HPV Sequences Research Articles

A Case of Condyloma Acuminatum on the Nipple Detected via Dermoscopy

Introduction Condyloma acuminatum (CA), also known as genital warts, is an anogenital warty growth caused by some types of the human papilloma virus (HPV). CA is usually found within the anal or genital area, but it occasionally presents in the oral, respiratory, conjunctival, nasal, or nipple region. We herein report a rare case of a 29-year-old Chinese woman with a tiny CA on her nipple, which was diagnosed through dermoscopy. With noninvasion and convenience, dermoscopy is valuable for the early diagnosis of tiny CA that is unable to be discerned by the naked eye. Case report A 29-year-old Chinese woman presented with a one-week history of a pink papule affecting her right nipple. The lesion was asymptomatic. Just two months ago, she had been diagnosed with CA caused by HPV-6 and HPV-18 and treated with carbon dioxide (CO2) laser in Chengdu Second People's Hospital. Physical examination revealed a pink papule with a size of millet at the center of her right nipple (Fig. 1A). Dermoscopy showed fingerlike patterns with hairpin and dotted vessels within the papillae (Fig. 1B). The acetowhitening test was positive, and real-time polymerase chain reaction (PCR) amplification positively detected HPV-18 sequences. In addition, a test for antihuman immunodeficiency virus antibody (HIV-Ab), the Treponema pallidum particle agglutination assay, and the tolulized red unheated serum test all produced negative results.Figure 1: Clinical and dermoscopic features of the lesion. (A) A pink papule with a size of millet at the center of the right nipple. (B) Dermoscopy shows fingerlike patterns with hairpin or dotted vessels within the papillae (×50).Based on these findings, we diagnosed this patient with CA on the nipple and started treatment with liquid nitrogen cryotherapy and topical 5% imiquimod cream. There has been no recurrence in the ensuing three months. Discussion CA is a sexually transmitted infection caused by certain types of HPV, most commonly HPV-6 and HPV-11. Suzanne et al.1 used PCR to assess the lesions of 472 patients with CA and detected a total of 14 HPV genotypes (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), of which HPV-6 and HPV-11 were the dominant types (94.7%) followed by HPV-16 and HPV-18. HPV-16 and HPV-18 accounted for 50% of the 22 CA patients, who were positive for a high-risk HPV genotype. Although CA usually occurs in the anal or genital area, it occasionally presents in the oral, respiratory, conjunctival, nasal, or nipple region. The anogenital HPV infection is almost acquired through sexual contact. The lesions are characterized by localized single or multiple papules without any symptoms in the early stage, which gradually develop into papillary, cristate, cauliflower-like, or crumby neoplasms. To date, a few cases of CA on the nipple have been reported. Wood2 reported the first case of CA on the nipple in 1978. Later, Kulke et al.3 cloned HPV-6 subtype from a CA located on the nipple, and Kowalzick et al.4 described a case of CA on the nipple associated with HPV-41. In 2014, Saeki et al.5 reported a case of CA on the nipple and areola that resembled seborrheic keratosis. This patient visited our clinic again for a new tiny papule on her nipple. If she had not been previously diagnosed with genital CA, we might have easily ignored such a tiny lesion. Then, dermoscopy showed typical wart manifestations, and HPV-18 sequences were positively detected by real-time PCR. However, there have been no reports of the correlation between HPV-18 and verruca vulgaris. In view of her history of CA caused by both HPV-6 and HPV-18, our patient was diagnosed with CA on the nipple. In most cases, clinicians familiar with the clinical manifestations of CA can easily make a diagnosis only based upon a physical examination. However, examination with a dermoscopy is helpful for diagnosing CA when the lesions are particularly small or atypical.6-7 The specific dermoscopic findings of CA include morphologic patterns (fingerlike, knoblike, mosaic-like) and multiple vascular features (glomerular/dotted, hairpin).6 As a noninvasive and convenient examination method, dermoscopy is valuable for the early diagnosis of tiny CA that is unable to be discerned by the naked eye.

High prevalence of human papillomavirus and European variants of HPV 16 infecting concomitantly to cervix and oral cavity in HIV positive women.

Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide, with a high prevalence and high transmissibility. High-risk HPV (hrHPV) infection is the primary cause of cervical cancer. The HPV variants present in the uterine cervix and oral cavity of HIV+ women have not been described.ObjectiveIdentify the prevalence of HPV infections in the uterine cervix and oral cavity and HPV16 variants in HIV+ women.MethodsA total of 174 HIV+ women attended an HIV+ specialized clinic in Mexico City. Cells were obtained from the oral cavity and cervix to extract DNA. Polymerase chain reaction (PCR) was used to amplify the HPV sequence with generic primers. We detected specific HPV types using the INNO-LiPA HPV Genotyping Extra II Kit (INNOGENETICS). The identification of variants was studied by sequencing the E6 gene with a Big Dye Terminator Kit and an Applied Biosystems 3500/3500xL genetic analyzer.ResultsHPV infection was very high in the uterine cervix (168/174, 96.6%) and oral cavity (161/174, 92.5%). The prevalence of HPV concurrent infections in the cervix and oral cavity was 155/174 (89.1%). We found hrHPVs to be more prevalent than low-risk HPVs (lrHPVs) in the oral cavity (90.2% versus 45.4%) and that infections simultaneously affected the cervix (94.3% versus 36.2%) and oral cavity (85.1% versus 20.1%). Surprisingly, only European variants of HPV type 16 were found in the uterine cervix of women and the oral cavity of all tested samples (52 oral cavity samples and 52 uterine cervix samples).ConclusionsThe high prevalence of HPV, multiple infections and presence of the EP350G intravariant in both anatomical regions are strongly related to the persistence of the virus, which is fundamental for the development of cancer. Therefore, it is very important to control and monitor this high-risk population as well as implement programs for the early detection of HPV and vaccination.

The HPV16 Genome Is Stable in Women Who Progress to In Situ or Invasive Cervical Cancer: A Prospective Population-Based Study.

The human papillomavirus (HPV) rate of evolution is essential for cancer-preventive strategies targeting HPV. We analyzed variability over time in a prospective, population-based nested case-control study of in situ (CIS) and invasive squamous cervical cancer (SCC). Among 757,690 women who participated in cervical screening in Sweden during 1969 to 2002, we identified 94 women who had HPV16 persistence in two serial cervical screening samples (median 24 months apart, range 0.5-178 months) and later were diagnosed with CIS (n = 59), SCC (n = 32), or remained healthy (n = 3). Whole-HPV16-genome sequencing and comparison of sequences in the serial samples revealed that all women had the same HPV16 lineage, particularly lineage A, in both serial smears. Fifty-six percent of women had an identical 7,906 base pair HPV16 sequence in both samples, and no woman had more than 15 nucleotide substitutions. The median substitution rate was 0 substitutions/site/year (95% confidence interval, 0-0.00008), with no variation between quartiles of follow-up. We concluded that in most women with HPV16 persistence preceding disease, the nucleotide substitution rate was not measurable within up to 15-years follow-up. This slow rate of evolution has important implications for both HPV-based screening and HPV vaccination. SIGNIFICANCE: These findings show there is no genomic variation over time in HPV16 infections progressing to cervical cancer, which could influence risk stratification of women when screening for cervical cancer and inform HPV vaccination strategies.

A novel sequencing-based vaginal health assay combining self-sampling, HPV detection and genotyping, STI detection, and vaginal microbiome analysis.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman’s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

Phylogeny and Classification of Human Papillomavirus (HPV)16 and HPV18 Variants Based on E6 and L1 genes in Tunisian Women with Cervical Lesions

Background:High-risk human papillomavirus (HPV) types are the main etiological factors for cervical cancer. HPV16 and HPV18 are generally the most common forms associated with development of high-grade cervical lesions. This study was undertaken to identify intratypic variants of HPV16 and HPV18 among women with cervical lesions in Tunisia.Materials and Methods:DNA was extracted from cervical samples collected from 49 women. using a PureLinkTM Genomic DNA mini Kit (Invitrogen). E6 and L1 open reading frames (ORF) were amplified by PCR and viral DNA amplicons were subjected to automated sequencing using Big Dye Terminators technology (Applied Biosystems). The obtained sequences were analyzed using an appropriate software program to allow phylogenetic trees to be generated.Results:HPV16 and HPV18 were detected in 15 and 5 cases, respectively. HPV16 E6 sequences clustered with the European German lineage (A2) whereas one isolate diverged differently in the L1 region and clustered with the African sub-lineage (B1). HPV 18 E6 sequences clustered with the European sub-lineage (A1) but L1 sequences clustered as a new clade which diverged from A1-A5.Conclusions:Our results suggest that the distribution of HPV16 and HPV18 sequences in women with cervical lesions in Tunisia is mainly related to European epidemiological conditions and point to the presence of recombinant HPV forms.

