Association of human papillomavirus type 16 with neoplastic lesions of the vulva and other genital sites by in situ hybridization.

The authors examined paraffin sections from 85 genital tract tissues from 49 cases for the presence of human papillomavirus (HPV) Types 6/11, 16, and 18 by stringent in situ hybridization using 35S-labeled viral DNA probes, and for viral capsid antigen by the immunoperoxidase test. The cases, selected mostly on the basis of vulvar pathology, were distributed as follows: early neoplasia (Group I, 6 cases); early neoplasia with viral cytopathic effect (CE) (Group II, 24 cases); and papillomavirus infection (PVI) (Group III, 19 cases). Available tissues from all affected sites were examined when the disease was multicentric. One or more viral DNAs were identified in 58% of 77 tissues from Groups II and III and in 2 of 8 tissues from Group I. HPV-6/11, HPV-16 and HPV-18 DNAs were detected, respectively, in 25, 24, and 2 tissues; 3 tissues were infected simultaneously with either two or three viruses. Viral DNA was identified at more than one site in 14 of 30 DNA-positive patients; in 10 of these, a single type was detected at all sites in the same patient. The viral DNA was localized mostly in areas showing viral cytopathology. The presence of HPV-16 correlated with neoplasia. HPV-16 DNA was identified in the 2 virus-positive tissues showing neoplasia, in 17 of 20 (85%) of the DNA-positive tissues showing neoplasia with CE, and in 5 of 25 (20%) of the DNA-positive tissues showing PVI. Conversely, HPV-6/11 was found in 25% of the DNA-positive tissues showing neoplasia with CE and in 80% of the cases of PVI. An HPV genome was identified in neoplastic cells in 14 instances; in all but 1 case, the genome was HPV-16. The association of HPV-16 with neoplasia was seen for both vulvar and cervical lesions. Viral antigen was detected in 83% of lesions associated with HPV 6/11 and in 62% of lesions associated with HPV-16.     

Detection of human papillomavirus DNA in anogenital exophytic lesions by in situ hybridization to paraffin sections

In situ hybridization was used to detect human papillomavirus (HPV) nucleic acids (type 6b, 11, 16, 18, 31, and 33) and immunohistochemistry was done to detect papillomavirus common antigen in paraffin sections of biopsy specimens. Two patients suffering from condyloma acuminatum contained HPV-6b and HPV-11. Both cases showed small foci of the antigen-positive cells. One patient having condyloma acuminatum with dysplastic features contained a small quantity of HPV-16 without any antigen-positive cells. One case of verrucous carcinoma showed neither HPV-DNA nor antigen. In situ hybridization is a powerful tool in the analysis of the pathogenesis of HPV-associated neoplasms.     

DETECTION OF HUMAN PAPILLOMAVIRUS DNA IN ANOGENITAL EXOPHYTIC LESIONS BY IN SITU HYBRIDIZATION TO PARAFFIN SECTIONS

In situ hybridization was used to detect human papillomavirus (HPV) nucleic acids (type 6b, 11, 16,18,31, and 33) and immunohistochemistry was done to detect papillomavirus common antigen in paraffin sections of biopsy specimens. Two patients suffering from condyloma acuminatum contained HPV-6b and HPV-11. Both cases showed small foci of the antigen-positive cells. One patient having condyloma acuminatum with dysplastic features contained a small quantity of HPV-16 without any antigen-positive cells. One case of verrucous carcinoma showed neither HPV-DNA nor antigen. In situ hybridization is a powerful tool in the analysis of the pathogenesis of HPV-associated neoplasms. ACTA PATHOL JPN 38: 1131∼1139, 1988.     

Presence and type of oncogenic human papillomavirus in classic and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma

The presence and type of oncogenic papillomavirus (HPV) in classic warty/basaloid vulvar intraepithelial neoplasia and in differentiated vulvar intraepithelial neoplasia and keratinizing vulvar squamous cell carcinoma was investigated using three techniques, that is, histology, in situ hybridization, and PCR-ELISA. HPV typing was performed using in situ hybridization and PCR-ELISA. Differentiated vulvar intraepithelial neoplasia and invasive keratinizing vulvar squamous cell carcinoma proved completely negative for HPV by PCR-ELISA, in situ hybridization, and histologic examination, while in classic vulvar intraepithelial neoplasia, a HPV positivity of 66.1% was found. HPV 16 was the predominant type, with HPV 35, 33, and 52 types found rarely and sometimes together with HPV 16. PCR-ELISA proved to be the most suitable method to detect and type mucosal oncogenic HPVs. The absolute absence of HPV DNA in differentiated vulvar intraepithelial neoplasias and in keratinizing vulvar squamous cell carcinoma suggests a strong HPV-independent pathway of malignant progression to invasive carcinoma.     

