E6 E7 Research Articles

Transcriptome-wide profiling for melanocytes derived from newborn and adult human epidermis with enhanced proliferation.

The senescence-associated protein p16INK4A acts as a limiter element in cell-cycle progression. The loss of p16INK4A function is causally related to cellular immortalization. The increase in p16INK4A levels with advancing age was demonstrated in melanocytes. However, the characteristic difference between young and senescent melanocytes affecting immortalization of melanocytes remains unclear. In this study, we generated 10 different cell lines in total from newborn (NB) and adult (AD) primary normal human epidermal melanocytes (NHEM) using four different methods, transduction of CDK4R24C and cyclin D1 (K4D), K4D with TERT (K4DT), SV40 T-antigen (SV40T), and HPV16 E6 and E7 (E6/E7) and performed whole transcriptome sequencing analysis (RNA-Seq) to elucidate the differences of genome-wide expression profiles among cell lines. The analysis data revealed distinct differences in expression pattern between cell lines from NB and AD although no distinct biological differences were detected in analyses such as comparison of cell morphology, evaluation of cell proliferation, and cell cycle profiles. This study may provide useful in vitro models to benefit the understanding of skin-related diseases.

The polymorphism analysis and therapy vaccine target epitopes screening of HPV-35 E6 E7 among the threaten α-9 HPV in Sichuan area

High-risk human papilloma virus (HR-HPV) persistent infection is closely associated with the development of cervical cancer and squamous intraepithelial lesion (SIL).The α-9 HPVs, which is predominantly composed of HR-HPV types, account for 75% of HR-HPV infection in Sichuan. The oncoproteins E6 and E7 of HPV play a crucial role in tumor initiation and progression. Notably, HPV-35 is the only HR-HPV type within the α-9 genus that is not included in the nine-valent HPV prophylactic vaccine. Cervical cell samples obtained from Sichuan were collected for HPV detection and genotyping. Among the 406 HPV-positive samples, 31 HPV-35 were detected, 24 HPV-35 E6 and 26 E7 were successfully amplified and sequenced, five nucleotide mutations in E6 and three in E7 were detected, T232C, T434G of E6 (W78R, I145R) and C67T, G84T of E7 (H23Y, L28F) were non-synonymy mutation. PAML 4.8 server was used to detect positive selection sites of HPV-35 E6, E7, and E6 is W78R. Phyre2 were used to predict and analyze protein structures, W78R made influences on protein structure. IEDB were used to screen epitopes vaccine target for HPV-35 affection therapy, and 5 HPV-35 E6 and 3 HPV-35 E7 most potential epitopes were obtained, the most potential peptides for therapy vaccine design were 79-91YRYSVYGETLEKQ, 45-60FACYDLCIVREGQPY, 124-135RFHNIGGRWTGR of E6; 3-19GEITTLQDYVLDLEPEA, 38-47TIDGPAGQAK, 70-88VQSTHIDIRKLEDLLMGTF of E7 and W78R mainly decreased the epitopes affinity.Conclusions Amino acid substitution in the positive selection sites of HPV-35 E6 and E7 genes have been found to influence protein structure and to decrease the overall affinity of antigen epitopes. This observation aligns with the evolutionary significance of positive selection site, which may confer advantages to the virus by making infected cells more challenging for the immune system to detect, thereby enhancing HPV’s adaptability to the host environment. The polymorphism analysis of HPV-35 E6, E7 contributes to the enrichment of α-9 HPV data in Sichuan China, which is instrumental in improving the effectiveness of clinical detection. Furthermore, these findings provide a relevant theoretical foundation for the prevention and treatment of HPV-related diseases.

