Human Papillomavirus E7 Genes Research Articles

Molecular detection of human papillomavirus DNA in dysplastic and non-dysplastic oral lichen planus patients

Introduction: Oral Lichen Planus (OLP) is a chronic inflammatory disease of cell-mediated dysregulation characterized by relapses and remissions. The aetiology of OLP has been extensively studied for decades. The pathogenesis is unclear with a debatable malignant transformation. The pathogenesis and malignant transformation of OLP may be affected by viruses such as Hepatitis C Virus (HCV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), and stress. HPV has been proved to be an etiological agent in oropharyngeal cancers and non-tobacco-associated leukoplakia. The role of human papillomavirus in the pathogenesis of OLP and its malignant transformation has to be studied extensively. Aim: This study aims to detect the presence of HPV DNA in the biopsy samples of dysplastic and non-dysplastic OLP and thus determine the role the virus played in the malignant transformation of OLP. Materials and Methods: Biopsy samples comprising 250 OLP tissues were collected. The DNA was extracted from the fixed tissue by using the Cetyltrimethylammonium bromide (CTAB) method. Polymerase Chain Reaction (PCR) was performed using primers to amplify the HPV E6 gene. Results: Hundred and three out of 250 (41.2%) OLP cases were positive for HPV DNA.

Expression of human papillomavirus 16 and 18 DNA in oral lichen planus using polymerase chain reaction.

Oral lichen planus (OLP) is a chronic inflammatory disease with cell-mediated immune dysregulation. The aetiology of OLP has been studied extensively for decades. Viruses like Hepatitis C Virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), and stress have been hypothesized to play a role in the pathogenesis and malignant transformation of OLP. HPV has been proved to be an etiological agent in oropharyngeal cancers and non-tobacco-associated leukoplakia. The role of human papillomavirus in the pathogenesis of OLP has to be studied extensively. This study aims to detect the presence of HPV 16 and HPV 18 DNA in the biopsy samples of OLP and also to determine the role played by the virus in the pathogenesis and malignant transformation of OLP. Biopsy samples comprising 30 OLP tissues were collected. The DNA was extracted by the cetyltrimethylammonium bromide method. Polymerase chain reaction was performed by using general primers to amplify the HPV E6 gene. Twelve out of 30 (40%) OLP cases were positive for HPV DNA. A significant relation was found between HPV, site (buccal mucosa) and the type (reticular) of the lesion (P = 0.007). However, the difference between the percentage of HPV positive males and females was statistically insignificant (P = 0.852). This study confirmed the presence of high-risk HPV 16 and HPV 18 DNA in OLP. The study showed a significantly higher expression of HPV in erosive OLP when compared to reticular OLP, suggesting a possible role of HPV in the malignant transformation of OLP.

Association between promoter methylation and gene expression of CGB3 and NOP56 in HPV-infected cervical cancer cells.

Overexpression of the E7 gene of human papillomavirus (HPV) type 16 is one of the primary causes of cervical cancer. The E7 protein can bind with DNA methyltransferase I and induce methylation of tumor suppressor genes, such as cyclin-A1 (CCNA1), leading to suppression of their expression, and thus, cancer progression. In the present study, the confirmation of methylation-related expression of chorionic gonadotropin subunit 3 (CGB3) and nucleolar protein 56 (NOP56) genes in 5-Azacytidine (5'-aza)-treated HPV16-positive SiHa and HPV16-negative C33A cell lines was shown. Using methylation-specific-PCR and quantitative PCR, the results showed that CGB3 and NOP56 methylation significantly decreased as the 5'-aza concentration was increased, and this was inversely associated with their expression. Moreover, overexpression of E7 contributed to the augmentation of CGB3 and NOP56 methylation levels in C33A cells, resulting in a decrease in their expression. This study extends on previous observations of E7 HPV16 oncogenic function in terms of methylation-repressing expression in more genes, which may be wholly applied to gene therapy in cervical cancer prevention.

Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer.

Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

Association between APOBEC3s and HPV16 E2 gene hypermutation in Uygur females with cervical cancer.

