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A 3-year-old African American girl taking sirolimus and tacrolimus for a small bowel transplantation presented with hypopigmented macules and papules throughout her trunk. A biopsy diagnosed epidermodysplasia verruciformis (EV) that was found to be associated with human papillomavirus (HPV) type 14 according to polymerase chain reaction analysis. There are few cases of acquired EV in the setting of organ transplantation. Although there is no standardized treatment for acquired EV, prevention and surveillance for transformation to squamous cell carcinoma are primary concerns., (© 2015 Wiley Periodicals, Inc.)

Invasive cervical carcinoma (ICC) remains a leading cause of female mortality in India. There is a paucity of data about human papillomavirus (HPV) type distribution among ICCs from Central India. Formalin-fixed, paraffin-embedded ICC specimens (n=270 patients) from Madhya Pradesh state were screened for HPV by GP5/6 primer-based polymerase chain reaction followed by cycle sequencing and NCBI BLAST search. HPV was detected in 93.3% of the tumors. Thirteen types were detected: HPV16 (73.7%), HPV18 (11.9%), HPV45 (2.2%), HPV66 (1.5%), HPV35 (1.1%), and HPV56 (0.7%). HPV31, 51, 58, 59, 67, 82 and JEB2 were each seen in 1 case (0.4%). HPV16 was more common among women less than 40 yr than 40 yr or older (P=0.006), and HPV18 was more common in women aged 40 yr or older (P=0.013). Squamous cell carcinomas were more frequently HPV-positive than adenocarcinomas (P=0.01). HPV18 was more common in adenocarcinoma than in squamous cell carcinoma (P=0.02). These data contribute to the pan-India profile of HPV-type relationship to ICC and show the potential of current vaccines to greatly relieve (by up to 85.6%) the ICC burden in Central India. The findings are also suggestive that next-generation HPV vaccines might be designed on a regional basis rather than from compounded global data.

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16(INK4a) immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16(INK4a) staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16(INK4a) negative and two HPV negative samples were p16(INK4a) positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16(INK4a) positive, PCR and CISH HPV positive/p16 (INK4a) negative, or PCR HPV positive/p16 (INK4a) and CISH negative, also requires further investigation.

Unlabelled: Barrett's esophagus (BE), a known precursor of esophageal adenocarcinoma has recently been associated with human papillomavirus (HPV). p16(INK4a) expression is a recognized surrogate marker of HPV infection in the cervix., Objectives: This study has assessed the possible role of human papillomavirus (HPV) infection in BE and esophageal adenocarcinoma, in the North American population by screening esophageal tissues for HPV by a combination of assays., Study Design: Formalin-fixed, paraffin-embedded blocks from cases of Barrett's esophagus (n=84), esophageal adenocarcinoma (n=36) and normal gastro-esophageal junction (n=29) were examined for HPV by PCR, chromogenic in situ hybridization, and p16(INK4a) immunohistochemistry., Results: HPV DNA was detected by PCR in 23 of 84 (27.4%) BE cases, 11 of 36 (31%) cases of adenocarcinoma and in 7 of 29 (24%) normal control cases (p=0.82). p16(INK4a) staining was positive in 10 (12%) cases of BE, 15 (42%) cases of adenocarcinoma and 6 (21%) cases of the control group. Positive p16(INK4a) staining was not statistically different between the three groups whether positive or negative for HPV DNA (p=0.91 and p=0.91 respectively). Similarly, negative p16(INK4a) staining did not show a difference between the three groups for whether positive or negative for HPV DNA (p=0.50 and p=0.28, respectively). HPV was not detected by CISH in the adenocarcinomas while in BE and control groups, CISH was non-contributory., Conclusions: These data suggest that while HPV is detectable in a subset of esophageal lesions and tumors, the HPV detected is unlikely to be of etiologic significance or a factor accounting for the increase in BE and esophageal adenocarcinoma cases in the United States., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The demonstration of human papillomavirus (HPV) in 99.7% of cervical carcinoma surgical specimens from around the world required investigations by multiple alternative polymerase chain reaction (PCR) assays. A similar approach may therefore be necessary to best characterize HPV prevalence and genotype distribution among cervical cytology samples. In an earlier study, 752 of 799 (94.1%) abnormal and 82 of 300 (27.3%) normal cytology specimens tested HPV positive after PCR using GP5+/6+primers. This study has reinvestigated the "HPV negative" abnormal samples (20 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 14 atypical squamous cells, cannot exclude HSIL, 6 high-grade squamous intraepithelial lesion) and an age-matched cohort of "HPV negative" normal (negative for an intraepithelial lesion or malignancy) samples by PCR using PGMY09/11, FAP59/64, and LCR-E7 primers. PGMY09/11-GP5+/6+ nested PCR was performed on samples that were HPV negative by PGMY09/11 PCR. After the first 3 assays, HPV was detected in 41 of 45 (91.1%) abnormal and in 10 of 47 (21.3%) normal samples (P<0.0001). Eighteen HPV genotypes were detected and in some samples the genotype that was identified differed between the tests. The nondetection of common HPV genotypes (eg, HPVs 6, 11, 16, and 18) was notable. High-grade histopathology was found for 2 patients with HPV52-positive cytopathology. Combined with our earlier study, HPV (40 different genotypes) is shown in 99.5% of abnormal samples (99.8% inclusive of the nested PCR data). These findings show that HPV genotype and prevalence estimates are dependent on the method(s) of detection and indicate that suboptimal analytical sensitivity for one or more of the less common high-risk HPV genotypes could lead to impaired clinical sensitivity. HPV may be causal in almost every instance of abnormal cervical cytology; however, passenger HPV that is incidental to an abnormality may also have been detected.