Human papillomavirus (HPV) genotyping using paired exfoliated cervicovaginal cells and paraffin-embedded tissues to highlight difficulties in attributing HPV types to specific lesions

Defining type-specific human papillomavirus (HPV) infections within cervical tissues is important for understanding the pathogenesis of cervical neoplasia and assessing the effectiveness of prophylactic vaccines with limited type-specific spectra. We compared HPV DNA-testing results from 146 matched exfoliated-cell and formalin-fixed-tissue specimens collected by cervicovaginal lavage (CVL) within 90 days of each other from women with histologically confirmed cervical intraepithelial lesions (CIN). The CVL specimens were HPV typed using a MY09/11 L1 consensus primer PCR method followed by dot blot hybridization. The tissue specimens were HPV typed using an SPF(10) line probe assay HPV detection system. Of the 146 specimen pairs with evidence of CIN in the tissue, 91.8% were positive for one or more HPV types in both the tissue and cellular specimens. Tissue sections were more likely to be HPV negative (P < 0.01). Typing directly from tissue sections resolved multiple infections detected in exfoliated cells to a single HPV type in only 46.9% of cases. Combined use of both specimen types to attribute lesions to HPV type 16 (HPV-16) and/or -18 led to 43.1% attributed to HPV-16 and/or -18 by both specimen types and 19.9% attributed to HPV-16 and/or -18 by one, but not both, specimen types. Unambiguous attribution of cervical lesions to a single, specific HPV type remains a difficult proposition. Use of multiple specimen types or the development of highly sensitive and robust in situ hybridization HPV-testing methods to evaluate the certainty of attribution of lesions to HPV types might provide insights in future efforts, including HPV vaccine trials.     

Cervical intraepithelial neoplasia 3, coinfected with HPV-16 and -18--case report.

Recently, detection of human papillomavirus (HPV)mRNA expression was made possible by in situ hybridization. We described a patient with cervical intraepithelial neoplasia (CIN) 3, showing a distinctive and rare form of co-infection with HPV type 16 and 18. HPV-16 was detected in high grade squamous intraepithelial neoplastic lesion (CIN 3) and HPV-18 was in low grade lesion just adjacent to the HPV-16 infected area. This case suggests that HPV infection may be one of the most responsible causative agents producing malignant transformation and two distinctive HPV types can also simultaneously infect the squamous epithelium of the uterine cervix.     

Detection of human papillomavirus DNA in genital lesions by using a modified commercially available in situ hybridization assay.

A modified, commercially available DNA-DNA in situ hybridization test that uses biotinylated probes for the identification of human papillomavirus (HPV) DNA types 6/11, 16/18, and 31/33/35 was evaluated. HPV DNA was detected in 314 of 787 (40%) histologically abnormal genital biopsy specimens by using the ViraType in situ assay (Life Technologies, Gaithersburg, Md.), in which the hybridization time was increased from 2 to 16 h. Ninety percent of positive condyloma acuminata specimens contained HPV type 6/11 DNA. The prevalences of HPV DNA for cervical intraepithelial neoplasia I, II, and III lesions by this in situ hybridization test were 42, 54, and 55%, respectively. The combined prevalence of HPV type 16/18 and 31/33/35 DNAs increased with the severity of the lesion, while the prevalence of type 6/11 DNA decreased. HPV type 6/11 DNA was found only in 1 of 16 (6%) positive cervical intraepithelial neoplasia III specimens. HPV type 16/18 and 31/33/35 DNA was detected in 11 of 16 (69%) and 4 of 16 (25%) in situ hybridization-positive cervical intraepithelial neoplasia III specimens, respectively. Thus, the observation that certain "higher-risk" HPV genotypes are associated with upper-grade cervical precancer lesions was confirmed by this commercial hybridization system. In general, the assay was found to be well suited for use in the clinical laboratory. The ViraType in situ procedure modified for a longer hybridization time may be helpful in identifying lesions containing higher-risk HPV strains.     

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia.

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.     