Inkjet Printing Based Droplet Generation for Integrated Online Digital Polymerase Chain Reaction.

We report on the development of a novel and flexible online digital polymerase chain reaction (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling, and a laser-induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning 4 orders of magnitude.

Integration of human papillomavirus 16 in esophageal carcinoma samples

BackgroundEsophageal carcinoma (EC) is one of the major cancers in China. In 1982, Syrjanen first hypothesized the relationship between human papillomavirus (HPV) infection and the development of esophageal cancer. Since then, many reports in the field have supported this viewpoint. This study investigated the etiological relationship between HPV infection and the occurrence of esophageal carcinoma at Tangshan City of the Hebei province in China.Methods189 samples of esophageal carcinoma patients were collected. DNA and RNA were isolated from samples, HPV DNA was detected by polymerase chain reaction (PCR) using My09/11 for HPV L1, and HPV16 was determined using type-specific primer sets for HPV16 E6. The HPV16 integration site was verified by amplification of papillomavirus oncogene transcripts, and HPV16 oncogene transcript products were ligated to the pMD-18 T vector and sequenced to confirm the physical location of HPV16 integration.Results168 HPV-positive samples were detected in 189 samples, and among them 76 specimens were HPV16 positive. Approximately 600 bp of the HPV16 oncogene transcript were detected in nine esophageal cancer samples. Sequence analysis revealed that HPV16 E7 integrated into human chromosome 2 in three samples, into human chromosome 5 in one sample, into human chromosome 6 in one sample, into human chromosome 8 in two samples, and into human chromosome 17 in two samples. The results verified that the integrated HPV16 E7 in five samples harbored one mutation of viral DNA compared with the HPV16 sequence provided in GenBank (K02718).ConclusionsThe high prevalence of HPV16 suggests that HPV16 may play an etiological role in the development of esophageal cancer. The integration of HPV16 into host cell chromosomes suggests that persistent HPV infection is key for esophageal epithelial cell malignant transformation and carcinogenesis.

Prevalence of fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell carcinomas of the head and neck

FGFR1 is a receptor tyrosine kinases involved in tumor growth signaling, survival, and differentiation in many solid cancer types. There is growing evidence that FGFR1 amplification might predict therapy response to FGFR1 inhibitors in squamous cell lung cancers. To estimate the potential applicability of anti FGFR1 therapies in squamous cell carcinomas of the head and neck, we studied patterns of FGFR1 amplification using fluorescence in situ hybridization (FISH). A tissue microarray was constructed from 453 primary treatment-naive squamous cell carcinomas of the head and neck regions with histopathological and clinical follow-up data [including oral cavity (n=222), oropharynx (n=126), and larynx (n=105)]. FGFR1 and centromere 8 copy numbers were assessed by dual-color FISH. FGFR1 amplification was defined as a copy number ratio FGFR1: centromere 8≥2.0. HPV sequencing and p16 immunohistochemistry (IHC) were applied to FGFR1-amplified cancers. FISH analysis was successful in 297 (66%) of the 453 cancers. FGFR1 amplification was found in 6% of analyzable tumors, and was more frequent in tumors of the oral cavity (13/133 amplified, 10%), than cancers of other localizations (1/79 oropharynx, 4/85 larynx; p=0.007 and 0.159, respectively). One out of 18 FGFR1 amplified cancers was HPV positive. No associations were found between FGFR1 amplification and tumor phenotype or p16 IHC. Head and neck cancers are recurrently affected by FGFR1 amplification, with a predominance in cancers of the oral cavity. Finding only one HPV positive and FGFR1 amplified cancer argues against a causal relationship between HPV and FGFR1 amplifications.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

ABSTRACTHuman papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially (P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related.IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency.

Genotyping of the human papilloma virus in a group of Mexican women treated in a highly specialist hospital: Multiple infections and their potential transcendence in the current vaccination programme

Background and objetiveHuman papilloma virus (HPV) is one of the main risk factors associated with the development of cervical cancer and its precursor lesions. It has been reported that HPV16 and 18 types cover approximately 70% of cervical cancer worldwide; however, significant variation in percentages of HPV infections could be related to specific populations. Materials and methodsPurified DNA of 67 cervical samples were analyzed by Linear Array® HPV genotyping kit. These analyzed samples correspond to 19 cervical tumours, 15 high-grade squamous intraepithelial lesions, 20 low-grade squamous intraepithelial lesions, and 13 cervical samples without injury were studied, all of them previously diagnosed. ResultsIn general, 16 different HPV types were found with differences in their frequencies, cervical invasive cancer being the richest in HPV sequences, followed by the low-grade squamous intraepithelial lesions and then high-grade lesions. HPV16 was the most frequently distributed type in neoplastic lesions of the cervix, followed by the HPV52, suggesting viral type variability, probably associated to the geographical region studied. ConclusionsThe results could indicate variability in HPV presence in Mexico, underlining the important role for HPV52 among others in the Mexican population. This would also potentially have an impact on the current anti-HPV vaccination schemes.

Genotipificación del virus del papiloma humano en un grupo de mujeres mexicanas atendidas en un hospital de alta especialidad: las infecciones múltiples y su potencial trascendencia en el esquema actual de vacunación

Background and objetiveHuman papilloma virus (HPV) is one of the main risk factors associated with the development of cervical cancer and its precursor lesions. It has been reported that HPV16 and 18 types cover approximately 70% of cervical cancer worldwide; however, significant variation in percentages of HPV infections could be related to specific populations. Materials and methodsPurified DNA of 67 cervical samples were analyzed by Linear Array® HPV genotyping kit. These analyzed samples correspond to 19 cervical tumors, 15 high-grade squamous intraepithelial lesions, 20 low-grade squamous intraepithelial lesions, and 13 cervical samples without injury were studied, all of them previously diagnosed. ResultsIn general, 16 different HPV types were found with differences in their frequencies, cervical invasive cancer being the richest in HPV sequences, followed by the low-grade squamous intraepithelial lesions and then high-grade lesions. HPV16 was the most frequently distributed type in neoplastic lesions of the cervix, followed by the HPV52, suggesting viral type variability, probably associated to the geographical region studied. ConclusionsThe results could indicate variability in HPV presence in Mexico, underlining the important role for HPV52 among others in the Mexican population. This would also potentially have an impact on the current anti-HPV vaccination schemes.

HPV16 E6 mutations and p53 codon72 polymorphism among women with cervical intraepithelial neoplasia 2 and 3 in China.

To study the distribution of HPV16 E6 gene mutations and p53 codon72 polymorphism among women with HPV16+ cervical precancerous lesions and explore their relationship with the risk of cervical intraepithelial neoplasia (CIN) 2, 3. Materials and Meth- ods: This study analyzed a total of 112 cases of exfoliated HPV16+ cervical cell specimens which were divided into group I (normal and CIN1, 55 cases) and group2 (CIN2, 3, 57 cases). Among the 112 specimens, 85 cases were successfully amplified for HPV E6 gene by PCR and the PCR products were sequenced directly. P53 codon72 region was also amplified from the 112 specimens and the PCR products were sequenced directly and compared with the standard sequence. Among the 85 amplified HPV sequences, point mutations such as T178G, T350G, G132A, A442C, T310G, G94T, C551A, etc. were found, among which, T178G showed the highest rate (51.76%). The rate of HPV16 E6 mutation T178G in CIN2, 3 group was significantly higher than that in normal and CINI group, i.e., in the 112 amplified p53 codon72 sequences, the distribution of Pro/Pro genotype in normal, and CIN1 group was significantly different from that in CIN2, 3 groups, and the disease risk of Pro/Pro genotype was much higher than that of Arg/Arg and Arg/Pro genotypes. HPV16 E6 T178G mutation increases the disease risk of CIN2, 3. Meanwhile, compared with Arg/Arg and Arg/Pro genotypes, p53 codon72 Pro/Pro genotype more associated with the disease risk of CIN2, 3.