The present study aimed to investigate whether apolipoprotein B mRNA-editing enzyme catalytic polypeptides (A3) are involved in the regulation of cervical cancer development and human papilloma virus (HPV)16 sustained infection in Uighur females. Cervical tissues of Uygur patients with HPV16 with cervical lesions were collected. Expression levels of A3C, A3F and A3G were detected using reverse transcription-quantitative PCR and western blotting. A model of SiHa cells with high expression levels of A3C, A3F and A3G was constructed. Hypermutation was detected using the differential DNA denaturation PCR and positive samples were amplified and sequenced. There were significant differences in A3 expression levels in cervical lesions of different grades. A3C and A3F mRNA and protein expression in cervical cancer tissues were significantly lower, whereas the A3G mRNA and protein expression levels were significantly higher compared with the cervicitis and cervical intraepithelial neoplasia (CIN) I–III groups. Hypermutation rates were increased with cervical lesion development. C>T and G>A base substitutions were detected in all hypermutation samples and numbers of C>T and G>A base substitutions in single samples in the cervical cancer group were significantly higher compared with those in the CIN I–III and cervicitis groups. Following transfection of A3F and A3G, HPV E2 mRNA and protein expression levels were significantly decreased in SiHa cells. Numerous C>T and G>A base substitutions were detected in the HPV E2 gene in A3G and A3C overexpressing SiHa cells. A3 family proteins inhibit viral replication during HPV16 infection and regulate the HPV16 integration by inducing C>T and G>A hypermutations in the HPV16 E2 gene, thus affecting the cervical cancer pathogenesis and development.

Reprogrammed CRISPR-Cas13a targeting the HPV16/18 E6 gene inhibits proliferation and induces apoptosis in E6-transformed keratinocytes.

The long-lasting infection of high-risk type (type 16 or type 18) human papillomavirus (HPV) is the main cause of gynecological and urinary malignancies. Given the high mortality rate after surgery, the development of a new molecular therapy would be of value to clinicians. The aim of the present study was to achieve targeted inactivation of the viral E6 gene in keratinocytes using the reprogrammed CRISPR-Cas13a system. To accomplish this, a reprogrammed CRISPR-Cas13a system, targeting both the HPV16/18 E6 genes was constructed using a guide RNA expressing vector. The expression levels of E6 protein were measured using western blot analysis after transfection of the vector into E6-transformed keratin oocytes. Cell proliferation was analyzed using CCK-8 assays and cell apoptosis was evaluated using Hoechst 33258 staining and ELISAs examining caspase-3 levels. The results indicated that both the HPV16/18 E6 genes can be inactivated using the CRISPR-Cas13a system. Furthermore, silencing E6 inhibited cell proliferation (14±1.8% on average) and induced apoptosis (80.2±3.2% on average) in E6-transformed keratinocytes but not in normal keratinocytes. In conclusion, results of the present study demonstrate that the reprogrammed CRISPR-Cas13a system has the potential for inactivating HPV E6 gene functions.

Heat Increases the Editing Efficiency of Human Papillomavirus E2 Gene by Inducing Upregulation of APOBEC3A and 3G

Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3 proteins have been identified as potent viral DNA mutators and have broad antiviral activity. In this study, we demonstrated that apolipoprotein B mRNA-editing catalytic polypeptide 3A (A3A) and A3G expression levels were significantly upregulated in human papillomavirus (HPV)-infected cell lines and tissues. Heat treatment resulted in elevated expression of A3A and A3G in a temperature-dependent manner in HPV-infected cells. Correspondingly, HPV-infected cells heat-treated at 44 °C showed accumulated G-to-A or C-to-T mutation in HPV E2 gene. Knockdown of A3A or A3G could promote cell viability, along with the lower frequency of A/T in HPV E2 gene. In addition, regressing genital viral warts also harbored high G-to-A or C-to-T mutation in HPV E2 gene. Taken together, we demonstrate that apolipoprotein B mRNA-editing catalytic polypeptide 3 expression and editing function was heat sensitive to a certain degree, partly explaining the mechanism of action of local hyperthermia to treat viral warts.

Curcumin improves the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cells via the NF-κB-p53-caspase-3 pathway.

