Identification Of Human Papillomavirus Research Articles

Identification of human papillomavirus DNA gene sequences in human breast cancer

Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at −70°C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast.

Molecular diagnosis of human papillomavirus (HPV) infections

Human papillomaviruses (HPVs) comprise more than 100 genotypes. The mucosal types can be divided into high-risk and low-risk (LR) types depending on the associated disease risk. HPV infection is mainly diagnosed by molecular methods, since reliable serological tools are not available and culture of the virus is not possible. Accurate molecular diagnostic techniques that can be used to inform patient management and follow-up after treatment are now available for detection and identification of HPV. The diagnosis of HPV infections in patients at risk of disease in a clinical setting requires a different approach from that used for epidemiological studies, vaccination trials and natural history studies. This review describes the different molecular methods available for HPV detection and genotyping and their possible clinical utility.

E2 sequence variations of HPV 16 among patients with cervical neoplasia seen in the Indian subcontinent

Objectives. Specific nucleotide variations in the E2 DNA sequence were looked for in samples with an intact human papillomavirus (HPV) 16 episomal E2 DNA. Methods. Ninety-two women, 76 with invasive cervical carcinoma and 16 with cervical intraepithelial neoplasia (CIN) were recruited. HPV DNA typing was performed by polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP). Intact episomal E2 DNA of HPV 16 was detected by PCR. Important nucleotide variations in samples with amplifiable E2 DNA were detected by RFLP. Nucleotide sequencing was performed on representative samples to confirm RFLP findings. Results. A total of 89 (96.7%) women were positive for HPV DNA. Of these, 56 (63%) were positive for HPV 16, and of these, 38 (68%) were positive for intact episomal HPV 16 E2 DNA while 18 (32%) were negative. Samples with intact episomal HPV 16 E2 DNA sequences were grouped into four different digestion profiles I to IV based on RFLP patterns. Digestion patterns revealed absence of any sequence variations in samples with digestion profile I and presence of a 2983 A-G variation in those with profile II. Samples with digestion profiles III and IV revealed three variations in the hinge region (3516 C-A, 3538 A-C, 3566 T-G) and two in the DNA binding domain (3684 C-A, 3694 T-A) of the E2 sequence. Sequencing performed on representative samples confirmed RFLP findings. Conclusions. PCR-RFLP helped in the identification of important HPV 16 E2 sequence variations, circumventing the need for sequencing. The presence of the nucleotide variations in positions that could alter the biological and immunological functions of the E2 protein combined with its increased occurrence in this study bring out the importance of these variations.

Identification of human papillomaviruses in tumors of the oral cavity in an Indian community

Oral cancers and other squamous cell cancers of the head and neck are common cancers in India, primarily due to tobacco chewing/smoking and alcohol consumption. Recent reports indicate involvement of human papillomavirus (HPV), HPV 16, in a subset of squamous cell carcinoma of head and neck (SCCHN) cases. To investigate the types of HPVs present in 83 oral cancers and 19 other head and neck tumors, degenerate primers directed to consensus regions in the HPV L1 open reading frame (ORF) were employed to amplify genomic DNA from tumor and when available, the adjacent normal mucosa. PCR-amplified products were cloned and sequenced. Similar studies were done on exfoliated buccal cells of 102 individuals visiting a dental hospital for dental complaints. HPV was detected in 32 out of 102 patients (31%), in either the tumor or the adjacent normal mucosa, while 5% (5/102) of the comparative group were found to be HPV-positive. Sequence analysis revealed a number of cutaneous HPVs, predominantly HPV types of the genus Beta-Papillomavirus, in the oral cavity. Multiple HPV infections were also commonly observed in patients (14/102; 14%). HPV 16 and 18 were each detected in 6 patients (6/102; 6%). Neither high-risk HPVs nor multiple infections were observed in the mouthwash samples of the comparative group. We report that the oral cavity harbors a variety of different HPVs. These viruses, in conjunction with the carcinogens present in tobacco could contribute to carcinogenesis.

