Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization.

We analyzed the distribution pattern of human papillomavirus (HPV) 6 and 11 DNA in vulvar lesions by in situ hybridization after amplification by the "hot start" polymerase chain reaction (PCR). HPV DNA was routinely detected in granular layer cells showing perinuclear halos and nuclear atypia by in situ hybridization with or without PCR. Cells that lack these changes rarely exhibited HPV DNA with standard in situ hybridization. After amplification, in situ analysis showed that many of the cells that lacked halos and atypia did contain HPV DNA and that the hybridization signal often localized to areas where there was a thickened granular layer. HPV DNA was not noted in the basal cells. The one copy of HPV 16 in SiHa cells was detectable after PCR with a single primer pair by in situ analysis only if the hot start modification was employed. Prior reports describing the PCR in situ methodology noted the need for from five to seven primer pairs. The hot start technique, which may be done by withholding the DNA polymerase until the temperature is sufficiently high to disfavor nontarget specific pathways, allowed the use of a single primer pair and showed that the degree of target-specific amplification, and not the size of the amplified product, determines the success of the PCR in situ technique.     

Survey of histologic specimens of human cancer for human papillomavirus types 6/11/16/18 by filter in situ hybridization

Histologic specimens (317) of genital and nongenital cancers and normal tissue were analyzed for the presence of the DNA of human papillomavirus (HPV) 6, 11, 16, and 18 by filter in situ hybridization performed on paraffin-embedded, formalin-fixed tissue (HISTOFISH). HPV DNA was found in 73 of 172 (42%) anogenital lesions and 17 of 116 (15%) nonanogenital carcinomas. No HPV DNA was found in normal mouse skin (five samples), human autopsy liver (two samples), or kidney (eight samples), or in carcinomas of the breast (three samples), bladder (five samples), or colon (nine samples). Of the nongenital tumors, HPV DNA was found in the carcinomas of the lung (2 of 5), anus (7 of 18), esophagus (9 of 39), buccal cavity (1 of 5), and larynx (5 of 50). HPV DNA was also detected in 2 of 11 histologically normal specimens of the cervix and 1 of 3 human skin lesions. The detection of HPV DNA in carcinomas of the lung, larynx, and esophagus as well as in the anogenital region confirms recent suggestions that HPV types 6, 11, 16, and 18 have a wider association with different types of cancer than previously believed. The study also shows that HISTOFISH is a useful method for detecting HPV-DNA in histologic specimens.     

Verruca vulgaris of the larynx. Demonstration of human papillomavirus types 6/11 by in situ hybridization.

Verruca vulgaris of the larynx (VVL) is a distinctly uncommon lesion related to the human papillomavirus (HPV). The clinical and pathologic features of a case involving the true vocal cords of a 37-year-old woman are presented and compared with the seven cases previously reported in the English language literature. Papillomavirus capsid antigen was detected in the excised tissue on immunostaining, and viral particles were seen by electron microscopy. In situ hybridization with biotinylated DNA probes clearly demonstrated HPV types 6/11. To our knowledge, this is the first case of VVL in which the virus associated with VVL has been genotyped. The results were unexpected because verruca vulgaris of the skin, lips, and oral cavity is associated with HPV types 2 and 4. This implies that verruca vulgaris can be caused by HPV types other than 2 and 4. In addition, since HPV types 6 and 11 are also the same genotypes associated with multiple papillomatosis of the larynx, it further indicates that VVL is virologically more related to multiple papillomatosis of the larynx than to its counterpart on the skin, lips, and oral cavity. The clinical and pathologic features that distinguish VVL from other similar lesions of the larynx are also discussed.     

Detection of human papillomavirus (HPV) DNA type 6/11 in a conjunctival papilloma by in situ hybridization with biotinylated probes.

Four cases of conjunctival papilloma in two different patients were examined by in situ hybridization for HPV DNA type 6/11, 16/18 and 31/33/51. Formalin-fixed and paraffin-embedded tissues were hybridized by biotinylated probes. One tumor and one of its recurrences showed nuclear positivity for HPV 6/11 in the superficial cells of the epithelium. The results suggest that HPV type 6/11 may be etiologic agent of conjunctival papillomas. The benign behavior of these neoplasms may be related to the etiologic role of this type of HPV.     

Laryngeal papillomatosis: correlation between severity of disease and presence of HPV 6 and 11 detected by in situ DNA hybridisation.

A technique using a biotin-streptavidin polyalkaline phosphatase complex was applied to routinely fixed and processed biopsy specimens of laryngeal papillomata from 45 patients taken over the past 20 years to detect human papilloma virus (HPV) types 6 and 11. Two thirds of both adult and juvenile onset cases were positive for HPV 6 or HPV 11 or both. Five specimens of normal vocal cord epithelium were negative for HPV 6 and 11. The detailed clinical history, endoscopic findings, success of treatment and eventual prognosis were compared with the HPV state of biopsy material for each patient. Patients with multiple confluent lesions when first seen, whose histology showed florid koilocytosis and who had strongly positive reactivity for HPV 6 or 11 present in the surface epithelial cell nuclei, had a poor prognosis requiring multiple endoscopies to control their disease.     