Genomic diversity and phylogenetic relationships of human papillomavirus 16 (HPV16) in Nepal

Objectives/BackgroundSequence variants in HPV16 confer differences in oncogenic potential; however, to date there have not been any HPV sequence studies performed in Nepal. The objective of this study was to characterize HPV16 viral genome sequences from Nepal compared to a reference sequence in order to determine their lineages. Additionally, we sought to determine if five High-grade Squamous Intraepithelial Lesion (HSIL) subjects were genetically distinct from the non-HSIL subjects. MethodsDNA was isolated from exfoliated cervical cells from 17 individuals in Nepal who were previously identified to be HPV16-positive. A custom HPV16 Ion Ampliseq panel of multiplexed degenerate primers was designed that generated 47 overlapping amplicons and covered 99% of the viral genome for all known HPV16 variant lineages. All sequence data were processed through a custom quality control and analysis pipeline of sequence comparisons and phylogenetic analysis. ResultsThere were high similarities across the genomes, with two major indels observed in the non-coding region between E5 and L2. Compared to the PAVE reference HPV16 genome, there were up to 9, 4, 38, 27, 8, 7, 52, and 32 nucleotide variants in the E6, E7, E1, E2, E4, E5, L2, and L1 genes in the Nepalese samples, respectively. Based on sequence variation, HPV16 from Nepal falls across the A, C, and D lineages in this study. We found no evidence of genetic distinctness between HSIL and non-HSIL subjects. ConclusionsThe evolutionary and pathological characteristics of the representative HPV16 genomes from Nepal seem similar to results from other parts of the world and provide the basis for further studies.

Molecular analysis of human Papillomavirus detected among women positive for cervical lesions by visual inspection with acetic acid/Lugol's iodine (VIA/VILI) in Libreville, Gabon.

BackgroundThe human papillomavirus (HPV) is the causative agent of cervical cancer, which is the leading cancer-related cause of death for women in Sub-Saharan Africa. In 2013, the Gabonese Ministry of Health and the Sylvia Bongo Ondimba Foundation implemented cervical cancer screening programs based on the detection of cancerous lesions by visual inspection with acetic acid and/or Lugol’s iodine (VIA/VILI). This pilot study was set up to determine the HPV profile and analyze the nucleotide sequence variation of HPV16 circulating in patients with cervical abnormalities detected by VIA/VILI testing.MethodsThe cervical abnormalities observed upon VIA/VILI were confirmed by liquid-based cytology for all tested women. Nested PCR using the MY09/11 and GP5+/6+ primer sets was used to detect HPVs present in the extracted DNA. HPV genotypes were determined after sequencing of amplicons based on a high-throughput sequencing approach. For isolates of the HPV16 genotype, the E6 gene and the long control region (LCR) were directly sequenced using Sanger method.ResultsThe study included 87 women who showed a positive VIA/VILI result. Cervical abnormalities were found in 40.23 % of women and 40 % were classified as high-grade lesions. The HPV detection rate was 82.9 % among women with abnormal cytology. Among all the identified high-risk HPV genotypes, HPV16, 18 and 33 were the most frequent. Multiple HPV infections were observed in 42.31 % of HPV-infected women. Analysis of the HPV16 sequence variation in the E6 gene and in the LCR showed that 85.3 and 14.7 % belonged to the African and European lineages, respectively. Among the African branch variants, Af2 was the most frequently identified in this study.ConclusionThis study offers the first report of the HPV detection rate and molecular epidemiology among Gabonese women with a positive result in a VIA/VILI screening test. Moreover, data on the HPV16 sequence variation confirm the predominance of African variants in high-grade lesions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13027-016-0098-1) contains supplementary material, which is available to authorized users.

The expression of miR-21 and miR-143 is deregulated by the HPV16 E7 oncoprotein and 17β-estradiol.

MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate their target mRNAs at a posttranscriptional level, thereby affecting crucial processes in cancer development. However, little is known about the molecular events that control expression of miRNAs in cervical cancer (CC). HPV16 E7 oncoprotein in conjunction with estrogen are sufficient to produce high grade cervical dysplasia and invasive cervical malignancies in a mouse model. In the present study, we determined the potential role that the E7 oncoprotein and 17β-estradiol (E2) play in the deregulation of miR-21 and miR-143 expression levels by these two risk factors. We found that, while the expression of miR-21 was upregulated and the expression of miR-143 was downregulated by the HPV16 E7 oncoprotein invivo, and invitro and that E2 treatment is also implicated in the deregulation of these important miRNAs invivo. Sustained upregulation of miR-21 resulted in suppression of PTEN expression, and repression of miR-143 increased the mRNA and protein levels from Bcl-2. These results suggested that HPV type 16 E7 oncoprotein and E2 play an important role in regulating miR-21 and miR-143 expression. We have observed similar results in CC patients containing HPV16 sequences, suggesting that these miRNAs could serve as diagnostic biomarkers in CC. The present study highlights the roles of miRNAs in cervical tissue and implicates these important molecules in cervical carcinogenesis.