This study investigated the role of human papillomavirus (HPV) in Schneiderian papillomas (SPs) to determine whether HPV is associated with the pathogenesis of particular histologic subtypes and whether p16(INK4a) can be used as a surrogate marker for HPV detection. Twenty-seven papilloma specimens (19 inverted [IPs], 6 exophytic [EPs], 1 oncocytic [OP] and 1 mixed) were collected from 23 patients. Purified SP DNA extracts were tested for HPV by PCR using GP5 +/GP6 + primers; HPV genotyping was performed by dot blot hybridization. PCR positive specimens were screened for HPV by biotinyl-tyramide-based chromogenic in situ hybridization (CISH). Immunohistochemsistry (IHC) for the HPV L1 capsid protein and for p16(INK4a) was performed on all specimens. HPV was detected by PCR in 16/27 (59.3%) SPs; 9/19 (47.4%) IPs; 6/6 (100%) EPs [p = 0.051], and 1/1 (100%) mixed SP. HPV was not detected in the single OP. High risk genotypes were detected in 4/9 IPs (44.4%) and 0/6 EPs (0%) [p = 0.10]. Seven of 16 PCR positive SPs were also CISH positive for HPV: 5/6 EPs (83.3%) and 1/9 IP (11.1%) [p = 0.01]. IHC for the L1 capsid protein was positive in 2 SPs (1 EP and 1 mixed). p16(INK4a) staining was seen in 14/16 (87.5%) PCR positive SPs and in 10/11 (90.9%) PCR negative SPs (p = 1.00). In summary, this study demonstrates a strong association between HPV and EPs, however, its role in IPs remains less well-defined. Further, p16(INK4a) is not a useful surrogate marker for HPV detection across the various SPs.

Aim: To determine if human papillomaviruses (HPVs) play a role in the histogenesis of adenosarcomas of the uterine cervix., Methods: Nine archival cases of primary cervical adenosarcoma were studied. The HPV status of the nine histologically proven tumours was investigated by non-isotopic in situ hybridisation (NISH) and PCR. NISH was performed using digoxigenin labelled probes to HPV types 6, 11, 16, 18, 31 and 33. PCR used GP5+/GP6+ primers to the HPV L1 gene., Results: Neither the benign epithelial components nor the malignant stromal components of the 9 neoplasms harboured nuclear NISH signals for the HPV types investigated. Amplimers of the HPV L1 gene were not detected by PCR in any of the tumours studied., Conclusion: HPVs do not appear to play an aetiological role in cervical adenosarcomas. This suggests that a different histogenetic pathway for this rare tumour type must exist.

Background and Aims: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities., Methods: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation., Results: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples., Conclusions: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.

