase PCR-TRAP-ELISA kit, and by polymerase chain reaction using human papillomavirus (HPV) DNA. The telomerase activity was significantly higher in the tumor than in the benign cervix (P < 0.001). There was no difference in telomerase activity in the AVM and PVM in patients with cervical carcinoma compared to the control group. Telomerase activity was associated with the presence of histologic malignancy in the PVM of patients submitted to radical hysterectomy (P = 0.03). This association was not observed with the presence of HPV in AVM or PVM in the study group. Telomerase activity is a marker of histologic malignancy in patients with SCC of the cervix. There was no association between the telomerase activity and the presence of HPV in vaginal margins of patients submitted to radical hysterectomy. © 2006, Copyright the Authors.

Human papillomavirus and Epstein-Barr virus infection, p53 expression, and cellular proliferation in laryngeal carcinoma

Laryngeal carcinomas are aggressive neoplasms with controversial association with the human papillomavirus (HPV) and Epstein-Barr virus (EBV). So far, the impairment of p53 protein function and its impact on cellular proliferation has not been studied adequately in these tumors. In this work, molecular biologic techniques were used to assess the frequency of HPV and EBV in 110 squamous cell carcinomas of the larynx. In addition, accumulation of p53 and Ki-67 cell proliferation antigen expression in malignant cells was assessed by immunohistochemical analysis. High-grade HPV was found in 37.3% of cases, and none had demonstrable EBV infection. Accumulation of p53 was found in 78.2% of the cases, and it was related to a high Ki-67 labeling index and higher histologic grade. The results demonstrate association of HPV with more than one third of laryngeal carcinomas studied, mainly glottic tumors. Tumors with increased cell proliferation were more frequently high grade, with p53 accumulation and lymph node metastasis. © American Society for Clinical Pathology.

Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil

There are few studies about the distribution of natural molecular variants of low-risk HPVs. Our aim was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. We also performed a phylogenetic analysis in order to compare nucleotide sequences identified in our study with previously reported isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world to determine the phylogenetic relationships of variants detected in Brazil. The complete coding region of the E6 gene of 25 samples was cloned and sequenced: 18 isolates of HPV-6 (72%) and 7 isolates of HPV-11 (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants was identified. It was not possible to correlate specific variants with disease severity. Phylogenetic trees for both HPV types were constructed enclosing both E6 sequences detected in our study and formerly published sequences. In both phylogenetic trees, the sequences from Brazil did not group together. We could not establish a geographical association between HPV-6 or HPV-11 variants, unlike HPV-16 and HPV-18. © 2013 Elsevier B.V.

Low frequency of human papillomavirus detection in prostate tissue from individuals from Northern Brazil

The presence of human papillomavirus (HPV) was evaluated in 65 samples of prostate tumours and six samples of prostates with benign prostatic hyperplasia from individuals from Northern Brazil. We used a highly sensitive test, the Linear Array HPV Genotyping Test, to detect 37 high and low-risk HPV types. In this study, only 3% of tumour samples showed HPV infection. Our findings support the conclusion that, despite the high incidence of HPV infection in the geographic regions studied, HPV was not associated with a higher risk of prostate cancer. To our knowledge, this is the first study evaluating the frequency of HPV detection in prostatic tissue of individuals from Brazil.

Determinants of baseline seroreactivity to human papillomavirus type 16 in the Ludwig-McGill cohort study

Background: Immunity plays an important role in controlling human papillomavirus (HPV) infection and associated lesions. Unlike infections caused by other viruses, natural HPV infection does not always result in a protective antibody response. Therefore, HPV antibodies are also considered markers of cumulative exposure. The aim of this study was to identify determinants of HPV16 seroreactivity at enrollment among women from the Ludwig-McGill cohort, a natural history study of HPV infection and risk of cervical neoplasia.Methods: HPV16 serology was assessed by ELISA for L1 and L2 capsid antigens, while HPV typing and viral load measurements were performed by PCR-based methods. The associations were analyzed by unconditional logistic regression.Results: Of 2049 subjects, 425 (20.7%) were strongly seropositive for HPV16. In multivariate analysis, seroreactivity was positively correlated with age, lifetime number of sexual partners, frequency of sex, and HPV16 viral load, and negatively associated with duration of smoking.Conclusions: HPV16 seroreactivity is determined by factors that reflect viral exposure.

Differences in transcriptional activity of human papillomavirus type 6 molecular variants in recurrent respiratory papillomatosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Implementing Human Papillomavirus Testing in a Public Health Hospital: Challenges and Opportunities

Objectives: Robust evidence now supports human papillomavirus (HPV) testing as a more effective option to screening and as more sensitive than cytology in detecting high-grade cervical intraepithelial neoplasia. Our goal was to analyze the performance of the Hybrid Capture II (HC2) assay for high-risk HPV (hrHPV) in women undergoing gynecological examination at a public health hospital as part of the evaluation of HPV screening as an alternative or complement to cytology. Study Design: This analysis is a subset of a cross-sectional study carried out at a large public hospital serving a predominantly low-resource population. A total of 705 women were enrolled; the sensitivity and specificity of each test were estimated and compared. Results: The analysis identified 272 hrHPV-positive women (mean age 36.3 years) and 433 hrHPV-negative women (mean age 41.2 years). HPV testing showed a significantly increased sensitivity of the HC2 assay versus cytology (84.5 vs. 69.7%; p < 0.0001) but a lower specificity (49.90 vs. 88.78%; p < 0.0001). Conclusion: The combination of both methods seems to be useful in improving detection of cervical lesions. Copyright (C) 2012 S. Karger AG, Basel

Longitudinal outcomes of high-risk human papillomavirus (HPV) infections as competing-risks events following cervical HPV test at baseline visit in the *NIS-LAMS** cohort

Background: The complex natural history of human papillomavirus (HPV) infections following a single HPV test can be modeled as competing-risks events (i.e., no-, transient- or persistent infection) in a longitudinal setting. The covariates associated with these compet ng events have not been previously assessed using competing-risks regression models. Objectives: To gain further insights in the outcomes of cervical HPV infections, we used univariate- and multivariate competing-risks regression models to assess the covariaies associated with these competing events. Study Design and Methods: Covariates associated with three competing outcomes (no-, transient- or persistent HR-HPV infection) were analysed in a sub-cohort of 1,865 women prospectively followed-up in the NIS (n = 3,187) and LAMS Study (n = 12,114). Results: In multivariate competing-risks models (with two other outcomes as competing events), permanently HR-HPV negative outcome was significantly predicted only by the clearance of ASCUS+Pap during FU, while three independent covariates predicted transient HR-HPV infections: i) number of recent (< 12 months) sexual partners (risk increased), ii) previous Pap screening history (protective), and history of previous CIN (increased risk). The two most powerful predictors of persistent HR-HPV infections were persistent ASCUS+Pap (risk increased), and previous Pap screening history (protective). In pair-wise comparisons, number of recent sexual partners and previous CIN history increase the probability of transient HR-HPV infection against the HR-HPV negative competing event, while previous Pap screening history is protective. Persistent ASCUS+Pap during FU and no previous Pap screening history are significantly associated with the persistent HR-HPV outcome (compared both with i) always negative, and ii) transient events), whereas multiparity is protective. Conclusions: Different covariates are associated with the three main outcomes of cervical HPV infections. The most significant covariates of each competing events are probably distinct enough to enable constructing of a risk-profile for each main outcome.

Covariates of high-risk human papillomavirus (HPV) infections are distinct for incident CIN1, CIN2 and CIN3 as disclosed by competing-risks regression models

Background: In addition to the oncogenic human papillomavirus (HPV), several cofactors are needed in cervical carcinogenesis, but whether the HPV covariates associated with incident i) CIN1 are different from those of incident ii) CIN2 and iii) CIN3 needs further assessment. Objectives: To gain further insights into the true biological differences between CIN1, CIN2 and CIN3, we assessed HPV covariates associated with incident CIN1, CIN2, and CIN3. Study Design and Methods: HPV covariates associated with progression to CIN1, CIN2 and CIN3 were analysed in the combined cohort of the NIS (n = 3,187) and LAMS study (n = 12,114), using competing-risks regression models (in panel data) for baseline HR-HPV-positive women (n = 1,105), who represent a sub-cohort of all 1,865 women prospectively followed-up in these two studies. Results: Altogether, 90 (4.8%), 39 (2.1%) and 14 (1.4%) cases progressed to CIN1, CIN2, and CIN3, respectively. Among these baseline HR-HPV-positive women, the risk profiles of incident GIN I, CIN2 and CIN3 were unique in that completely different HPV covariates were associated with progression to CIN1, CIN2 and CIN3, irrespective which categories (non-progression, CIN1, CIN2, CIN3 or all) were used as competing-risks events in univariate and multivariate models. Conclusions: These data confirm our previous analysis based on multinomial regression models implicating that distinct covariates of HR-HPV are associated with progression to CIN1, CIN2 and CIN3. This emphasises true biological differences between the three grades of GIN, which revisits the concept of combining CIN2 with CIN3 or with CIN1 in histological classification or used as a common end-point, e.g., in HPV vaccine trials.

Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA. E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants. (C) 2012 Elsevier Inc. All rights reserved.

Expression of BAG-1 and PARP-1 in Precursor Lesions and Invasive Cervical Cancer Associated with Human Papillomavirus (HPV)

Cervical cancer remains persistently the second most common malignancies among women worldwide, responsible for 500,000 new cases annually. Only in Brazil, the estimate is for 18,430 new cases in 2011. Several types of molecular markers have been studied in carcinogenesis including proteins associated with apoptosis such as BAG-1 and PARP-1. This study aims to demonstrate the expression of BAG-1 and PARP-1 in patients with low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs) and invasive squamous cell carcinomas (SCCs) of the uterine cervix and to verify a possible association with HPV infection. Fifty samples of LSILs, 50 samples of HSILs and 50 samples of invasive SCCs of the uterine cervix were analyzed by immunohistochemistry for BAG-1 and PARP-1 expression. PCR was performed to detect and type HPV DNA. BAG-1 expression levels were significantly different between LSILs and HSILs (p = 0,014) and between LSILs and SCCs (p = 0,014). In regards to PARP-1 expression, we found significant differences between the expression levels in HSILs and SCCs (p = 0,022). No association was found between BAG-1 expression and the presence of HPV. However, a significant association was found between PARP-1 expression and HPV positivity in the HSILs group (p = 0,021). In conclusion our research suggests that BAG-1 expression could contribute to the differentiation between LSIL and HSIL/SCC whereas PARP-1 could be useful to the differentiation between HSIL HPV-related and SCC. Further studies are needed to clarify the molecular aspects of the relationship between PARP-1 expression and HPV infection, with potential applications for cervical cancer prediction.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Abstract Background Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.