Interactions of two large antiviral polyamides with the long control region of HPV16.

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5'-W2GW7-3'; W=A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3nM-20nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5'-W2GW5GW4-3' via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicates a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

Genetic Variation of high-risk HPV-16 From a Cervical Cancer Biopsy Collected in Pakistan

Aim: HPV-16 is one of the causative agents of cervical cancer. The HPV-16 genome is characterized widely to assist mutagenesis studies, phylogenetic analysis and protein annotations. This is the first report of HPV-16 sequence analysis from Pakistan. Materials & methods: The gene and long control region specific primers were used to generate the targeted amplicons that were then cloned and sequenced. Results: The complete genome analysis revealed nine changes both at amino acid and nucleotide level including seven novel and two previously reported mutations. The evolutionary relationship of HPV from Pakistan and reported sequences shows that strain reported in this study segregates with European taxa. Conclusion: This report lays foundation to conduct HPV genome mutation analysis in Pakistan.

Historia natural de la infección del cérvix uterino por el virus papiloma humano

La asociación epidemiológica entre infección por virus papiloma humano (VPH) y carcinoma cervical cumple con los criterios epidemiológicos establecidos para causalidad. La prevalencia de infección por VPH en mujeres jóvenes ha sido estimada entre 20 y 46% en diversos países, pero resultados recientes de estudios en los EEUU sugieren que 60% de las jóvenes en secundaria está infectada con VPH en algún momento, sin conocerse el impacto a largo tiempo, en carcinogénesis. El cáncer cervical es considerado como una consecuencia tardía de la infección persistente del epitelio cervical por ciertos tipos de VPH, por fallas en la respuesta inmune o la habilidad del virus de evadirla. Por otro lado, las lesiones cervicales en la mayoría de mujeres infectadas con VPH regresiona, sugiriendo una respuesta inmune mediada por células activa. Estas mujeres también son inmunes a reinfección con el mismo tipo de VPH. En conjunto, más de 95% de los cánceres cervicales contienen secuencias VPH. La mayoría ocurre en la zona de transformación cervical, que consiste de epitelio metaplásico escamoso. Los VPH-16 y VPH-18 son los encontrados con más frecuencia y están en 50 a 70% de los cánceres cervicales invasores y sus lesiones precursoras.

Mutation Detection of E6 and LCR Genes from HPV 16 Associated with Carcinogenesis

Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from Cordoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions.

Integration of the full-length HPV16 genome in cervical cancer and Caski and Siha cell lines and the possible ways of HPV integration.

Integration of high-risk human papillomavirus (HPV) into the host genome is a key event for cervical carcinogenesis. Different methods have been used to explore the physical states of the HPV genome to reveal the mechanisms for malignant transformation of the infected cells. Consensus has been reached that, although variable portions of the HPV genome are deleted in the integrated HPV sequences, common disruption of the viral E2 gene has been demonstrated in different studies. The head-to-tail concatemers of the full-length HPV16 genome is another typical integration pattern of HPV16, typically found in Caski cell lines, but its prevalence in cervical cancer has never been tested. Here, by introducing a modified PCR, we identified this head-to-tail concatemers of full-length HPV genomes in advanced cervical cancer with HPV16 single positive. Our results show that more than half of the cases contain this integrated head-to-tail concatemers of full-length HPV16 genomes. Further studies in two cervical cell lines, Caski cells and Siha cells, revealed a correlation between the prevalence of the spliced variants of integrated HPV16 sequences and the full-length transcription of the integrated head-to-tail concatemers of the full-length HPV16 genome. Based on these results, we propose that HPV16 integrated into host cells by two mechanisms: one mechanism is shared by other DNA virus and cause integration of the head-to-tail concatemers of the viral genome; another is related to the reverse transcription process, which the integrated HPV sequence is generated by the reverse transcription of the viral mRNA.