Background: A survey of the distribution of human papillomavirus (HPV) types across the spectrum of cervical cytologic categories defined by the Bethesda 2001 guidelines was conducted with the objective of examining how HPV detection by polymerase chain reaction (PCR) analysis may benefit the management of patients who have abnormal Papanicolaou (Pap) test results., Methods: DNA samples from women with no intraepithelial lesion or malignancy (NLM) (n = 300 samples); atypical squamous cells of undetermined significance (ASC-US) (n = 200 samples); low-grade squamous intraepithelial lesion (LSIL) (n = 200 samples); atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion (ASC-H) (n = 200 samples); and high-grade squamous intraepithelial lesion (HSIL) (n = 200 samples) were tested for HPV using a modified general primer (GP)5+/GP6+ PCR assay and dot-blot hybridization with type-specific oligonucleotide probes (PCR assay analytical sensitivity: 1-100 copies of HPV, depending on the HPV type, in a background of 100 ng human DNA)., Results: HPV was detected in 27% of NLM samples, in 89.5% of ASC-US samples, in 97.5% of LSIL samples, in 93% of ASC-H samples, and in 96.5% of HSIL samples. Thirty-seven different HPV types were identified in total. One or more of 13 high-risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) were detected in 53% of samples that were diagnosed as ASC-US (59.0% of patients younger than age 30 yrs; 45.5% of patients age 30 yrs and older), in 55.5% of samples that were diagnosed as LSIL (60.0% of patients younger than age 30 yrs; 44.0% of patients age 30 yrs and older), in 80% of samples that were diagnosed as ASC-H, and in 87.5% of samples that were diagnosed as HSIL (P < 0.001). HPV-16 was detected in 17.5% of ASC-US samples, in 15.5% of LSIL samples, in 48.5% of ASC-H samples, and in 49.0% of HSIL samples (P < 0.001). Among abnormal smears, HR HPV was significantly more common in women younger than age 30 years compared with women age 30 years and older (P < 0.002). Follow-up biopsy data were obtained for 359 patients. A "benign" biopsy result was recorded for 47 of 64 women (73.5%) with ASC-US, 30 of 66 women (45.5%) with LSIL, 39 of 87 women (45.0%) with ASC-H, and 26 of 142 women (18.0%) with HSIL and was most common in women age 30 years and older (P < 0.0001). Cervical intraepithelial neoplasia (CIN) Grade I (CIN-I) was found in 14.0% of women with ASC-US, in 39.5% of women with LSIL, in 8.0% of women with ASC-H, and in 7.0% of women with HSIL. CIN-II was diagnosed in 9.5% of women with ASC-US, in 13.5% of women with LSIL, in 19.5% of women with ASC-H, and in 24.0% of women with HSIL. CIN-III was identified in 2 women (3.0%) with ASC-US, in 1 woman (1.5%) with LSIL, in 24 women (27.5%) with ASC-H, and in 71 women (50.0%) with HSIL., Conclusions: HR HPV testing by PCR of samples diagnosed according to the Bethesda 2001 guidelines may benefit the management of patients with ASC-US or patients with LSIL, especially among women age 30 years and older, by allowing exclusion from referral for biopsy of women who are negative for HR HPV types. However, the small numbers of women who had CIN-III detected after a diagnosis of ASC-US or LSIL limited the assessment of test sensitivity.

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16 immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16 immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P<0.001). p16 immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P<0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration. Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.

Human papillomaviruses (HPVs) are accepted as a necessary cause of cervical neoplasia. However, the benefits of testing simply for high-risk HPV types are limited because of their high prevalence in intraepithelial lesions of all grades, the majority of which regress if left untreated. One factor considered to be of key importance for the progression of intraepithelial lesions to invasive disease is integration of HPV into the host cell genome. Although questions remain about the prevalence of integration amongst pre-invasive lesions, sensitive in situ hybridization techniques utilizing tyramide reagents may aid determination of the significance of HPV infection by enabling routine detection of both high-risk HPV and its physical status. This will provide important data relevant not only to our understanding of the biology of HPV-associated neoplasia, but also potentially to clinical testing for HPV., (Copyright 2003 John Wiley & Sons, Ltd.)