Effective PCR Systems for Rapid Identification of Human Papillomaviruses (HPVS)

ABSTRACTThe main approaches for rapid and precise identification of human papillomaviruses (HPVs) are molecular virology techniques. We have adapted effective polymerase chain reaction (PCR) systems that successfully identify numerous clinically significant HPV types: 6, 11, 16, 18, 31, 33, 39, 40, 42, 45, 51–59, 66, 68. Standard (uniplex) and multiplex variants of PCR were applied in our laboratory to identify HPVs by direct amplification of specific viral sequences in cervical scrapes. Introduction of the molecular virology techniques as PCR in the routine activity of virology labs increases the opportunity for objective estimate of the individual carcinogenic risk and promotes the quality of treatment approaches.

Epidemiology of human papillomavirus infections: new options for cervical cancer prevention.

In the last two decades, the cervical cancer puzzle has become a coherent description that includes the identification of human papillomavirus (HPV) as the sexually transmitted etiologic agent and the characterization of the major determinants of HPV acquisition. Triage studies have consistently shown that HPV testing is more sensitive that repeated cytology in identifying underlying high-grade lesions in women with atypical squamous cells of undetermined significance (ASCUS). Studies that reflect primary screening conditions have shown that the sensitivity of HPV tests is higher than standard cytology in detecting high-grade lesions whereas the specificity is similar only in women aged 30-35 and above. HPV vaccines have an intrinsic attraction as a preventive strategy in populations with limited resources. However, vaccines designed to widespread use are still in development and testing phases. Time is ripe for exploring in depth the clinical implications of current achievements and to devise novel strategies for the prevention of cervical cancer. This paper is available too at: http://www.insp.mx/salud/index.html.

Diagnosis of human papillomavirus infection by dry vaginal swabs in military women

Objective: Human papillomavirus (HPV) assays are likely to be used with increasing frequency in clinical management of women with abnormal Papanicolaou smears and in cervical cancer screening. Our objective was...

Clinical and Prognostic Significance of Human Papillomavirus in a Chinese Population of Cervical Cancers

Objective: To investigate the clinical and prognostic significance of human papillomavirus (HPV) in a Chinese population of cervical cancers. Methods: We studied 121 cervical cancer tissue samples from patients treated at our hospital. Identification and typing of HPV were done by polymerase chain reaction (PCR) using consensus primers MY11 and MY09 followed by direct DNA sequencing. The results were correlated with various clinical and prognostic parameters. Results: We found HPV DNA in 95 (78.5%) cases, including HPV-16 in 59 (48.8%) and HPV-18 in 14 (11.6%) cases. χ² analysis revealed no significant correlation between the presence of HPV DNA and age at diagnosis, clinical stage, histologic type, tumor grading, 2-year and 5-year survival rate. Of the factors evaluated, age at diagnosis and histologic type were found to have a statistically significant relationship with HPV type. The mean age of the HPV-18 group was 48.6 years compared to 57.1 years for the HPV-16 group (p = 0.045) and 58.2 years for the HPV-negative group (p = 0.04). HPV-18 was detected more often in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). Conversely HPV-16 was detected significantly more often in SCC (p < 0.0001). The HPV-negative group also had a higher incidence of SCC (p = 0.007). HPV-18-positive patients seemed to have more nodal involvement than both HPV-16-positive patients (45.5 vs. 20.8%) and HPV-negative patients (45.5 vs. 18.2%); however, it did not reach statistical significance. Conclusions: These observations suggest that the presence of HPV DNA does not bear any clinical or prognostic significance in a Chinese population of cervical cancers. HPV-18 is found more often in younger patients and is associated with AC.

HPV infection and cervical carcinoma--epidemiology, detection and immunology

Certain molecularly defined types of human papillomaviruses (HPV) induce, by interaction with additional cofactors, cervical cancer. In comparison to infection with HPV, all risk factors identified to date are less important. In addition to HPV a not yet classified genetic predisposition is important. Identification of HPV in cervical smears is done by detection of viral DNA. PCR based methods have the highest specificity. Currently, first vaccination studies with therapeutic vaccines are performed and phase I/II trials for prophylactic vaccination show promising results.