Genital human papillomavirus genotypes in northwestern Tanzania

Using MY09-MY11 PCR and human papillomavirus (HPV) typing by reverse blot hybridization, we found a 34% cervical HPV prevalence among 561 pregnant women in Tanzania. One hundred three of 123 women (84%) with typeable samples harbored high-risk oncogenic strains. HPV type 16 (HPV-16) was the most prevalent subtype (18%) among HPV-infected women and among women with cervical neoplasia (3 of 19). A multivalent vaccine for HPV-16, -18, -31, -33, and -35 would be necessary to prevent 50% of the neoplasia in this population.     

Detection of human papillomavirus genotypes with liquid bead microarray in cervical lesions of northern Chinese patients

Human papillomavirus (HPV) is the main cause of cervical cancer. Blending multiplex polymerase chain reaction (PCR) amplification and multiplex hybridization to liquid bead microarray (LBMA), we detected and identified 25 common HPV genotypes using type-specific primers for HPV E6 and E7 genes in cervical lesions of northern Chinese patients. Of the 511 cervical samples, 349 (68.3%) were found to be HPV positive by HPV-LBMA. The distribution was 22 HPV positive of 100 in the control group (22%), 41 of 80 with chronic cervicitis (51%), 80 of 99 with cervical intraepithelial neoplasia (CIN) I (81%), 46 of 56 with CIN II (82%), 67 of 74 with CIN III (90%), and 93 of 102 with invasive cervical carcinoma (91%). HPV-16 was the most frequent genotype in the CIN and cervical cancer groups. The most common genotypes were HPV-16 (28%), HPV-58 (14%), HPV-52 (14%), HPV-18 (8%), and HPV-33 (7%) in the CIN group, and HPV-16 (63%), HPV-52 (9%), HPV-18 (7%), HPV-58 (7%), and HPV-33 (5%) in the cervical cancer group. HPV-LBMA found multiple genotypes in 1 of 22 control (4%), 64 of 193 CIN (33%), and 22 of 93 cervical cancer (24%). The HPV-LBMA results were compatible with those of PCR and DNA sequencing. HPV-LBMA is a simple, high-throughput method that provides useful information on viral genotype and multiple HPV infections in cervical lesions. In northern China, the most common high-risk HPV genotypes seem to be HPV types 16, 58, 52, 18, and 33. Genetic information on HPV in cervical specimens could provide particular benefits in the management of cervical lesions.     

Vulvar carcinomas: search for sequences homologous to human papillomavirus and herpes simplex virus DNA

Ten cases of intraepithelial carcinoma, five with Bowenoid features and five with early invasion, and ten cases of invasive vulvar carcinoma were examined by in situ hybridization and Southern blot analysis using DNA probes for human papillomavirus (HPV) types 6, 11, 16, 18 and 31. HPV DNA was detected in 90% of the intraepithelial cases and in 10% of the invasive cases. All positive cases showed the presence of DNA of HPV type 16. The cases with intraepithelial lesions revealed a strong correlation between the presence of HPV type 16 DNA, cigarette smoking habit, other potential cofactors such as herpes simplex (HSV) DNA sequences and the use of contraceptive drugs, and clinicopathologic features of Bowen's type in situ squamous cell carcinoma. Similar associations were not observed among the cases with invasive disease. While HPV-16 is associated with differentiated Bowenoid type vulvar intraepithelial neoplasia, which appears to be the most common form of early carcinoma of the vulva, the same association was not seen with respect to advanced vulvar invasive squamous cell carcinoma.     

Detection of high-risk human papillomavirus subtypes in cervical glandular neoplasia by in situ hybridization

In situ hybridization (ISH) was performed on paraffin-embedded tissues to detect multiple high-risk human papillomavirus (HPV) subtypes in 27 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma (CA) specimens. These results were compared with those of HPV detection by HPV-PCR genotyping and p16 immunohistochemistry in the same specimens. Of the 27 cases, 17 (63%) showed HPV-DNA by HPV-ISH, including 3 metastatic lesions. HPV-DNA was detected in 18 cases (67%) by PCR. The concordance rate between HPV-ISH and HPV-PCR genotyping was 74% with moderate agreement (Kappa value, 0.41). HPV-16 was identified in 5 cases, HPV-18 in 2 cases, and HPV-45 in 1 case. Combining the results of HPV-ISH and HPV-PCR/genotyping, 22 cases (81.5%) were considered HPV positive. Immunohistochemical staining of p16 indicated that 25 (93%) cases were positive; however, 4 of these cases were HPV-negative by both PCR and ISH. Combining HPV-ISH and HPV-PCR/genotyping techniques demonstrated a high sensitivity of HPV detection in FFPE tissues from cervical glandular neoplasias. In contrast, p16 immunohistochemistry seemed to have a low specificity for determining HPV status in cervical glandular neoplasia. HPV-ISH is useful for recognizing the distribution of HPV in AIS and CA tissues and visualizing signal patterns, and may be a useful tool to confirm the cervical origin of neoplasias and metastatic lesions.     

Identification of genital tract papillomaviruses HPV-6 and HPV-16 in warts of the oral cavity

Warty lesions of the oral cavity were examined for etiologic association with genital tract papillomaviruses HPV-6, HPV-11, and HPV-16. DNAs extracted from ten oral biopsies were screened for HPV genomic sequences by Southern transfer hybridization with 32P-labeled viral DNA probes. Nonstringent hybridization with an HPV-6 probe revealed papillomavirus DNA sequences in four of seven tissues with histologic evidence of papillomatosis, in none of two tissues without histologic evidence of papillomatosis, and in one tissue that was not examined by histology. Stringent hybridization tests with HPV-6 and HPV-16 probes identified the genome in one tissue as being HPV-16, in a second tissue as being HPV-6 subtype a, and in a third tissue as HPV-6 (subtype unidentified); papillomavirus DNA sequences in two tissues are as yet not identified. An additional case of HPV-6 or HPV-11 related oral cavity lesion was diagnosed by in situ hybridization of paraffin sections with a 35S-labeled, mixed HPV-6 + HPV-11 probe. The hybridization in the positive section was extensive and confined to epithelial nuclei. The oral lesions associated with genital tract papillomaviruses were asymptomatic, multiple or single, and were located in different parts of the oral cavity, for example, on the gingivae, on the tongue, on the lip, on the tonsillar pillar, and on the floor of the mouth.     

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.     

Association between HLA-DRB1 polymorphism, high-risk HPV infection and cervical neoplasia in southern Chinese

Multiple determinants are involved in the progression of human papillomavirus (HPV)-infected cervical lesion to invasive cancer. Human leukocyte antigen (HLA) polymorphism seems to play a role. This study examined the association between HLA-DRB1 polymorphism, high-risk HPV infection, and the development of cervical neoplasia in southern Chinese. Three hundred and seventy women with cervical neoplasia (43 cervical intraepithelial neoplasia grade II, 154 grade III, and 173 invasive cancers) and 323 controls were recruited for HLA-DRB1 typing by a sequence-based approach. Cervical specimens were collected for HPV detection by a consensus primer-based polymerase chain reaction, and with the type of HPV identified by hybridization with type-specific oligonucleotide probes. A protective effect of HLA-DRB1*12 for cervical neoplasia was observed, and with stronger associations when subgroup analyses were carried out for patients infected with HPV16 and HPV58. The protective effect of HLA-DRB1*13 that had been reported from other populations was not observed. The data obtained in this study showed that HLA-DRB1*03 conferred a higher risk for HPV18-infected, but not for HPV16-, HPV52-, or HPV58-infected cervical lesions. Although, HPV52 was reported as uncommon worldwide, it was found to be the second most prevalent type in the southern Chinese population. However, no additional risk association was observed when subgroup analyses were performed for HPV52-infected patients. The current study shows that, among southern Chinese, the outcome of HPV-infected cervical lesions is associated with HLA-DRB1 polymorphism. These associations often vary with the type of HPV infection.     

Human papillomavirus infection of morphologically normal cervical epithelium adjacent to squamous dysplasia and invasive carcinoma

We have begun a systematic study of human papillomavirus (HPV) infection in colposcopically and/or morphologically normal epithelium of the uterine cervix. Paired biopsies were taken from the lesions (cervical intraepithelial neoplasia [CIN], condyloma, invasive carcinoma) and from the normal-appearing adjacent epithelium 3 to 5 mm from the edge of the lesion. Myometrium or ectocervical epithelium from patients who had undergone hysterectomy for reasons other than genital dysplasia or malignancy served as controls. One biopsy was examined histologically. DNA from the second biopsy was digested with Pst I, and the presence or absence of HPV was determined by Southern blotting using HPV-16 DNA as a probe. HPV was not detected in any of the 12 control samples. Of 30 patients with CIN and/or condyloma, five of 18 who were HPV-positive had either HPV-16 (three cases) or virus resembling HPV-31 (two cases) in the lesion and adjacent epithelium. Of seven patients with invasive carcinoma, four had HPV in the lesion and adjacent epithelium; two of these four patients had typical HPV-16. Such infection of apparently normal epithelium has major implications for our understanding of the pathogenesis, treatment, and follow-up of patients with cervical neoplasia.     

Human papillomavirus type 16 DNA is integrated into chromosome region 12q14-q15 in a cell line derived from a vulvar intraepithelial neoplasia

The SK-v cell line, established from a precancerous lesion (a vulvar intraepithelial neoplasia), contains 10 to 20 copies of the human papillomavirus type 16 (HPV16) genome, and was previously shown to derive from a clone of cells present in the patient's lesions. By in situ hybridization the integrated HPV16 DNA sequences were localized to a single site in chromosome region 12q14-q15. The localization of viral sequences to a single nonrearranged chromosome 12 suggests that integration occurred at this site in the patient's premalignant lesions. The INT1 and GLI protooncogenes are located in this chromosomal region. No detectable modification of the structure and expression of these genes was observed by blot hybridization experiments.     

Human papillomavirus genotype spectrum in Czech women: correlation of HPV DNA presence with antibodies against HPV-16, 18, and 33 virus-like particles.

Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV-genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus-primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot-blot hybridisation with type-specific oligonucleotide probes and thermocycle DNA sequencing. Twenty-two different HPV types were detected, HPV-16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV-DNA-positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low-grade lesions, 58.0% in high-grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV-specific IgG antibodies was tested by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HPV-16, -18, and -33. Antibodies were significantly more prevalent in HPV-DNA-positive than in HPV-DNA-negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV-16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV-16 or HPV-33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV-18-DNA-positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV-specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV-16, -18, and -33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age.     

Clinical and histologic features of vulvar carcinomas analyzed for human papillomavirus status: evidence that squamous cell carcinoma of the vulva has more than one etiology.

The association between the human papillomavirus (HPV) and malignant neoplasms of the uterine cervix is well established; however, its role in the pathogenesis of vulvar cancer has not been well defined. This study correlates the clinical and histopathologic features of 21 invasive carcinomas of the vulva with the presence of HPV DNA as detected by Southern blot and polymerase chain reaction (PCR) analysis. By one or both techniques, HPV DNA was detected in 10 of the 21 tumors analyzed; all HPVs containing tumors hybridized with HPV-16 probes, although PCR also detected HPV-6 in two of the HPV-16-containing tumors. No HPV-18 DNA was detected in any tumor by PCR or Southern blot hybridization. Both the invasive cancer and the surrounding intraepithelial disease tended to display histopathologic features that usually could distinguish HPV-associated cancers from those without HPV DNA. The intraepithelial lesions associated with HPV-containing tumors were of the bowenoid type with koilocytosis, while tumors lacking HPV generally demonstrated a simplex type of intraepithelial lesion. Invasive tumors with no viral DNA were more frequently keratinizing than the HPV-containing cancers. Race, parity, hormonal therapy, and alcohol use did not affect the HPV status; however, HPV DNA was more prevalent in the tumors of younger women and in those with a history of tobacco use. Human papillomavirus status had no impact on the stage of disease or its prognosis. These findings identify two subsets of vulvar carcinoma cases based on HPV hybridization data and the histopathologic characteristics of the tumor.     

High frequency of human papillomavirus 6/11, 16, and 18 infections in precancerous lesions and squamous cell carcinoma of the conjunctiva in subtropical Tanzania

Dysplastic lesions and epithelial neoplasms of the conjunctiva account for approximately 2% of all malignant tumors in subtropical Tanzania. We examined the pathophysiologic role of human papillomavirus (HPV) in the development of conjunctival carcinoma in subtropical Tanzania, which has a high HPV prevalence. Tissue samples from 14 patients were obtained from the cancer registry archives at the medical center of the university in Dar es Salaam, Tanzania. A highly sensitive nonradioactive in situ hybridization technique (ImmunoMax) was applied to paraffin-embedded tissue samples to identify HPV DNA in conjunctival epithelial dysplasia and epithelial neoplasms. In each case, conventional morphologic evaluation revealed a transitional lesion extending from koilocytic dysplasia to severe dysplasia or invasive squamous cell carcinoma. Highly specific, morphologically easily distinguishable labeling of HPV-6/11, HPV-16, and HPV-18 was found in most cases. Coinfections were observed frequently. The signals showed varying intensities and different patterns of distribution. In general, higher signal intensity was found in dysplasia grades 1 and 2 and in well-differentiated areas of the invasive component of conjunctival carcinoma compared with less differentiated areas. This observation underlines the central role of HPV-16 and HPV-18 in the oncogenesis of conjunctival cancers in subtropical Tanzania.