Abstract 5531: Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine model

Abstract Granulocyte macrophages colony stimulating factor (GM-CSF) also known as CSF2 is a popular cytokine responsible for homeostasis. According to TCGA databank CSF2 expression upregulated in human HPV+ (Human Papilloma Virus) Head and Neck Small Cell Carcinoma (HNSCC) compared to normal tissue. The mechanism of CSF2 in tumor progression and chemo resistance has not been well studied. Previously, our lab generated a murine model of HPV+ HNSCC that is partially responsive to cisplatin and radiation (like human disease). The role of our study is to evaluate the importance of Csf2 with cisplatin treatment (CT) in HPV + HNSCC in the murine model. Using CRISPR-Cas9 technology we knocked out the expression of the Csf2 ligand in our parental mEERL (murine E6 E7 Ras Luciferase) tumor cell line and implanted both the parental mEERL tumor and the Csf2 KO mEERL tumor in the oral cavity of C57BL6/J mice. When tumors reached a palpable size, we administered Cisplatin (20mg/m2, 3 doses total). We observed slow tumor growth in absence of Csf2 without treatment. On the other hand, we observed tumor regression in both tumor models with cisplatin treatment. Post treatment, mice with Csf2 KO tumors had a significantly prolonged rate of survival. To evaluate the role of Csf2 in the Tumor Microenvironment (TME), we collected the tumors 96 hours post CT and characterized the TME using multicolored flow cytometry. The results showed that post treatment mice with Csf2 KO tumors increased the number of the Natural Killer cell population and a surge of M-MDSC (Ly6Chi) population. The synergistic effect of knocking out Csf2 and cisplatin treatment in our tumor model appears to be driven by chemokine and cytokine changes in the TME. Simultaneously, we observed significantly lower levels of lung metastasis in Csf2 KO tumor bearing mice compared to those with parental mEERL tumors. Whereas we have seen increased metastasis in ipsilateral lymph node of Csf2 KO mice. This metastasis data mimics human disease where many patients have regional lymph node involvement, but few develop distant metastasis. These results indicate that GM-CSF (CSF2) in the TME could be an excellent target that would cause less metastasis and improved overall survival. Further investigations are needed to understand the underlying mechanism of immune response in the absence of CSF2 in the TME. Citation Format: Payal Ghosh, Craig Welbon, William C. Spanos. Loss of csf2(gm-csf) with cisplatin treatment decreases metastasis and improves survival in a hpv positive head and neck cancer murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5531.

Recent Updates on the Management of Human Papillomavirus-related Cancers

Abstract: Cervical cancer incidence and mortality rates have been steadily decreasing in developed nations owing to the excellent screening programs executed. However, it still remains one of the most prevalent tumors in developing nations, contributing significantly to cancer-related mortality in females. The major causal factor in the genesis of cervical cancer is recognized to be human papillomavirus (HPV) infection. The female population, particularly in poor countries, is highly susceptible to HPV infections and cervical cancer as a result of the increasing costs posed by widespread cervical screening and HPV vaccination methods. Understanding the roles of HPV oncoproteins (E6 and E7) and non-coding RNAs, along with their many cellular targets, can help us develop targeted drug therapy to manage cervical cancer. In the hunt for novel ways for effective disease control and prevention, new insights and methodologies in molecular biology keep evolving continuously. In the recent past, newer studies have revealed deeper knowledge of HPV-activated molecular signaling pathways as well as prospective targets for early diagnosis, prevention, and therapy of HPV-related malignancies. Also, there has been much new research conducted on genome-editing tools for HPVinduced cervical cancer treatment in conjunction with other treatment strategies, such as immunotherapy and therapeutic vaccines.

Multiple infections containing the top five prevalent HPV genotypes and their impact on cervical lesions in Changzhou, China

ABSTRACT Human papillomavirus (HPV) infection is the primary cause of cervical cancer and its precursor lesions. The overall prevalence of HPV genotypes in Changzhou has previously been reported. However, the distribution of multiple HPV infections and their roles in cervical injury have less been investigated. We aimed to assess the prevalence of multiple HPV infections among the people in Changzhou. Furthermore, we analyzed whether multiple HPV infections comprising the top five prevalent HPVs were more associated with abnormalities in E6 and E7 (E6/E7) mRNA, liquid-based cytology, and cervical histopathology than a single infection. In the current study, HPV 16, 52, 58, 53, and 81 were the top five prevalent HPV types, both in single and multiple infections. Compared to a single infection, multiple infections containing HPV 16/52/58 were closely linked to positivity for E6/E7 mRNA. In addition to HPV 16, multiple infections containing the remaining top four HPVs conferred a significant advantage on atypical squamous cells of undermined significance or worse in comparison to a single infection. Furthermore, women with multiple infections containing the top five prevalent HPV types were more likely to develop cervical intraepithelial neoplasia grade II or worse than those with a single HPV infection. Our results demonstrate the superiority of multiple HPV infections containing the top five prevalent HPV types in cervical disease progression, which should be closely monitored. These findings are conducive for formulating regional preventive strategies for cervical cancer screening and vaccination in Changzhou.