Morphometric Characterization of HPV-Related Lesions of Uterine Cervix

This study addresses three issues related to human papillomavirus (HPV) cervical lesions: identification of HPV in histologic normal tissue, identification of HPV subtypes with risk for cervical cancer, and histologic differences between HPV not associated to cervical intraepithelial neoplasia (CIN1) lesions (condyloma) and HPV associated to CIN1 lesions. The authors histologically classified 48 cervical biopsy slides into three groups: normal (n = 22), condyloma (n = 20), and CIN1 (n = 6). Morphometric analyses of nuclear and cytoplasmic ratio for area, length, and diameter of 25 cells per case were performed. Histologic reports and in situ hybridization for HPV subtype were compared to morphometric data to assess correlation among them. Using image analysis, the authors correctly classified all cases except two into histologic or in situ hybridization diagnosis. Morphometry helped identify viral changes in cells that appeared histologically normal, HPV subtype at risk in condyloma lesions, and condyloma from those combining HPV lesions and CIN1. There were enough data supporting morphometric distinction of HPV-related cervical lesions assessed by nuclear and cytoplasmic ratio.

Morphometric Characterization of HPV-Related Lesions of Uterine Cervix

Objectives. This study addresses three issues related to human papillomavirus (HPV) cervical lesions: identification of HPV in histologic normal tissue, identification of HPV subtypes with risk for cervical cancer, and histologic differences between HPV not associated to cervical intraepithelial neoplasia (CIN1) lesions (condyloma) and HPV associated to CIN1 lesions. Materials and Methods. The authors histologically classified 48 cervical biopsy slides into three groups: normal (n = 22), condyloma (n = 20), and CIN1 (n = 6). Morphometric analyses of nuclear and cytoplasmic ratio for area, length, and diameter of 25 cells per case were performed. Histologic reports and in situ hybridization for HPV subtype were compared to morphometric data to assess correlation among them. Results. Using image analysis, the authors correctly classified all cases except two into histologic or in situ hybridization diagnosis. Morphometry helped identify viral changes in cells that appeared histologically normal, HPV subtype at risk in condyloma lesions, and condyloma from those combining HPV lesions and CIN1. Conclusions. There were enough data supporting morphometric distinction of HPV-related cervical lesions assessed by nuclear and cytoplasmic ratio.

Genital Human Papillomavirus Infection and Associated Penile Intraepithelial Neoplasia in Males Infected with the Human Immunodeficiency Virus

To investigate the association between human papillomavirus (HPV) infections and penile intraepithelial neoplasia (PIN) in genital lesions from human immunodeficiency virus (HIV)+ men. For comparison, we also investigated the same association using specimens from HIV- men. Imprint smears from penile lesions were obtained from 70 men (mean age, 30 years) who visited a dermatologist. Thirty of them were known to be HIV seropositive. Two study groups were formed: one of 40 HIV- and another of 30 HIV+ males. The smears were examined cytologically for HPV identification or PIN, immunocytochemically for HPV detection and by in situ hybridization for HPV typing. The rates of detecting HPV infection cytologically were higher among men with HIV infection (50%) than among their HIV-seronegative counterparts (30%). There was immunocytochemical evidence of HPV in HIV-infected men in a greater proportion (50%) than in HIV noninfected men (37.5%). By in situ hybridization it was found that there was a higher prevalence of potentially oncogenic HPV (16/18, 31/33/35): 75% in moderate or severe dysplasia (PIN 2 and 3) and 66.6% in HIV+ men as compared with HIV- men (10-16.6%). HIV-seropositive males showed an unbalanced distribution of HPV, with a predominance of "high-risk" HPV types. This suggests that immunodepression encourages infection by this oncogenic virus, thereby contributing to the frequency of precancerous lesions in HIV+ men.

Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer.

The expression of p53 and bcl-2 proteins by immunohistochemistry and the identification of human papillomavirus (HPV) infection by a non-isotopic polymerase chain reaction (PCR)based method were investigated in 30 patients with head and neck cancer. Ten cases were HPV-positive (33%), mostly as double or multiple infections by high- or intermediate-risk types. Twenty-one patients were p53-positive (70%), 9/10 with HPV-positive tumours and 12/20 with HPV-negative tumours; this difference was not statistically significant. Only four cases were bcl-2-positive, irrespective of the presence of either HPV or p53. No correlation was found between these biological factors and tumour stage, differentiation grade, and alcohol or tobacco use. Our findings indicate that p53 is involved in the majority of cases, bcl-2 is rare, and high-risk HPV could play a key role, especially in tumours of tongue and tonsil. In conclusion p53 and bcl-2 protein expression and the presence of HPV infection are independent events in these malignancies.