Recent advances in the understanding of HPV-associated cervical squamous intraepithelial lesions, specifically with respect to HPV DNA integration into basal cervical epithelial cells, need to be incorporated into strategies for diagnosing and classifying these lesions. The biology and evolution of HPV-associated cervical squamous intraepithelial lesions is reviewed, along with recent developments using tyramide-based in situ hybridization and MIB-1 immunoreactivity. It is proposed that HPV DNA integration into the basal cells of cervical squamous epithelium precedes the transformation of low-into high-grade lesions. The HPV DNA tyramide-based in situ hybridization system may prove to be a powerful diagnostic/prognostic tool in this regard. It is also proposed that the presence of mitoses (especially atypical forms) in the upper layers may be a discriminatory hallmark in the morphologic distinction between low- and high-grade lesions. Further, since the biologic changes manifest between these two lesions are reflected in their respective phenotype, it appears plausible to adopt the Bethesda System two-tiered/binary classification of LGSIL and HGSIL for histopathologic diagnoses.

Purpose of Investigation: The aim of this study was to examine whether HPV testing specificity for cervical intraepithelial neoplasia (CIN) grades 2 or 3 could be improved by restricting the range of HPV types classified as 'high-risk'., Methods: DNA was extracted from 28 CIN I, nine CIN II and 13 CIN III formalin-fixed, paraffin-embedded biopsies. HPV type was determined by General Primer mediated 5+/6+ PCR assay., Results: The prevalence of specific HPV types among the different grades of CIN and the relationship to the referral smear diagnosis was examined. HPV type-16 alone was more highly associated with CIN grade (p < 0.0001; Specificity = 0.93; Sensitivity = 0.68) than was the group of HPV types collectively classed as high-risk (p = 0.025; Specificity = 0.23; Sensitivity = 1.00)., Conclusions: These data suggest HPV testing specificity could be improved simply by including a separate test for HPV-16. In conjunction with previous studies, the data also suggests redefinition of the high-risk HPV category to take into account the differing degrees of oncogenicity of high-risk HPV types.

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide-based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus. Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P =.025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P <.0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus-positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P <.0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas. Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide-based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.

Carcinosarcomas (malignant Müllerian mixed tumors [MMMTs]) of the uterine cervix are rare neoplasms. This report describes the morphology, immunohistochemical profiles, and human papillomavirus (HPV) status of eight cervical MMMTs. Patients' ages ranged from 32 to 93 years (mean, 61 years). Seven cases showed in situ squamous cell carcinoma (SCC). The invasive epithelial component (EC) was composed of combined adenoid basal carcinoma, basaloid SCC, and adenoid cystic carcinoma (ACC) in two cases. Keratinizing SCC, large cell nonkeratinizing SCC, undifferentiated carcinoma, and basaloid SCC predominated in the remaining tumors, one of which had admixed ACC. The sarcomatous component (SC) was homologous and spindled with admixed myxoid areas in three lesions. The ECs and SCs in six MMMTs showed dual immunostaining with epithelial membrane antigen and the pan-keratin marker, MNF116. The SC was vimentin-positive in seven cases. Five tumors had a vimentin-positive EC. The SC was positive for muscle specific actin and/or smooth muscle actin in seven lesions, of which four were desmin-positive. Polymerase chain reaction (PCR) using GP5+/GP6+ L1 consensus primers detected HPV DNA in all eight cases. Nonisotopic in situ hybridization with digoxigenin-labeled probes to HPV types 6, 11, 16, 18, 31 and 33 demonstrated integrated HPV 16 in three cases, not only in the EC, but also in nuclei of the SC. This is the first study to implicate HPV in the evolution of cervical MMMTs. The above observations lend support to a metaplastic theory of histogenesis.

Aim: To investigate the prevalence of human papillomavirus in transitional cell carcinoma of the urinary bladder in South Africa., Methods: Ninety-one archival samples of bladder transitional cell carcinoma were subjected to polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) for the detection of human papillomavirus 6, 11, 16, 18, 31, and 33 genotypes., Results: HPV was detected in only one case with PCR. HPV was not detected in any of the cases subjected to the NISH system., Conclusion: This study shows that although HPV has been shown to be associated with uterine cervical and esophageal squamous cell carcinomas in South Africa, this virus is not present in the transitional cell carcinoma of the urinary bladder in this geographical location. It is suggested that other factors, including nitrosamine exposure, p53 mutation, and additional unknown chromosomal events, may play a role in the carcinogenesis of this neoplasm in the bladder.

The aim of this study was to identify the role of bcl-2 protein expression in precancerous lesions of the cervix in patients from Johannesburg, South Africa and to correlate this expression with human papillomavirus (HPV) status. Archival cervical biopsy specimens (n = 107) of normal squamous epithelia (n = 18), pure HPV squamous epithelial lesions (n = 15), cervical intraepithelial neoplasia (CIN) I lesions (n = 17), CIN II lesions (n = 26), and CIN III lesions (n = 31) underwent bcl-2 immunohistochemical analysis with use of the streptavidin-biotin complex/horseradish peroxidase system and nonisotopic in situ hybridization for the detection of HPV DNA. Although 45 (61%) of the 74 CIN lesions demonstrated bcl-2 protein expression in the epithelia, most seemed to be in a patchy basal cell distribution, with a 1+ to 2+ intensity. Furthermore, comparison of bcl-2 immunoreaction between the low and high grades of the CIN lesions did not reveal significant differences. In addition, there was no apparent link between the presence of HPV DNA and bcl-2 expression in the CIN lesions. In contrast to previous studies that showed an increase in bcl-2 immunostaining intensity with increasing severity of CIN, only 4 (5.4%) of our 74 CIN specimens satisfied this pattern. Hence, we suggest that bcl-2 protein expression might not play a significant role in the majority of CIN lesions in this population group and that it might not correlate with HPV status.

Esophageal squamous cell carcinoma (ESCC) demonstrates wide regional variation in incidence and causal associations. Human papillomavirus (HPV) has been implicated in ESCC, particularly the sub-types 16 and 18. Transforming proteins E6 and E7 from these high risk sub-types, interact with p53 protein and Rb protein respectively, leading to loss of function of these tumor suppressor gene products. These interactions further lead to inactivation of the growth suppressive effects of the p53 and Rb proteins, resulting in abnormal proliferative states. p53 protein expression has been found in both HPV-positive and -negative tumors, indicating that HPV and p53 protein expression are not mutually exclusive and can occur together in the same tumor. It has been observed that HPV plays a more significant role in esophageal carcinogenesis in geographic areas with a high prevalence of the disease. A variation in the association between HPV and ESCC worldwide may be due to environmental and geographic factors, or to genetic susceptibility to esophageal HPV infections. Variations in the sensitivity of techniques used in the detection of the virus and in the methodology for processing the tumor tissues, may also be responsible for global differences. Esophageal carcinogenesis is a complex multistep process with a multifactorial etiology. Infection with oncogenic HPV types may be an integral part in a multistep process that leads to ESCC.

This study investigated the role of human papillomavirus (HPV) in adenoid basal carcinoma, a rare neoplasm of the uterine cervix. Nine archival paraffin-embedded tumors were analyzed with non-isotopic in situ hybridization (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin-labelled probes. The polymerase chain reaction (PCR) was performed on each of the cases using E6 consensus primers to HPV. A total of 67% of adenoid basal carcinomas harbored the HPV genome with NISH, of which 3 were PCR-positive. Integrated HPV 16 DNA was demonstrated in 4 of the 6 NISH positive cases. Two cases showed integrated HPV 33. HPV DNA was not detected in the three remaining cases. These results show that the integrated high-risk HPV, in particular type 16, is associated with this uncommon cervical tumor.

Aims: To determine the human papillomavirus DNA status of schistosomal associated squamous cell carcinoma of the urinary bladder in South Africa., Methods: Twenty five archival samples of bladder squamous cell carcinoma associated with Schistosoma haematobium were subjected to non-isotopic in situ hybridisation and the polymerase chain reaction for the detection of human papillomavirus 6, 11, 16, 18, 31, and 33 genotypes., Results: Using these two techniques, none of the 25 cases was shown to harbour human papillomavirus DNA., Conclusions: This study abrogates the role of human papillomavirus in schistosoma associated bladder carcinoma in South Africa. It is suggested that other factors including nitrosamine exposure, p53 mutation, and additional unknown chromosomal events play a major role in the development of this parasite associated neoplasm.

Aim: To investigate the role of human papillomavirus (HPV) in adenoid cystic carcinoma of the uterine cervix., Methods: Eleven archival, paraffin wax embedded specimens were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin labelled probes. The polymerase chain reaction (PCR) was carried out on each of the cases using consensus primers to HPV., Results: A total of eight adenoid cystic carcinomas harboured the HPV genome by NISH, of which five were PCR positive. Integrated HPV 16 DNA was demonstrated in seven of the eight NISH positive cases. One adenoid cystic carcinoma showed integrated HPV 31. HPV DNA was not detected in the three remaining cases., Conclusions: Integrated high risk HPV genome, in particular type 16, is associated with this uncommon type of primary cervical cancer.

Objective: To determine the human papillomavirus (HPV) subtypes in vulvovaginal warts in prepubescent children., Design: Histopathology case series., Setting: Outpatient and gynaecology clinics of hospitals in the greater Johannesburg area., Patients: All cases of vulvovaginal warts diagnosed in children under the age of 12 years received at the South African Institute for Medical Research, Johannesburg, during the period 1 January 1991 to 31 December 1993., Main Outcome Measures: Positivity for "genital' HPV types 6, 11, 16, 18, 31, 33 and 35 using non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR)., Results: Eight of the 9 vulvovaginal warts contained HPV 11 when assessed by means of NISH (89%). PCR amplified HPV DNA in all 9 (100%) of the biopsies., Conclusion: Detection of genital subtypes of HPV in childhood condylomata acuminata points strongly to sexual abuse, but should only be used as a guide to further investigation by a multidisciplinary team.

Using morphological criteria, the presence of human papillomavirus (HPV) in oesophageal carcinomas has been inferred in patients from Finland and South Africa. However, studies to demonstrate the viral antigen in tissue sections of these tumours have proved disappointing. This study investigates 48 archival oesophageal carcinoma biopsies from South Africa for the presence of HPV DNA using non-isotopic in situ hybridization (NISH) with HPV DNA probes to HPV 6, 11, 16, 18, 31, and 33. HPV DNA sequences were detected in 25/48 (52 per cent) oesophageal cancers. HPV 16 was present in 84 per cent of the HPV-positive cancers. A NISH type 2 signal pattern (punctate/dot) was present in all HPV-positive tumours. This signal pattern was previously shown to represent integrated HPV DNA within host chromosome. Integrated HPV DNA in oesophageal cancers has also been demonstrated in patients from China and Japan. In addition, the prevalence of HPV DNA in oesophageal cancers from high-risk countries like South Africa (52 per cent) and China (49 per cent) would appear to be consistent.

It has been proposed that wild-type p53 cell-regulating functions are annulled in human cervical carcinomas, either by mutations in the human papillomavirus (HPV)-negative cases or as a consequence of their complexing with HPV E6. The aim of this study was to test this hypothesis on 39 fresh cervical biopsies by p53 immunocytochemistry (ICC) with antibody PAb 240 and with NISH (non-isotopic in situ hybridization) and PCR (polymerase chain reaction) for HPV detection. p53 protein was present in the basal layer of pure wart virus infection; the basal to middle third of CIN (cervical intraepithelial neoplasia); in 19/22 (86 per cent) HPV-positive cervical carcinomas, ten of which contained integrated HPV; and in 4/8 (50 per cent) HPV-negative cervical carcinomas. Dual detection of p53 antigen and HPV 16 DNA in the same sections demonstrated either p53 protein or integrated HPV 16 alone in the majority of cells. Co-localization of both signals was only evident in isolated cells. These data suggest that PAb 240 immunoreactivity is not mutant-specific. They are, however, consistent with the conformation hypothesis which proposes that wild-type p53 changes from a suppressor (PAb 240-negative) to a promoter (PAb 240-positive) form during cell growth response. Hence, according to this hypothesis, p53 protein expression may represent either the wild-type promoter form or mutant p53 protein, both of which share the same conformation. This may explain co-localization of p53 and HPV in some tumours. However, the absence of p53 protein in 50 per cent HPV-negative squamous cell carcinomas suggests that not all HPV-negative tumours accumulate p53 protein.

The presence of human papillomavirus (HPV) in cervical cells is closely related to the development of cervical carcinoma. Detection of virus may be by Southern blot, dot blot or the highly sensitive polymerase chain reaction. Whatever method is employed, there are problems of false negatives due to poor clinical samples in which the DNA may be degraded or is absent altogether. Here we describe a new method of dual labelling for dot blots using a 32P-labelled probe for HPV and a 35S-labelled probe for human actin genes. The samples were counted on a Beta-plate flat-bed scintillation counter and the data analysed to separate the activities of the two isotopes. The counts from the actin probe show whether human DNA is present or not and false negatives from this cause may thereby be eliminated. The counts due to HPV when compared with those for actin give a quantitative measure of HPV abundance for the particular sample and this may have clinical relevance.

Aims: To determine which type of human papillomavirus (HPV) is associated with cervical adenocarcinoma and whether the virus was integrated or episomal in two continents., Methods: Biopsy specimens from the UK (n = 16) and South Africa (n = 22) were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, 33, and 35 on archival biopsy specimens using digoxigenin labelled probes., Results: A total of 20 adenocarcinomas (53%) from both groups contained HPV DNA. In the UK group, seven and four cases contained HPV 18 (44%) and 16 (25%) respectively. In the South African group, nine cases contained HPV 18 (41%) while HPV DNA was not detectable in the other 13 cases. Hence HPV 18 was present in 80% of HPV positive adenocarcinomas., Conclusions: The HPV 16 or 18 genome was integrated in all viral positive cases. In two cases HPV 18 was also present in an episomal form. These data indicate that HPV integration is common to cervical adenocarcinoma in two continents by the same methodology. The lower prevalence of HPV 18 detection in the South African group may have been due to the presence of other or unsequenced HPV types.

Aims: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis., Methods: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear., Results: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration., Conclusions: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.

In South Africa asymptomatic wart virus infection diagnosed by morphological criteria occurs in 16-20% of all ethnic groups; the incidence in black women is 66%. To identify human papillomavirus (HPV) types the prevalence of HPV in cervical intraepithelial neoplasia (CIN) in South African women (n = 72) with age matched British women (n = 73) was compared by non-isotopic in situ hybridisation (NISH) using digoxigenin labelled probes for HPV 6, 11, 16, 18, 31, 33 and 35 on archival biopsy specimens. A higher proportion of British biopsy specimens (68%) contained HPV than those from South Africa (50%) in CIN 2 and 3; this difference was due to HPV 16. Thirty six per cent of the positive biopsy specimens from South African women also contained HPV 33/35 compared with 16% in the United Kingdom. There was no difference in HPV detection with age in either group. These data indicate that HPV types vary geographically, with "minor" HPV types being more common in South Africa. Three qualitatively distinct NISH signals were observed; a diffuse (type 1) signal in superficial cells, mainly koilocytes; a punctate signal (type 2) in basal/"undifferentiated" cells in CIN 3; and combined type 1 and 2 signals in CIN with wart virus infection (type 3). The punctate signal may represent HPV integration.

In a previous study three types of HPV signal were described in CIN. It was suggested that a type 1 signal represented episomal HPV while a type 2 signal represented integrated HPV; and a type 3 signal was indicative of both episomal and integrated HPV. To test this hypothesis 91 squamous cell cancers (SCC) of the cervix from Britain and South Africa were examined for HPV 6, 11, 16, 18, 31, 33, 35. Of the South African group (n = 69) 64% contained HPV types 16 (n = 29) and 18 (n = 15). The SCC in the British group (n = 22) contained HPV 16 and HPV 33 in 12 and three cases, respectively. Of the HPV positive biopsy specimens, 86% showed a type 2 signal in keratinising and non-keratinising tumours and the remainder a type 3 signal. Type 3 signal was present only in keratinising tumours. The presence of punctate signal in 100% of HPV containing SCC, together with localisation of HPV signal to sister chromatids in tumour cell mitotic figures in vivo, provides further evidence for type 2, and the punctate component of type 3 signal representing viral integration.