Detection and Partial Molecular Characterization (E6–E7 Region-Early Genes) and Prevalence of Human Papillomavirus (HPV) Causing Cervical Cancer in and Around Tirupati Region, Andhra Pradesh

Detection and Partial Molecular Characterization (E6–E7 Region-Early Genes) and Prevalence of Human Papillomavirus (HPV) Causing Cervical Cancer in and Around Tirupati Region, Andhra Pradesh

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

AGO-accessible anticancer siRNAs designed with synergistic miRNA-like activity

Small interfering RNAs (siRNAs) therapeutically induce RNA interference (RNAi) of disease-causing genes, but they also silence hundreds of seed-matched off-targets as behaving similar to microRNAs (miRNAs). miRNAs control the pathophysiology of tumors, wherein their accessible binding sites can be sequenced by Argonaute crosslinking immunoprecipitation (AGO CLIP). Herein, based on AGO CLIP, we develop potent anticancer siRNAs utilizing miRNA-like activity (mi/siRNAs). The mi/siRNAs contain seed sequences (positions 2–7) of tumor-suppressive miRNAs while maintaining perfect sequence complementarity to the AGO-accessible tumor target sites. Initially, host miRNA interactions with human papillomavirus 18 (HPV18) were identified in cervical cancer by AGO CLIP, revealing tumor-suppressive activity of miR-1/206 and miR-218. Based on the AGO-miRNA binding sites, mi/siRNAs were designed to target E6 and E7 (E6/E7) transcript with seed sequences of miR-1/206 (206/E7) and miR-218 (218/E7). Synergistic anticancer activity of 206/E7 and 218/E7 was functionally validated and confirmed via RNA sequencing and in vivo xenograft models (206/E7). Other mi/siRNA sequences were additionally designed for cervical, ovarian, and breast cancer, and available as an online tool (http://ago.korea.ac.kr/misiRNA); some of the mi/siRNAs were validated for their augmented anticancer activity (206/EphA2 and 206/Her2). mi/siRNAs could coordinate miRNA-like activity with robust siRNA function, demonstrating the potential of AGO CLIP analysis for RNAi therapeutics.

Analysis of Codon Usage Patterns in the Human Papillomavirus Oncogenes

Background: Persistent high-risk genital human papillomavirus (HPV) infection is a major cause of cervical cancer in women. The products of the viral transforming genes E6 and E7 in the high-risk HPVs are known to be similar in their amino acid composition and structure. We performed a comparative analysis of codon usage patterns in the E6 and E7 genes of HPVs. Methods: The E6 and E7 gene sequences of eight HPV subtypes were analyzed to determine their nucleotide composition, relative synonymous codon usage (RSCU), effective number of codons (ENC), neutrality, genetic variability, selection pressure, and codon adaptation index (CAI). Additionally, a correspondence analysis (CoA) was performed. Results: The analysis to determine the effects of differences in composition on the codon usage patterns revealed that there may be usage bias for ‘A’ nucleotides. This was consistent with the results of the RSCU analysis, which demonstrated that the selection of A/T-rich patterns and the preference for A/T-ended codons in HPVs are influenced by compositional constraints. Moreover, the results reveal that selection pressure plays an important role in the CoA results for the RSCU values, Tajima’s D tests, and neutrality tests. Conclusion: The results of this study are consistent with previous findings that most papillomavirus genes are under purifying selection pressure, which limits changes to the encoded proteins. Natural selection and mutation pressures resulting in changes in the nucleotide composition and codon usage bias in the two tumor genes of HPV act differently during the evolution of the HPV subtype; thus, throughout the viral life cycle, HPV can constantly evolve to adapt to a new environment.

HPV E2, E4, E5 drive alternative carcinogenic pathways in HPV positive cancers

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.

P16/ki67 and E6/E7 mRNA Accuracy and Prognostic Value in Triaging HPV DNA-Positive Women.

The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis. Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided. 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both). p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

Alternative mRNA splicing can attenuate the pathogenicity of presumed loss-of-function variants in BRCA2

PurposeCurrent interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants in the canonical splice site sequences ofBRCA2, to be associated with high cancer risk. However, some variant alleles produce alternative transcripts that encode (partially) functional protein isoforms leading to possible incorrect risk estimations. For accurate classification of variants it is therefore essential that alternative transcripts are identified and functionally characterized. MethodsWe systematically evaluated a large panel of human BRCA2 variants for the production of alternative transcripts and assessed their capacity to exert BRCA2 protein functionality. Evaluated variants included all single-exon deletions, various multiple-exon deletions, intronic variants at the canonical splice donor and acceptor sequences, and variants that previously have been shown to affect messenger RNA (mRNA) splicing in carriers. ResultsMultiple alternative transcripts encoding (partially) functional protein isoforms were identified (e.g., ∆[E4–E7], ∆[E6–E7], ∆E[6q39_E8], ∆[E10], ∆[E12], ∆E[12–14]). Expression of these transcripts did attenuate the impact of predicted LoF variants such as the canonical splice site variants c.631+2T>G, c.517-2A>G, c.6842-2A>G, c.6937+1G>A, and nonsense variants c.491T>A, c.581G>A, and c.6901G>T. ConclusionThese results allow refinement of variant interpretation guidelines for BRCA2 by providing insight into the functional consequences of naturally occurring and variant-related alternative splicing events.

Safety and immunogenicity of VGX-3100 formulations in a healthy young adult population

ABSTRACT HPV remains the most common sexually transmitted disease worldwide, despite improvements in awareness, screening, prophylactic vaccination uptake, and surgical treatment. VGX-3100 is an immunotherapy that uses electroporation to introduce DNA encoding for modified HPV-16 and HPV-18, E6-and E7 proteins into myocytes to stimulate an effector T cell response. We now report immunogenicity and safety of VGX-3100 for a refrigeration-stable formulation, which improves patient-care setting usability. This multi-arm, double-blinded, randomized trial enrolled 235 healthy men and women to receive either a refrigerated (RF) or frozen formulation (FF) of VGX-3100. Three doses were administered intramuscularly with electroporation at 0, 4, and 12 weeks. Non-inferiority of RF to FF was assessed by comparing the proportion of subjects who achieved a ≥2-fold increase from baseline to Week 14 in Spot Forming Units/106 PMBCs using an interferon-γ enzyme-linked immunospot assay. There were no related SAEs. Injection site reactions were the most common adverse event (54%, RF; 66%, FF) the majority of which resolved within a few minutes following administration. The primary endpoint was met with 89.9% of RF recipients and 97.2% of FF recipients reaching a ≥2-fold rise in SFU/106 PBMC, 2 weeks following the last dose; RF was statistically non-inferior to FF (p = .022). A systemic, immunologic approach has the potential to fill a critical gap in the ability to treat men and women with high grade HPV diseases. These safety and immunogenicity data are supportive of the continued development of a refrigerated formulation of VGX-3100.

Summaries of safety labeling changes approved by FDA-Boxed warnings highlights, October-December 2018.

Journal Article Summaries of safety labeling changes approved by FDA—Boxed warnings highlights, October–December 2018 Get access American Journal of Health-System Pharmacy, Volume 76, Issue 7, 1 April 2019, Pages e6–e7, https://doi.org/10.1093/ajhp/zxz015 Published: 19 March 2019

Human Papillomavirus Genotypes and HPV16 E6/E7 Variants among Patients with Genital Cancers in Vietnam.

We previously reported human papillomavirus type 52 (HPV52) as the most prevalent high-risk genotype in non-cancer individuals in Vietnam. This study aimed to evaluate HPV genotypes and HPV16 E6 and E7 (E6/E7) gene variations in Vietnamese patients with genital cancers. Biopsy samples were collected from 124 Vietnamese patients with genital cancers (20 with vaginal, 50 with vulvar, and 54 with penile cancer). The HPV-DNA was amplified and genotyped, and HPV16 E6/E7 genes were compared with those previously reported for women with normal cervical cytology (N = 23). HPV-DNA was detected in 80.6% (100/124) of the cancer patients (80.0% of vaginal, 82.0% of vulvar, and 79.6% of penile), with HPV16/18 in 86.0% (86/100) and HPV52 in 7.0% (7/100) of the HPV-positive samples. The HPV-DNA prevalence and HPV genotype distribution did not significantly differ among the genital cancer patients (both P = 0.95). Significantly fewer instances of the HPV16 A4 sublineage (34.8% vs. 82.6%, P < 0.0001) and HPV16 E7 29S (36.4% vs. 87.0%, P = 0.0002) occurred in the cancer patients than in the women with normal cytology. Our results indicate that HPV16/18 accounts for more than 85% of genital cancers in Vietnam, and the HPV16 sublineage A4 containing E7 29S may be less oncogenic.

Foraminiferal palaeobathymetry and depositional sequences of the subsurface Eocene Apollonia Formation in North Western Desert, Egypt

Sea-level changes, palaeobathymetry, palaeo-productivity and depositional sequences (sequence stratigraphy) are evaluated using benthic foraminifers from two subsurface boreholes in the North Western Desert (Egypt) belonging to the Eocene Apollonia Formation. Five planktic foraminiferal biozones (E4–E5, E8–E10) have been identified. The absence of E6–E7 and upper Eocene zones are attributed to long-time unconformities (regional tectonics) during the Cretaceous/Palaeogene (K/Pg) and Eocene/Oligocene (E/O) boundaries with estimated time gaps of 16 and 13.9 Ma, respectively. Six benthic foraminiferal assemblages, spanning middle to outer neritic depths, are noted. They are represented by Bolivinoides decoratus, Eponides mariei, Bulimina quadrata, Anomalinoides zitteli, Uvigerina eocaena and Uvigerina pygmoides. Three depositional sequences based upon four sequence boundaries are also identified. Each depositional sequence can be classified into transgressive and highstand system tracts inferred from benthic foraminiferal palaeobathymetry, which are separated by maximum flooding surfaces (MFS), characterised by maximum abundance of buliminids and uvigerinids, and peak values of total foraminiferal numbers, planktic (P)% and diversity. Comparisons of our recorded sequences and their boundaries inside and outside Egypt reflect that the studied succession is affected by both tectonic activity that caused long time gaps and eustatic processes.

17β-estradiol and progesterone effect on human papillomavirus 16 positive cells grown as spheroid co-cultures.

Human papillomavirus (HPV) is the key epidemiologic factor of cervical cancer, but additional cofactors are mandatory. Estrogen has been considered as one of those. Here, the aim was to study the effects of steroid hormones on HPV16 E6-E7, estradiol receptors ERα and ERβ, and progesterone receptor (PR) in HPV16-positive cervical carcinoma cell lines SiHa and CaSki grown as epithelial and fibroblast spheroid co-cultures. The spheroid co-cultures were exposured to 17β-estradiol or progesterone from day 7 onwards. mRNA levels of HPV16 E6-E7, ERα, ERβ and PR normalized against GAPDH were analyzed with quantitative reverse transcription-qPCR (RT-qPCR). 17β-estradiol and progesterone decreased HPV16 E6-E7 mRNA expression in CaSki and increased in SiHA co-cultures. In CaSki co-cultures, ERβ expression was blocked after 17β-estradiol exposure while in SiHa cells it slightly increased ERβ expression. PR expression was seen only in CaSki spheroids and it vanished after exposure to steroid hormones. Fibroblasts expressed all three hormone receptors as monolayers but ERβ expression decreased and ERα and PR vanished after co-culturing. Cell culturing platform changes both oncogene and hormone receptor expression in HPV16 positive cervical cancer cell lines. This needs to be considered when in vitro results are extrapolated to in vivo situations.

The epithelial-mesenchymal transition (EMT) is regulated by oncoviruses in cancer.

The epithelial-mesenchymal transition (EMT), defined as transdifferentiation of epithelial cells into mesenchymal cells, is critical for embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. Recently, the role of EMT in carcinogenesis has attracted much attention. Oncoviruses, including human papillomaviruses (HPVs), Epstein-Barr virus (EBV), and hepatitis B and C viruses (HBVs, HCVs), are known to be involved in the etiology of cancer and have been found to play important roles in cancer metastasis, especially in the EMT process. The HPV encoded oncoproteins E6 and E7 (E6/E7), EBV latent membrane protein-1 and -2A, EBV nuclear antigen, HBV-encoded X antigen, and nonstructural HCV protein 5A are all involved in the regulation of EMT. This review primarily focuses on the role of oncoviruses and their encoded proteins or signaling pathways in the EMT process. Understanding their roles will help us in the development of effective strategies for prevention and treatment of virus-related cancers.-Chen, X., Bode, A. M., Dong, Z., Cao, Y. The epithelial-mesenchymal transition (EMT) is regulated by oncoviruses in cancer.

Molecular Detection of Oncogenic Human Papillomavirus (HPV) Genotypes in Vulva Cancers, Penile Cancers and Anal Cancers in Myanmar

Abstract 12 A causal role for human papillomavirus (HPV) associated cancers of vulva, penile, and anus is supported by evidence from molecular and epidemiologic investigations. This study detected the oncogenic HPV genotypes in vulva cancers, penile cancers and anal cancers by a cross-sectional descriptive study in 2013. A total of 100 paraffin embedded biopsy tissues of histologically confirmed vulva cancers, penile cancers and anal cancers within past five years during 2008 and 2012 were studied. Those cases were 61 vulva cancers from Central Women Hospital, Yangon and 30 penile cancers and 9 anal cancers from Yangon General Hospital. HPV-DNA testing and genotyping were performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Consensus sequence primer pairs within the E6 andE7 open reading were used to amplify oncogenic HPV genotypes (HPV-16,-18,-31,-33,-35,-52b,-58). Restriction enzymes were used for determination of specific HPV genotypes. HPV was identified in 36.1% of vulva cancers (22/61), 26.7% of penile cancers (8/30) and 44.4% of anal cancers (4/9). In vulva cancers, HPV-33 was the most common genotype (40.9%) followed by HPV-16 (31.8%), HPV-31 (22.7%), and HPV-18 (4.6%). In penile cancers, HPV-16 (62.5%) was the most common genotype followed by HPV-33 (25%) and HPV-18 (12.5%). Among anal cancers, the most frequent genotypes were HPV-16 (75%) and HPV-18 (25%). This study is the first report of evidence based oncogenic HPV genotypes in vulva cancers, penile cancers and anal cancers in Myanmar. This research provides the valuable information in understanding the burden of HPV associated cancers of the vulva, penile, and anus in Myanmar and the consideration of the effectiveness of prophylactic HPV vaccination in not only cervical cancer but also non-cervical cancers. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST: No COIs from the authors.

Genetic variability in E6, E7, and L1 genes of human papillomavirus genotype 52 from Southwest China

Human papillomavirus (HPV) is the major causative agent of cervical cancer, which accounts for the second highest cancer burden in women worldwide. HPV-52, the prevalent subtype in Asia, especially in southwest China, was analyzed in this study. To analyze polymorphisms, intratypic variants, and genetic variability in the E6–E7 (n=26) and L1 (n=53) genes of HPV-52, these genes were sequenced and the sequences were submitted to GenBank. Phylogenetic trees were constructed using the neighbor-joining and Kimura 2-parameters methods, followed by analysis of the diversity of secondary structure. Finally, we estimated the selection pressures acting on the E6–E7 and L1 genes. Fifty-one novel variants of HPV-52 L1, and two novel variants of HPV-52 E6–E7 were identified in this study. Thirty single nucleotide changes were observed in HPV-52 E6–E7 sequences with 19/30 non-synonymous mutations and 11/30 synonymous mutations (five in the alpha helix and five in the beta sheet). Fifty-five single nucleotide changes were observed in HPV-52 L1 sequences with 17/55 non-synonymous mutations (seven in the alpha helix and fourteen in the beta sheet) and 38/55 synonymous mutations. Selective pressure analysis predicted that most of these mutations reflect positive selection. Identifying new variants in HPV-52 may inform the rational design of new vaccines specifically for women in southwest China. Knowledge of genetic variation in HPV may be useful as an epidemiologic correlate of cervical cancer risk, or may even provide critical information for developing diagnostic probes.