Prevalence of Human Papillomavirus in Squamous Cell Carcinoma of the Head and Neck Determined by Polymerase Chain Reaction and Southern Blot Hybridization: Proposal for Optimized Diagnostic Requirements

Human papillomaviruses (HPV) are discussed as cofactors in the carcinogenesis of squamous cell carcinoma of the head and neck (SCCHN). The prevalence of HPV infection in SCCHN is the subject of controversy since reported HPV prevalences range from less than 10% to almost 100%, depending mainly on the detection method employed. This study presents a realistic approximation to the real prevalence of HPV in SSCHN by applying polymerase chain reaction (PCR) and Southern blot hybridization (SBH), which are the most sensitive and specific HPV detection methods. Diagnostic procedures were optimized by applying a ''hot-start'' PCR protocol followed by a confirmatory SBH of the PCR products to reactions using both type-specific and consensus primers and probes. DNA of 75 tumour samples and 22 normal mucosa samples of the same patients were investigated. In 14 cases genomic SBH using complete HPV genomes as probes was performed additionally. HPV DNA was detected in 17/75 (22.7%) SCCHN specimens. HPV 16 was identified in four cases, HPV 45 in three cases, and HPV 6 and 18 in one case each. Hot-start PCR and SBH are the most reliable HPV detection methods, as they minimize both false-positive and false-negative results. With these methods, a HPV prevalence of 23% was achieved, which can be assumed to be representative for comparable study populations. The significant number of positives detected only by consensus primer PCR, along with the identification of HPV 45, indicate that further HPV types may play an important role in the genesis of SCCHN.

Seven-Color Time-Resolved Fluorescence Hybridization Analysis of Human Papilloma Virus Types

Identification of human papilloma virus (HPV) types is important in order to determine the risk of cervical carcinoma in women. This requires a technique to probe individual samples for multiple virus specificities. Here we describe simultaneous multicolor analysis of amplification products for any of seven amplified HPV types 16, 18, 31, 33, 35, 39, and 45, associated with cancer of the cervix. A seminested polymerase chain reaction was performed in a single tube using a biotinylated inner primer. Sets of amplification products, immobilized on a 96-pronged manifold solid support, were rendered single stranded and probed with a mix of seven type-specific, differentially labeled oligonucleotides. These probes contained 10 or 20 lanthanide chelates at the 5′ ends with seven distinct combinations of europium, terbium, and samarium ions. The seven viral strains were correctly identified by time-resolved fluorescence measurement of the specifically hybridized probes. Using this assay format, simultaneous detection of any of seven or even more target variants is possible.

Human papillomavirus expression and p53 gene mutations in squamous cell carcinoma.

To determine the incidence of human papillomavirus (HPV) infection and p53 gene mutation expression in squamous cell carcinomas (SCCs) of the oral cavity and tonsils, to correlate the presence of HPV and p53 gene mutation with known clinical and pathological features of SCC, and to determine whether infection with HPV or the presence of p53 gene mutations are independent prognosticators of patient survival. To accomplish this goal, 58 patients with SCCs of the oral cavity and 42 patients with SCCs of the tonsils were randomly examined. The cases examined met the criteria of 5-year clinical follow-up, availability of complete staging information and treatment history, and the presence of paraffin-embedded tumor specimens. Immunohistochemical tests were performed to identify the mutant p53 protein. Human papillomavirus identification was accomplished with polymerase chain reaction, with confirmation via restriction fragment length polymorphisms. The incidence of p53 gene mutation expression for this series was 66%. Human papillomavirus infection was found in 11 patients (11%). There was a trend toward increased p53 gene mutation expression with advancing stage of tumor in the oral cavity cancer group, although this was less evident in the tonsil cancer population. The p53 gene mutation status was found not to correlate with the histological grade of the tumor, patient age or sex, recurrence rates, or survival status. Like p53 expression, there were no correlations found between the presence of HPV and age, sex, histological grade, or recurrence rates. However, a correlation did exist between HPV and survival status in the tonsil cancer group, with improved survival noted among patients with tonsil cancers infected with HPV compared with those not infected with HPV. A significant correlation existed with both p53 gene mutation status and HPV status with respect to alcohol and tobacco use. The presence of the p53 gene mutation positively correlated with increased tobacco and alcohol use, whereas infection with HPV predicted a significantly lower rate of alcohol and tobacco consumption. Human papillomavirus infection is an independent risk factor for the development of oral cavity and tonsil SCCs in those patients with a relatively low alcohol and tobacco use history. Conversely, there is a strong association between heavy alcohol and tobacco use and mutation of the p53 gene. Neither p53 gene mutation nor HPV infection serve as prognosticators of tumor behavior in SCCs of the oral cavity or tonsils, with the exception of improved survival noted among patients with tonsil cancers infected with HPV.