Paclitaxel, isolated from Taxus brevifolia, is considered to be an efficacious agent against a wide spectrum of human cancers, including human cervical cancer. However, dose-limiting toxicity and high cost limit its clinical application. Curcumin, a nontoxic food additive, has been reported to improve paclitaxel chemotherapy in mouse models of cervical cancer. However, the underlying mechanisms remain unclear. In this study, two human cervical cancer cell lines, CaSki [human papilloma virus (HPV)16-positive] and HeLa (HPV18-positive), were selected in which to investigate the effect of curcumin on the anticancer action of paclitaxel and further clarify the mechanisms. Flow cytometry and MTT analysis demonstrated that curcumin significantly promoted paclitaxel-induced apoptosis and cytotoxicity in the two cervical cell lines compared with that observed with paclitaxel alone (P<0.05). Reverse transcription-polymerase chain reaction indicated that the decline of HPV E6 and E7 gene expression induced by paclitaxel was also assisted by curcumin. The expression levels of p53 protein and cleaved caspase-3 were increased significantly in the curcumin plus paclitaxel-treated HeLa and CaSki cells compared with those in the cells treated with paclitaxel alone (P<0.01). Significant reductions in the levels of phosphorylation of IκBα and the p65-NF-κB subunit in CaSki cells treated with curcumin and paclitaxel were observed compared with those in cells treated with paclitaxel alone (P<0.05). This suggests that the combined effect of curcumin and paclitaxel was associated with the NF-κB-p53-caspase-3 pathway. In conclusion, curcumin has the ability to improve the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cell lines via the NF-κB-p53-caspase-3 pathway. Curcumin in combination with paclitaxel may provide a superior therapeutic effect on human cervical cancer.

Abstract 4147: Mate pair sequencing reveals that human papillomavirus integration into the human genome in oropharyngeal squamous cell carcinoma is rare and different from cervical cancer

Abstract In spite of decreases in the incidence of smoking in the United States over the past several decades, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing. This is presumably due to an increase in the incidence of human papillomavirus (HPV)-positive OPSCC. The model for how HPV contributes to cancer development is based upon studies characterizing cervical cancer. In cervical cancer almost all invasive cancers have HPV integrated into the human genome with loss of episomal copies of HPV. The site of integration is usually within the HPV E2 gene which functions as a repressor of the two key HPV oncoproteins, E6 and E7. The resulting over-production of E6 and E7 inactivates two key pathways which results in genomic instability and additional down-stream alterations that eventually leads to invasive cervical cancer. In order to definitively test whether this model is indeed true for OPSCC we performed mate-pair next generation sequencing on both tumor and adjacent normal tissues from 20 OPSCC patients: 7 were HPV16-positive and had high expression of the E6 and E7 transcripts; 6 were HPV-positive but with low E6 and E7 transcription; and as a control we also analyzed 7 HPV16-negative tumors. This high throughput technology was able to successfully detect all HPV integration events into the human genome as well as HPV bridged coverage from these samples with high specificity. Just a single HPV-positive tumor with high oncogene expression had HPV integration events (two), but this patient also had a very high copy number of episomal HPV sequences. A single HPV-positive tumor with low oncogene expression had one HPV integration event, but no episomal HPV sequences present. One of the HPV16-negative tumors had a single HPV26 integration with no episomal HPV sequences. None of the matched normal samples had either HPV integrations or an appreciable amount of episomal HPV sequences present. Thus, only 21.4% of HPV-positive OPSCCs had integrations event. All four integrations were validated by PCR and Sanger sequencing which revealed that none of them interrupted the HPV E2 gene. All four of the integrations occurred next to human genes that have been found to be altered in different cancers. Our results suggest that the role that HPV plays in OPSCC may be quite distinct from what is observed in cervical cancer. The results also demonstrate that not all HPV-positive OPSCCs have HPV in the same physical state. It will thus be interesting to compare the distinct groups of HPV-positive OPSCCs with respect to overall clinical prognosis. Citation Format: Ge Gao. Mate pair sequencing reveals that human papillomavirus integration into the human genome in oropharyngeal squamous cell carcinoma is rare and different from cervical cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4147. doi:10.1158/1538-7445.AM2014-4147

MiR-122 directly inhibits human papillomavirus E6 gene and enhances interferon signaling through blocking suppressor of cytokine signaling 1 in SiHa cells.

Human Papillomavirus (HPV) 16 infection is considered as one of the significant causes of human cervical cancer. The expression of the viral oncogenes like E6 and E7 play an important role in the development of the cancer. MiR-122 has been reported to exhibit a strong relationship with hepatitis viruses and take part in several tumor development, while the effects of miR-122 on HPV infection and the HPV viral oncogenes expression still remain unexplored. In this study, using RNAhybrid software, the potential binding sites between miR-122 and HPV16 E6 and E7 mRNAs were identified. Over and loss of miR-122 function showed that miR-122 could directly bind with HPV16 E6 mRNA and significantly inhibit its expression in SiHa cells, which was further confirmed by constructing the miR-122-E6-mu to eliminate the miR-122 binding effects with E6. The increase of the expression of type I interferon (IFN) and its classical effective molecules and the phosphorylation of signal transducers and activators of transcription (STAT1) protein indicated that miR-122 might enhance type I interferon in cervical carcinoma cells, which explained the significant reduction of HPV16 E7 and E6*I mRNA expression. This might be due to the binding between miR-122 and suppressor of cytokine signaling 1 (SOCS1) mRNA, which is the suppressor of interferon signaling pathway. Moreover, it was identified that the miR-122 binding position was nt359-nt375 in SOCS1 mRNA. Taken together, this study indicated that HPV16 could be effectively inhibited by miR-122 through both direct binding with E6 mRNA and promoting SOCS1-dependent IFN signaling pathway. Thus, miR-122 may serve as a new therapeutic option for inhibiting HPV infection.

Electrochemical DNA biosensor for the detection of human papillomavirus E6 gene inserted in recombinant plasmid

In the current study, we describe a novel, simple, inexpensive, sensitive, specific, stable and label-free electrochemical DNA biosensor used to identify a target gene cloned into a plasmid. The biosensor was designed with a 23-mer oligonucleotide of guanine-free, which was immobilized on the pencil graphite electrode (PGE) for E6 gene detection from human papillomavirus 16 type (HPV16). The E6 gene was used due to its clinical importance. The optimal probe concentration was obtained in 500nM. The hybridization detection showed a good linearity in the range of 40–5,000pg/μL with a detection limit of 16pg/μL. The electrochemical method showed higher sensitivity and specificity when compared with the agarose gel electrophoresis assay. This technology could be postulated as a new and attractive alternative for cloning analysis in plasmids.

The timing of TGF-β inhibition affects the generation of antigen-specific CD8+ T Cells

BackgroundTransforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.MethodsBALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.ResultsInhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).ConclusionsThese studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.

Abstract 4776: Expression of Human Papillomavirus E6 and E7 genes in colorectal cancer.

Abstract Purpose: The role of human papillomavirus (HPV) has been well established in the pathogenesis on cervical cancers and anogenital. However, HPV-DNA has also been detected in tumor tissues of other malignancies such as head and neck cancer, esophageal cancer, and some skin cancers. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. Both E6 and E7 can form specific complexes with tumor suppressor gene p53 and pRB respectively. However, the role of HPV in the pathogenesis of colorectal cancer is still undetermined. Our objectives were (1) to evaluate the presence of HPV infection in patients with CRC; (2) to type HPV positive cases using primer specific for HPV-16, and (3) to determine the mRNA expression levels of the viral oncogenes HPV-16 E6 and HPV-16 E7. Experimental Designs: To determine the association of HPV and CRC, we conducted an age-and-gender matched case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases) and without CRC (controls). Presence of L1, E2, and E6 genes was analyzed to determine whether a full-length HPV genome existed in tumor and normal tissue samples. Human HPV-16 DNA detection was performed by a nested PCR strategy using the PGMY09/PGMY11 as outer primer, and GP5+/GP6+ as inner primers. We detected the expression of E6 and E7 mRNA in the HPV-16 (+) cases. Hypothesis testing was performed using Pearson Chi-square analysis of proportions and conditional logistic regression using SPSS 17. Results: A total of 54 (ß-actin+) CRC patients (mean age 61 ±11 years, 27 males) and 36 controls (mean age was 60 ± 9 years, 18 males) were evaluated. HPV DNA was identified in 25 of 54 (46.3%) patients with CRC and in 1 of 36 (2.8%) controls (OR = 25.58; 95% CI 3.22 to 203.49, p &amp;lt; 0.001). HPV-16 was identified in 18 of 25 (72.0%) HPV-positive CRC cases, and in one (2.8%) control was HPV (+). All 18 HPV-16 CRC cases showed expressed E6 mRNA. Six of 18 (33.3%) HPV-16 positive cases expressed E7 mRNA. Among the 12 CRC cases where both tumor and adjacent tissue was available, 2 of 9 (16.7%) exhibited HPV-16 DNA in both tumor and tumor-adjacent tissues. Conclusion: These results support that colorectal HPV infection is common among patients with CRC. The high presence of HPV in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Moreover, our finding of highly frequent active expression of E6 and E7 viral oncogenes in the host strongly suggests that HPV oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: Wesley Villavicencio-Torres, Mercedes Y. Lacourt-Ventura, Sharon C. Fonseca-Williams, Maria Del Mar Gonzalez- Pons, Yaritza Diaz-Algori, Carlos Torres-Ramos, Raul D. Bernabe-Dones, Marcia R. Cruz-Correa. Expression of Human Papillomavirus E6 and E7 genes in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4776. doi:10.1158/1538-7445.AM2013-4776