In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia

The authors examined 18 cervical intraepithelial neoplasms (CIN) for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and DNA-DNA in situ hybridizations for HPV DNA sequences and compared the epithelial distribution of HPV 16 DNA sequences with HPV 6/11 sequences in selected condylomas. Fifteen of the 18 CIN lesions contained HPV 16 DNA as determined by Southern blot hybridization. With the use of biotinylated HPV 16 DNA probes, 10 of the 18 were positive by in situ hybridization, 9 of which were also positive by Southern blot hybridization. In situ hybridization to HPV 16 probes was found primarily in areas of CIN which contained either maturation or koilocytotic atypia, although in two cases hybridizing sequences were detected in superficial cells from epithelium with no discernible maturation. Staining in both condylomas and CIN lesions varied in distribution and intensity. However, in some CIN lesions staining from cell to cell varied considerably. This greater variability in staining appeared to correlate with greater morphologic variations which characterize CIN, and which may influence greater variation in HPV DNA replication. Thus, some differences in patterns of hybridization for HPV DNA between CIN and condylomas may be explained by morphologic differences in the two classes of lesions. Differences in viral gene expression between condylomas and CIN and their relationship to morphologic findings remain to be clarified.     

HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Comparison of Southern transfer hybridization and dot filter hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35

A commercial dot filter hybridization kit (Virapap Kit) was compared with Southern transfer hybridization for the detection of seven types of human papillomavirus (HPV) in cervical specimens from 450 consecutive females attending a sexually transmitted diseases clinic. In comparison with Southern transfer hybridization, performed with the same probes used in the dot filter kit, the sensitivity, specificity, and positive and negative predictive values of dot filter hybridization were 90%, 94%, 74%, and 98%, respectively. Among patients with cervical cytologic dysplasia, HPV DNA was detected in 44% by dot filter hybridization and in 35% by Southern transfer hybridization. Although 26% of specimens positive by dot filter hybridization were not confirmed by Southern transfer hybridization, cervical dysplasia was detected in 5 (25%) of 20 with HPV DNA detected by dot filter hybridization alone, compared with 25 (8%) of those with no definitive evidence of HPV by either method (P = 0.009) and with 16 (30%) of 53 with HPV DNA detected by both methods (P = 0.7). The kappa statistic for interobserver and intraobserver reproducibility for interpretation of blots was similar for the two methods. The dot filter hybridization method evaluated appears to be a satisfactory alternative to Southern transfer hybridization for detection of HPV DNA.     

Demonstration of human papillomavirus types 6 and 11 in juvenile laryngeal papillomatosis by in-situ DNA hybridization

A study is reported in which an in situ hybridization technique for the demonstration of human papillomavirus (HPV) employing a biotin--streptavidin polyalkaline phosphatase complex has been successfully applied to formalin-fixed paraffin-processed tissue obtained from ten patients with juvenile laryngeal papillomatosis. In all cases, positive results were obtained for either HPV type 6 or 11. Normal vocal cord epithelium was negative.     

Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 35S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.     

Simultaneous in situ hybridization for DNA and RNA reveals the presence of HPV in the majority of cervical cancer cells.

Thirteen cases of invasive squamous cell carcinoma of the uterine cervix containing HPV types 16 or 18 DNA sequences, as detected by Southern blot analysis, were investigated by in situ hybridization on routine paraffin sections, using 35S nick-translated DNA probes. Simultaneous in situ hybridization for DNA and RNA showed that in ten out of 13 cases (77%) the percentage of tumor cells containing HPV 16 or 18 varied from 75 to 100%. In one case, harboring both in situ and invasive carcinoma, the same type of HPV DNA was detected in both components. This finding suggests that neoplastic cells retained the viral genome during progression to invasiveness.     

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.     

Assessment of immunohistochemistry for p16INK4 and high-risk HPV DNA by in situ hybridization in esophageal squamous cell carcinoma

The role of high-risk human papillomavirus (HPV) in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remains unclear. p16(INK4) is used as a surrogate marker to detect HPV-related tumors but has had discrepant results in ESCC. In this study, 32 cases of ESCC were examined to determine the relationship between p16(INK4) expression and high-risk HPV. All the tumors were stained by immunohistochemistry for p16(INK4). Tumors having p16(INK4) nuclear and/or nuclear and cytoplasmic expression were considered positive. Tumors positive for p16(INK4) expression were tested for high-risk HPV by in situ hybridization (ISH). In all, 20 cases of ESCC (63%) showed only cytoplasmic staining for p16(INK4), and 11 cases (34%) showed both cytoplasmic and nuclear staining for p16(INK4); 4 cases (13%) showed no staining for p16(INK4). None of the p16(INK4) -positive cases were positive for high-risk HPV by ISH. These results indicate that p16(INK4) expression in ESCC does not correlate with the presence of high-risk HPV DNA by ISH. High-risk HPV does not seem to play a major role in the carcinogenesis of ESCC in low-risk areas.     