Human Papillomavirus Identification and Typing of Both Sexual Partners

To investigate the efficacy of diagnostic methods for human papillomavirus (HPV) infection. The diagnostic methods were morphologic and immunohistochemical techniques and in situ hybridization. The first ones consisted of (1) a cytologic smear on the females, (2) cytologic examination of a urethral brushing on the males, (3) colposcopy on the females, and (4) histologic examination of the uterine cervix. A synchronous HPV infection was found in 50% of the sexual partners. HPV was detected also in 30% of cervical biopsies by immunohistochemistry. In situ hybridization was performed on both the cervical biopsies and urethral samples from the males. By this method the same subtype of the virus was observed in both sexes. The types of HPV were identified in 45% of 20 females and 80% of 20 males. Analytically, in men, 7 cases of HPV 6/11 types, 3 cases of 16/18, 5 cases of 31/33/35 and 1 case each of 16/18 and 31/33/35 were observed. In women, in 4 cases subtypes 6/11, in 2 cases types 16/18 and in 3 cases types 31/33/35 were identified. Male partners of women with HPV infection should be investigated in order to produce greater insight into HPV prevalence and the mechanisms of viral transmission.

Evaluation of a solution chemiluminescent hybridization assay for identification of human papillomavirus from paraffin‐embedded tissues

AbstractAims To evaluate the use of the Digene Hybrid CaptureTM system, a chemiluminescent hybridization assay for the analysis of low and intermediate/high risk human papillomavirus (HPV) DNA, on paraffin‐embedded tissues and to investigate possible reasons for reaction failure.Methods Fifty cervical biopsies were tested by an in situ hybridization (ISH) method using probes for HPV 6/11. 16/18, 31/33/51 and by the Digene Hybrid CaptureTM system.Results The ISH was able to detect HPV in 23 out of the 50 biopsies. Eight samples were positive with the HPV 6/11 probe, 4 with the 16/18 probe. 6 with the 31/33/51 probe and 5 had mixed infections. With the Hybrid CaptureTM system, having taken the mixed infections into account. 40 samples gave concordant results, while total and partial discordances were observed in 6 and 4 samples respectively. The relative sensitivity of the new assay was 91.6% and specificity 84.2% for low risk HPVs; and 80% and 94.3% respectively for intermediate/high risk HPVs. Agreement rates on HPV positivity for either low risk or intermediate/high risk types were 86% and 94% respectively in comparison with ISH results.Conclusion The Hybrid CaptureTM system is simple, reliable in use and suitable for routine discrimination of HPVs in tissue sections from paraffin‐embedded blocks. It might thus be of clinical value in the diagnosis and prognosis of HPV infection.

Evaluation of a solution chemiluminescent hybridization assay for identification of human papillomavirus from paraffin-embedded tissues

Aims To evaluate the use of the Digene Hybrid Capture™ system, a chemiluminescent hybridization assay for the analysis of low and intermediate/high risk human papillomavirus (HPV) DNA, on paraffin-embedded tissues and to investigate possible reasons for reaction failure.Methods Fifty cervical biopsies were tested by an in situ hybridization (ISH) method using probes for HPV 611, 1618, 31/33/51 and by the Digene Hybrid Capture™ system.Results The ISH was able to detect HPV in 23 out of the 50 biopsies. Eight samples were positive with the HPV 611 probe, 4 with the 1618 probe, 6 with the 31/33/51 probe and 5 had mixed infections. With the Hybrid Capture™ system, having taken the mixed infections into account, 40 samples gave concordant results, while total and partial discordances were observed in 6 and 4 samples respectively. The relative sensitivity of the new assay was 91.6% and specificity 84.2% for low risk HPVs; and 80% and 94.3% respectively for intermediate/high risk HPVs. Agreement rates on HPV positivity for either low risk or intermediate/high risk types were 86% and 94% respectively in comparison with ISH results.Conclusion The Hybrid Capture™ system is simple, reliable in use and suitable for routine discrimination of HPVs in tissue sections from paraffin-embedded blocks. It might thus be of clinical value in the diagnosis and prognosis of HPV infection.

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR.

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes.