Molecular variants of human papillomavirus type 16 and 18 and risk for cervical neoplasia in Portugal

Persistent high-risk human papillomavirus (HPV) infection is considered as the central cause of invasive cervical cancer. Specific HPV 16 and 18 sequence variations were associated with an increased risk for progression. The purpose of this study was to analyze intratypic variations of HPV 16 and 18 within the E6 gene, MY09/11 and LCR regions, and to evaluate the risk of these variants for cervical neoplasia among Portuguese women. Cervical samples from 187 HPV 16-positive and 41 HPV 18-positive women with normal epithelium, cervical intraepithelial neoplasia, or invasive cervical cancer were amplified by type-specific PCR, followed by sequence and phylogenetic analysis. Sixteen new HPV 16 and 18 patterns are described in this paper. European HPV 16 variants were the most frequent (74.3%), particularly Ep-T350 (44.4%), followed by African (16.1%), and Asian-American (9.6%). Non-European HPV 16 variants were more frequent in pre-invasive lesions than in normal tissue and low-grade lesions. However, when analyzed separately, only African variants were associated significantly with an increased risk for cervical cancer. For HPV 18, the AsAi variant showed a trend, which was not statistically significant to an enhanced oncogenicity. European variants seemed to be significantly associated with a lower risk for cervical cancer development. The distribution of HPV 16 and 18 variants was not related to age or race among women living in the same geographical region. Knowledge of variants will be important for risk determination as well as for designing primers or probes for HPV detection methods, and for appropriate cervical cancer prevention strategies.     

Cervicovaginal, oral, and serum IgG and IgA responses to human papillomavirus type 16 in women with cervical intraepithelial neoplasia

Oncogenic human papillomaviruses (HPVs) are obligate mucosal pathogens and typically cause localized infections. The mucosal surface of the genital tract also provides the first line of defense against genital HPV infection. Although local antibody production following HPV-infection has been demonstrated, their role in protection from cervical disease is unclear. This study evaluated oral and cervical HPV infection and the associated linkage between HPV-16 oral, cervical and serum antibody responses in 103 women with varying grades of cervical intraepithelial neoplasia (CIN). We found that HPV-16 was the most prevalent cervical HPV infection (30/103, 29.1%) but was only detected in 1.1% (1/91) of the oral samples. Both the frequency and magnitude of HPV-16-specific cervical IgA was significantly elevated in women with CIN 2/3 compared with women with CIN 1 (P = 0.0073 frequency; P = 0.0045 magnitude). Women with cervical HPV-16 infection had significantly higher magnitude and frequency of cervical HPV-16 IgA responses than women without cervical HPV-16 DNA (P = 0.0002 frequency; P = 0.0052 magnitude). Despite our contention that mucosal HPV-16 antibody responses within distinct mucosal compartments may be linked, the concordance analysis carried out within and between mucosal compartments and serum suggests that no such linkage exists and that these compartments may be functioning independently of one another. An HPV-16 specific antibody response in one mucosal compartment in women with CIN is therefore not predictive of a response at another.     

Genotyping of human papillomavirus in paraffin embedded cervical tissue samples from women in ethiopia and the Sudan

Cervical cancer is the most frequent female malignancy in most developing countries. Previous studies have demonstrated a strong association of human papillomavirus (HPV) infection with dysplasia and carcinoma of the uterine cervix. The objective of this study was to identify the prevailing HPV genotypes responsible for the development of cervical cancer among women in Ethiopia and the Sudan. A molecular characterization of HPV was done on 245 paraffin embedded cervical biopsy samples collected from the two countries. Amplification of HPV and subsequent genotyping was done using SPF10 primers and Line probe assay. Of samples collected from Ethiopian patients, 93% (149/160) and 13% (21/160) had high risk and low risk HPV genotypes, respectively. Among samples collected from the Sudan, 94% (80/85) harbored high risk and 11.7% (10/85) low risk HPV genotypes. Human papillomavirus 16 was the most frequent genotype identified in samples from Ethiopia (91%, 136/149) and the Sudan (82.5%, 66/80). HPV 52, 58, and 18 were the second, third and fourth common genotypes identified in Ethiopia, whereas HPV 18, 45, and 52 were the second, third, and fourth genotypes identified in samples collected from the Sudan. Thus, individuals living in different geographical localities should receive vaccines based on the specific genotypes circulating in the area and a vaccine targeting HPV 16, 18, 45, 52, and 58 may be optimal for the control of cervical cancer in the two countries. J. Med. Virol. 85:282–287, 2013. © 2012 Wiley Periodicals, Inc.     

Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.     

Sequence variation of human papillomavirus type 16 E7 in preinvasive and invasive cervical neoplasias

Variation in the nucleotide sequence of the HPV 16 E7 gene in preinvasive cervical intra-epitherial neoplasia (CIN) and invasive cervical carcinoma specimens was analyzed. Direct DNA sequencing of PCR-amplified products with primers different from those used for PCR with 5-end labeling generated distinct sequence ladders with a low background, even in specimens containing relatively low copy numbers of HPV. Of 14 cervical neoplasias, 11 cases showed sequence diversity from prototype HPV16, and a total of 22 nucleotide exchanges were detected. Nine of these led to single amino acid exchanges: [Thr⁵] to [Lys⁵] in one case and [Asn²⁹] to [Ser²⁹] in eight cases. The [Ser²⁹] E7 was distributed uniformly among invasive carcinomas and precancerous legions, and was also found in a normal cervix. The [Lys⁵] E7 and [Ser²⁹] E7 had transforming potential similar to the prototype E7 assessed by cooperation with the activatedras gene in rat embryo fibroblasts.     

A pilot study on the distribution of human papillomavirus genotypes and HPV-16 variants in cervical neoplastic lesions from Ecuadorian women

Human papillomaviruses (HPVs) are the cause of cervical intraepithelial neoplasia and invasive carcinomas of the uterine cervix. The distribution of specific HPV genotypes varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. The aim of this study was to determine the spectrum of HPV genotypes and HPV-16 variants among women with cervical lesions living in Ecuador. A total of 71 cases have been analyzed, including 32 chronic cervicitis, 29 cervical intraepithelial neoplasia grade 1, and 10 cervical intraepithelial neoplasia grade 2-3. HPV sequences were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Overall, 31 (43.7%) cases were HPV positive with prevalence rates of 37.5%, 44.8%, and 60% in patients with chronic cervicitis, cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 2-3, respectively. Among the positive cases, the most common genotypes were HPV 16 (64.5%) and HPV 81 (29%) followed by HPV 31, 53, 56, and 58, in descending order of prevalence. Seventeen (85%) HPV-16 isolates were classified as European and three (15%) as African-1 variant on the basis of nucleotide signature present within the MY09/MY11 L1 sequence. The results suggest that HPV 16 has a very high prevalence among women with cervical lesions in Ecuador; therefore, an effective HPV-16 based vaccine should prevent the development of cervical cancer in a large proportion of Ecuadorian women.     

Effect of naturally acquired type-specific serum antibodies against human papillomavirus type 16 infection

BackgroundWhile vaccine-induced antibodies are known to confer protection against incident human papillomavirus (HPV) infection, there is inconsistent data regarding the protective effect of naturally acquired anti-HPV antibodies.ObjectivesTo estimate the protective effect of naturally acquired anti-HPV16 serum antibodies against incident anogenital infection with HPV16 in females aged 20–64 years and to assess whether antibodies influence the persistence/clearance of anogenital HPV16 infection.Study design4432 women attending the organized national cervical cancer screening program in Slovenia were initially enrolled. 2199 and 1848 women had valid HPV DNA results obtained using PCR-based assays and HPV antibody serotyping results obtained using pseudovirion-based serological assay, at baseline and at three-year follow-up, respectively.ResultsBaseline HPV16 seroprevalence was 2.4-fold higher among HPV16 DNA-positive women (55.7% vs. 23.2%; p < 0.01). Baseline HPV16 DNA-positive/seronegative women frequently acquired anti-HPV16 antibodies during follow-up (OR = 8.2; 95% CI: 3.8–17.8). Baseline anti-HPV16 antibodies persisted at follow-up, irrespective of baseline HPV16 DNA status (OR = 40.6; 95% CI: 30.3–54.5). Baseline HPV16 DNA-negative/seropositive women were less likely to acquire HPV16 infection at follow-up (unadjusted OR = 0.2; 0.1–0.9). However, the age-adjusted association was non-significant (adjusted OR = 0.3; 0.1–1.2). The tendency for protective effect was stronger among women older than 25 years (OR = 0.2; 0.03–1.8). Baseline anti-HPV16 antibodies were not associated with persistence/clearance of HPV16 infection at follow-up (OR = 0.8; 0.3–1.9).ConclusionsNaturally acquired anti-HPV16 serum antibodies appeared to protect against anogenital HPV16 infection, but this association was at least partially confounded by age. Baseline anti-HPV16 serum antibodies did not influence persistence/clearance of HPV16 infection at follow-up.     

Association of human papillomavirus type 16 and its genetic variants with cervical lesion in Korea

Persistent human papillomavirus type 16 (HPV16) is the major risk factor for cervical cancer. HPV16 intratypic variants differ in their geographical distribution and oncogenic potential. This study aimed to analyze the distribution of HPV16 variants and their association with cervical lesion histopathology in Korean women. In total, 133 HPV16‐positive cervical samples from women admitted to Seoul National University Boramae Hospital were analyzed by sequencing E6, E7, and L1 genes and the long control region (LCR), and the variant distribution according to cervical lesion grade was determined. Isolates were grouped into a phylogenetic lineage, and A1‐3, A4, C, and D sublineages were detected in 54.1, 37.8, 0.7, and 7.4% of samples, respectively. The most commonly observed LCR variations were 7521G>A (91.5%), 7730A>C (59.6%), and 7842G>A (59.6%). Furthermore, A4 or D sublineage‐positive women had a higher risk for cervical cancer than women who were positive for A1–3. Among HPV phylogenetic clusters, A1–3 was the predominant sublineage, and within A1–3, the 350G polymorphism was highly frequent. These results differed from those of previous studies in Korea and other Asian countries. The findings suggest that cervical neoplasia incidence in HPV16‐infected patients could be affected by the distribution of HPV16 variants in the population.     

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Nine different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2, followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin.     

Follow-up of antibody responses to human papillomavirus type 16 E7 in patients treated for cervical carcinoma

A synthetic peptide comprising amino acids 6-35 of HPV-16 E7 was used in an ELISA to screen sera taken from 31 cervical carcinoma patients. Sera obtained before and during treatment, and in follow-up, were tested for the presence of antibodies to this peptide. Sixteen patients with negative pretreatment serum determination remained negative during treatment and follow-up. Of the 15 patients with positive pretreatment sera, 12 showed a decrease in anti-E7 6–35 antibody level during treatment. During follow-up an increase in anti-E76–35 antibody level was observed in 6 out of 7 patients with progressive or recurrent disease, whereas all patients who remained in complete remission showed stable or further decreasing antibody levels. During the course of disease of the 15 seropositive patients, serum anti-E76–35 antibody levels were compared with serum squamous cell carcinoma antigen (SCC-Ag) profiles, a clinically useful tumor marker in the management of cervical cancer patients. Similar patterns were observed in 10 out of 15 patients. The results of this study suggest that in a subset of cervical cancer patients, anti-E76–35 antibody response against HPV-16 E7 at least partially depends on the presence of viable tumor lesions, and that to some extent the anti-E7 profile reflects the course of disease. © 1995 Wiley-Liss, inc.     

Genetic variability and functional implication of HPV16 from cervical intraepithelial neoplasia in Shanghai women

Human papillomavirus (HPV)16 gene mutation is usually associated with persistent HPV infection and cervical intraepithelial neoplasia (CIN). However, the functional implications of HPV16 mutations remain poorly understood.145 LCR/E6/E7 of the HPV16 isolates were amplified and sequenced, and HPV16 integration status was detected. In total, 89 SNPs (68 in the LCR, 13 in E6, 8 in E7) were discovered, 11 of which were nonsynonymous mutations (8 in E6, 3 in E7). The H85Y and E120D variants in E6 were significantly reduced in the high-grade squamous intraepithelial lesion (HSIL) group compared to the <HSIL group (P = .046 and .005), conversely the N29S in E7(P = .01). Amino acid substitutions (D32N/E, E36Q, H85Y, and E120D in E6 and N29H/S and R77C in E7) were predicted to have an effect on conserved structural and functional residues, and five amino acid substitutions (H85Y, E36Q, I34L, and D32E in E6; R77C in E7) would potentially change the secondary structure. “6329G>T,” a potential binding site for TATA-binding protein, is the most common in LCR variants. A4 (Asian) was associated with an increased risk of HSIL compared to A1–3(P = .009). The H85/E120 in E6 and N29 in HPV16 E7 might play a critical role in carcinogenesis by disrupting p53 and Rb degradation due to affecting their interaction, respectively. In a word, the findings in this study provide preventative and therapeutic interventions of HPV16 -related cervical lesions/cancer.     

子宫颈癌、子宫颈上皮内病变组织中SIRT1、HPV 16/18E6的表达及其意义

目的检测子宫颈癌及癌前病变中SIRT1的表达情况,探讨其与临床病理特征的关系。同时检测早期癌蛋白中HPV16/18E6的表达,分析两者的相关性。方法应用免疫组化En Vision法检测30例子宫颈炎、100例子宫颈上皮内病变(高级别、低级别各50例)、30例子宫颈鳞状细胞癌组织中SIRT1和HPV 16/18E6的表达。结果子宫颈癌组织中SIRT1阳性率为93.33%(28/30),高于子宫颈炎组织(13.33%,4/30),差异有统计学意义(P0.05)。子宫颈高级别上皮内病变SIRT1阳性率(88%,44/50)高于低级别上皮内病变(14%,7/50),差异有统计学意义(P0.05)。但高级别上皮内病变与子宫颈癌SIRT1阳性率差异无统计学意义(P0.05);低级别上皮内病变与子宫颈炎中SIRT1阳性率差异无统计学意义(P0.05)。在子宫颈癌中,SIRT1的阳性率与临床分期和组织学分级有关,晚期、分化差的子宫颈癌阳性率高于早期、分化好的子宫颈癌,差异有统计学意义(P0.05)。在子宫颈癌中SIRT1的阳性率(93.33%,28/30)高于HPV 16/18E6(30%,9/30),在子宫颈高级别上皮内病变中SIRT1的阳性率(88%,44/50)高于HPV 16/18E6(28%,14/50),但在低级别上皮内病变中SIRT1的阳性率(14%,7/50)低于HPV 16/18E6(36%,18/50),两者差异有统计学意义(P0.05)。结论 SIRT1的阳性率随着病变程度增加而升高,提示SIRT1与细胞恶性转变有关,其过表达可能促进上皮向高级别内病变乃至子宫颈癌进展。     

Variation in human papillomavirus type-16 viral load within different histological grades of cervical neoplasia

The objective of this study was to investigate variation in human papillomavirus (HPV) type-16 load within histologically defined grades of cervical intraepithelial neoplasia. Two hundred and thirty-seven liquid based cytology samples were collected from women attending colposcopy clinics, DNA was extracted, and presence of virus determined by PCR-enzyme immunoassay. Quantitative real-time PCR was used to determine viral load for 70 HPV-16 positive single infections. Viral load was expressed as the ratio of copies of the viral L1 gene to copies of the human beta-globin gene. Measurements varied from 0.019 to 4,194 HPV genomes per cell. Our data demonstrate that in cervical neoplasia, HPV load tends to correlate with disease severity, but that the number of viral genomes/cell varies considerably within histological grades. This variation within disease grades currently limits the clinical utility of viral load measurement.     

Extended human papillomavirus genotype distribution in cervical intraepithelial neoplasia and cancer: Analysis of 40 352 cases from a large academic gynecologic center in China

Our aim was to conduct a large epidemiologic analysis of the distribution of human papilloma virus (HPV) genotypes associated with cervical neoplasias and cancers at a major Chinese gynecologic center. The pathologic database was searched for cervical histopathologic diagnoses with prior HPV genotyping from liquid cervical cytology specimens obtained ≤6 months before biopsy. HPV testing was performed by using the Tellgenplex HPV27 or YanengBio HPV23 genotyping assays. A total of 40 352 cases meeting study criteria were identified. High risk human papillomavirus (hrHPV) was detected in 94.1% of squamous cancers compared to in only 83.3% of cervical adenocarcinomas. The prevalence of multiple HPV infections was highest in cervical intraepithelial neoplasia 1 (CIN1) (33.8%) and decreased with increasing severity of squamous lesions. The distribution of HPV genotypes was similar between CIN1 and histopathologic-negative cases. HPV16 was one of the three most common hrHPV genotypes before all histopathologic abnormalities, ranging from 72.0% for cervical cancers, 38.7% for CIN2/3/AIS, 13.1% for CIN1, and 9.1% for biopsy-negative cases. HPV16 and HPV18 accounted for over 87.2% of detected hrHPV genotypes for all glandular intraepithelial neoplastic lesions and cancers, whereas squamous lesions did not show this pattern. 80.3% of cervical cancers were associated with genotypes covered by HPV16/18 vaccines and 89.6% with genotypes covered by 9-valent vaccination.