Abstract 552: Characterization of HPV infection in Hispanic colorectal cancer patients

Abstract Purpose: The association between Human Papillomavirus (HPV) infection and the development of cervical cancer and anogenital cancer has been established. Moreover, molecular studies have detected HPV DNA in tumor tissues of head and neck cancer, esophageal cancer, and some skin cancers. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. Both E6 and E7 can form specific complexes with tumor suppressor gene p53 and pRB respectively. However, the role of HPV infection in colorectal cancer (CRC) and its mechanisms of carcinogenesis have not been properly elucidated. Our objectives were to evaluate the presence of HPV infection in patients with CRC; to probe the viral integrations status of HPV 16 in CRC and its adjacent normal colonic mucosa, and to determine the mRNA expression levels of the viral oncogenes HPV-16 E6 and HPV-16 E7. Experimental Designs: To determine the association of HPV and CRC, we conducted an age-and-gender matched case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases) and without CRC (controls). Presence of L1, E2, and E6 genes was analyzed to determine whether a full-length HPV genome existed in tumor and normal tissue samples. Head-to-tail junctions of HPV-16 genomes were analyzed to determine the integration the HPV-16 genome in the HPV-16 (+) cases. Moreover, we determined the mRNA expression levels of E6 and E7 in the HPV-16 (+) cases. Hypothesis testing was performed using Pearson Chi-square analysis of proportions using SPSS 17. Results: A total of 45 (ß-actin+) CRC patients (mean age 61 ±11 years, 24 males) and 47 controls (mean age was 60 ± 9 years, 18 males) were evaluated. Tumors were located in proximal colon (n=7) and distal colon (n=38). HPV DNA was identified in 19 of 45 (42.2%) patients with CRC and in 2 of 47 (4.3%) controls (OR = 16.4; 95% CI 3.5-76.3, p &amp;lt; 0.001). HPV-16 was identified in 12 of 19 (63.2%) HPV (+) CRC patients and in none (0%) HPV(+) controls. The twelve HPV-16 CRC cases showed integration of HPV-16. Three of 12 (25%) HPV16 (+) cases expressed E6 and E7 mRNA. Among the 12 CRC cases where both tumor and adjacent tissue was available, 2 of 9 (16.7%) exhibited HPV-16 DNA in both tumor and tumor-adjacent tissues.Conclusion: These results suggest that colorectal HPV infection is common among patients with CRC. The presence of HPV in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Moreover, our finding of highly frequent integration of viral DNA and the active expression of E6 and E7 viral oncogenes in the host strongly suggests that HPV oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 552. doi:1538-7445.AM2012-552

Oncolytic adenovirus armed with human papillomavirus E2 gene in combination with radiation demonstrates synergistic enhancements of antitumor efficacy

High-risk human papillomavirus (hr-HPV) E6 and E7 oncogenes are associated with resistance to radiotherapy in cervical cancer. Efforts have been taken to employ HPV E2, a crucial negative transcriptional modulator of HPV E6 and E7 oncogenes, and also an apoptosis-inducing agent, for therapeutic intervention. Despite being conceptually attractive, the potency and feasibility of current hr-HPV E2-based therapies remain limited. Here, we designed a novel recombinant adenovirus, named M5, with a 27-bp deletion in E1A conserved region-2 by which to realize tumor-specific replication, and a total HPV type 16 (HPV16) E2 gene complementary DNA inserted into the E3 coding region. In this design, M5 exploited the adenovirus E3 promoters to express HPV16 E2 gene in a viral replication-dependent manner and preferentially silenced the hr-HPV E6 and E7 oncogenes in HPV-positive cervical cancer cells. In vitro and in vivo assays confirmed that M5 exhibited potent antitumoral efficacy. Moreover, the effects of combined treatment with M5 and radiation treatment resulted in synergistically enhanced potency (P<0.01). The increase in killing efficacy of M5 was also found in HPV-negative cervical cancer cells, for which the pro-apoptotic activity of HPV16 E2 was thus responsible. Our results indicated that the use of M5 that locally delivers HPV16 E2 to cancers has broad therapeutic windows and that the combination therapy with radiation for cervical cancer will be the more effective way of improving survival.

State of HPV16 integration in Lithuanian women with cervical neoplasia

AbstractCervical cancer morbidity and mortality in Lithuania is one of the biggest in the European Union. The main risk factor of cervical cancer is human papillomavirus (HPV). The deletion of the HPV E2 gene influences HPV DNA integration into the cell genome, as well as a rapid progression of cervical lesions. The purpose of this study is to determine HPV, its types, and HPV 16 integration in different grades of cervical intraepithelial neoplasias (CIN). 253 women with cytological lesions were involved in the study. After a histology, 31 women were diagnosed with CIN I, 35 with CIN II, and 51 with CIN III. The biggest prevalence of HPV infection was detected in women younger than 25 years old (69.7%) and in women with CIN II (90.9%). HPV 16 was detected in 67.8% of all cases, with the highest prevalence in CIN III (84.4%). A partial integration form was detected in 65.0% of HPV 16 infected women, a complete virus integration in 26.5%, and an episomal form in 8.4% of cases. Our study concludes that in all the cases confirmed using a histology, the partial virus integration form of CIN was identified the most. It was less frequently detected in CIN I cases (60.0%), but more frequently in CIN II and CIN III cases (72.8 and 69.3%, respectively).

Abstract 5359: HPV 16 infection and physical status in Hispanic patients with colorectal cancer: A case-control study

Abstract Purpose: The association between human papillomavirus (HPV) infection and the development of cervical cancer and anogenital cancer has been established. In addition, molecular studies have detected HPV DNA in tumor tissues of head and neck cancer, oral cancer, esophageal cancer, and some skin cancers as well as lung cancer. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. However, the role of HPV infection in colorectal cancer (CRC) and its mechanisms of carcinogenesis have not been properly elucidated. Our objectives were to evaluate the presence of HPV16 infection in patients with CRC and to probe the viral integrations status of HPV 16 in CRC and its adjacent normal colonic mucosa. Experimental Designs: To determine the association of HPV 16 and CRC, we conducted a case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases), and compared the prevalence of HPV 16 with normal colorectal mucosa from individuals without CRC (controls). Detection of L1, E2, and E6 genes, which cover the two ends and middle part of the virus genome, we were able to analyze whether a full-length HPV genome existed in the tissue samples. Head-to-tail junctions of HPV genomes were analyzed to determine the physical status the HPV genome in the HPV (+) cases. Hypothesis testing was performed using Fisher exact and analysis of proportions using STATA 10.0. Results: A total of 21 (ß-actin+) CRC patients (mean age 63 ±11 years, 8 males) and 20 controls (mean age was 63 ± 9 years, 3 males) were evaluated. Tumors were located in rectum (n=3), sigmoid (n=6), descending (n=7) and ascending (n=5) colon. HPV 16 DNA was identified in 13 of 21 (62%) patients with CRC and in none of 20 (0%) controls (p = 0.0002). HPV 16 DNA was observed in the all rectal cancer cases and in 10 (55.5%) of colon cancer cases. Among the 9 cases where both tumor and adjacent tissue was available, 2 of the 9 (22%) exhibited HPV16 DNA in both tumor and tumor-adjacent tissues. Nine of the ten (90%) tumors and all adjacent normal colonic mucosa (5/5) contained the HPV integrated form. Conclusion: These results suggest that colorectal HPV16 infection is common among patients with CRC. Furthermore, our finding of frequent integration of viral DNA in the host genome suggests that integration HPV 16 could be a common event in CRC patients. The presence of HPV 16 in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Taken together our findings suggest that HPV 16 oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5359.