Identification of human papillomavirus types in male urethral condylomata acuminata by in situ hybridization

An in situ hybridization technique was applied under stringent conditions to paraffin sections of urethral condylomata from male patients to determine the presence of DNA sequences of human papillomavirus (HPV) types 6, 11, 16, and 18. The material consisted of 15 classical condylomata acuminata, two flat condylomata, and five recurrent lesions. HPV DNA sequences could be identified in all 15 condylomata acuminata; in 13 lesions, two types of viral DNA were observed (types 6 and 11 in 12, types 6 and 18 in one). In the remaining two condylomata acuminata, only HPV type 11 was present. One of the two flat condylomata was negative with all the probes, and one was borderline-positive for HPV 6. Four of five recurrent lesions contained the same types of viral DNA as the primary lesions, albeit with slight differences in the intensity of viral expression. One lesion was negative with all probes. We conclude that urethral condylomata in males contain the same types of HPV as seen in other anogenital lesions of both sexes and that infection with two viral types is common. In situ hybridization with HPV DNA probes is applicable to archival material and therefore may prove to be of value in future epidemiologic studies comparing lesions in sexual partners. The determination of viral type may have therapeutic implications.     

Detection of HPV16 and 18 by in situ hybridization in precancerous and cancerous lesions of cervix

Fifty cervical biopsies from women with preinvasive and invasive malignancies of uterine cervix and ten normal cervical biopsies were examined for the presence of human papilloma virus (HPV) 16 and 18 DNA sequences by in situ hybridization (ISH) method with biotinylated DNA probes. The overall positivity of HPV DNA was 48% (24/50). The positivity of HPV 16 DNA for low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) were 33.33%, 45.45%, 42.30% respectively. The positivity for HPV 18 DNA for LSIL, HSIL and SCC were 0%, 18.18%, 30.76% respectively. Two cases of cervical adenocarcinomas showed positivity for HPV 18 DNA only.     

Multiple HPV infection: microanatomy by in situ hybridization and immunohistochemistry.

Specific human papillomavirus (HPV) types have been shown to be associated with proliferative epithelial lesions with variable biological consequences in infected patients. Simultaneous infection by more than one HPV type has been infrequently reported, and its clinical significance is unknown. We have examined four biopsies of cervical and vulvar tissue, each with evidence of infection by two different HPVs. Using both in situ hybridization and immunohistochemical techniques, we determined the cellular distribution of the viral infections. Using biotinylated type-specific probes and stringent conditions we were able to demonstrate that in each case the two HPVs occupied distinct, non-overlapping foci within the lesions. The condylomatous tissues contained DNA from HPV types that are associated with high-grade neoplasia and invasive cancer (16 and 18), as well as types commonly associated with benign proliferative lesions. Immunohistochemical analysis of the lesions with antibody to bovine papillomavirus capsid antigen failed to detect HPV in regions shown by in situ hybridization to contain HPV 16 and 18 DNA, whereas type 6 and 11 infected areas were readily identified. These results provide indirect evidence of viral interference between HPV types and indicate that interference may limit the number of HPV types that produce active infections within a single cell.     

Oligonucleotide primers for DNA amplification of the early regions 1, 6, and 7 from human papillomavirus types 6, 11, 16, 18, 31, and 33

Summary Human papillomavirus (HPV) type-specific sequences required for polymerase chain reaction (PCR) mediated amplification of HPV DNA sequences are presented. One primer pair within the E1 open reading frame (ORF) was shared by HPV 6, HPV 11, HPV 16, and HPV 31, whereas the other primer pair within the E1 ORF was specific for HPV 16. Eight primer pairs from the E6 and E7 ORFs specifically detected HPV 6, HPV 16, HPV 18, and HPV 33 sequences. This system has been used for detection of HPV DNA in biopsies, cytological smears and sections of formalin-fixed tissues.     

Seroprevalence of HPV vaccine types 6, 11, 16 and 18 in HIV-infected women from South Africa, Brazil and Botswana

Background

Many resource limited settings (RLS) suffer from high rates of both cervical cancer and HIV. Limited HPV serology data are available from RLS; such data could help describe local patterns of HPV infection and predict vaccine efficacy.

Objectives

To determine seropositivity to HPV types 6, 11, 16 and 18 in HIV-infected women from South Africa (SA), Botswana and Brazil.

Study design

HPV serotyping for high-risk types 6, 11, 16 and 18 was performed on samples collected from HIV-infected women from 2003–2010 using competitive Luminex Immuno Assay (HPV-4cLIA). We examined the association between seropositivity to these HPV types and country of enrollment, CD4, HIV-1 RNA level, and Pap smear.

Results

HPV serology results were available for 487 HIV-infected women (157, 170 and 160 from SA, Botswana and Brazil respectively). Approximately 65% of women had serum antibodies to one of the 4 HPV types and less than 3% of women had antibodies all 4 serotypes. Approximately 30% women demonstrated antibodies to type 16 HPV. Rates of seropositivity to HPV 11, and HPV 16+18 varied significantly between countries. Statistical difference was also shown in women in different age categories in the different countries. There was no difference in serology results compared by CD4 count, HIV viral load or Pap smear results.

Conclusions

These data suggest that the quadrivalent vaccine may be effective in preventing HPV infection in these countries.