Gene characterization of E6 and E7 gene of human papillomavirus of 15 cervical cancer in Beijing

Conducting the gene characterization of the E6 and E7 gene of human papillomavirus 16 (HPV16) isolated from 15 cases of cervical cancer at Beijing. Overlapping primers were designed according to the full-length genomes of E6 and E7 from the GenBank and PCR was used to amplify the E6 and E7 fragments. TA clone was used to select a purified clone in order to have better and valuable sequencing results. Nucleotide and amino acid sequence were analyzed by the Sequencer, Bioedit, Mega et al. 8 of 15 (8/15) cervical samples contained HPV E6 and E7 gene, and 4 had Asian type like and 4 had Europe prototype like. There were two nucleotide mutation at E6 position 178 (T-->G,D25E) and at E7 position 647 (A-->G, N29S) in 4 Asian type like viruses. There were one nucleotide mutation at E6 position 335 (C-->T, H78Y) in 1 of 4 Europe prototype like virus. In the cervical cancer samples, 8 of 15 contained the HPV16E 6 and E7 gene. HPV16 E6 and E7 can not be detected in denosquamous carcinoma and adenocarcinoma. HPV16 is the main etiology of the cervical carcinoma. The HPV16 infectious ratio of squamous carcinoma is more than the ratio of adenocarcinoma. 178th nucleotide in E6 gene is the very important site to distinguish the Asia and the Europe prototype strain like. 178 nucleotide in E6 and 647 nucleotide in E7 are the frequent mutation site in cervical carcinoma. Analysis based on the E6 and E7 gene sequence of HPV 16 isolates suggests that naturally occurring sequence variants of E6 and E7 gene may have identify the oncogenic properties.

B-cell Depletion Using an Anti-CD20 Antibody Augments Antitumor Immune Responses and Immunotherapy in Nonhematopoetic Murine Tumor Models

The role played by B cells in cancer biology is complex and somewhat controversial. Previous studies using genetically engineered mice suggest that B cells may be immunosuppressive and inhibit tumor rejection. However, the effects of B-cell depletion employing an antibody in mice bearing solid tumors has not been tested owing to difficulties in making an effective antimouse CD20 antibody (similar to rituximab). Injection of a newly developed antimouse CD20 antibody was effective in depleting circulating B cells from blood and lymph nodes, although depletion was less complete in the spleen. B-cell depletion slowed the growth of new solid tumors (not expressing CD20) and retarded the growth of established tumors but did not induce tumor regression. However, when the antibody was combined with an active immunotherapy approach using an adenovirus vaccine expressing the human papilloma virus-E7 gene (Ad.E7) in mice bearing TC1 tumors (murine lung cancer cells expressing human papilloma virus-E7), we noted enhanced antitumor effects and increased numbers of tetramer+/CD8+ T cells within the spleens and activated CD8+ T cells within tumors. B-cell depletion using an anti-CD20 antibody was thus effective in retarding tumor growth in multiple solid tumor models and augmenting immunotherapy in a tumor vaccine model. These studies raise the possibility that B-cell depletion may be a useful adjunct in human immunotherapy trials.

Epidemiologic modeling of cervical dysplasia with molecular and cytopathological markers

Objectives Conventional cervical screening is insufficient at identifying patients who are likely to progress from cervical dysplasia to carcinoma. Traditional epidemiologic studies have identified potential factors to aid in the discrimination between those lesions likely to progress from those likely to regress; however, there is still much to be learned. To examine the role of traditional epidemiologic factors in conjunction with molecular markers of human papillomavirus activity, we studied a group of women attending colposcopy clinics in Houston, TX, and Vancouver, BC, between October 2000 and July 2003. Methods Quantitative real-time PCR was used to measure mRNA expression of the human papillomavirus E7 gene, and quantitative cytology was used to gather information about the DNA index and chromatin features of the cells from these women. Logistic regression was used to establish predictor variables for histologic grade based on the epidemiologic risk factors and the molecular markers. Results The most predictive factors were mRNA level, DNA index, parity, and age. The ROC curve for the individual logits indicated excellent discrimination. Conclusion In accordance with other authors, these results suggest that molecular markers of the malignant process should be included in analyses looking to predict the progression potential of cervical lesions.