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.     

Dermatologic manifestations of HPV in HIV-infected individuals

Dermatologic human papillomavirus (HPV) infection in HIV patients manifests as both anogenital and nongenital skin disease. Anogenital HPV-related disease includes benign condyloma acuminata, the most common cutaneous manifestation of genital HPV infection; intermediate malignancy or premalignant conditions including giant condyloma acuminata (also called Buschke-Loewenstein tumor), anal intraepithelial neoplasia, penile intraepithelial neoplasia, and vaginal or vulvar intraepithelial neoplasia; and frankly malignant disease including Bowen’s disease and invasive anal, penile, or vulvar carcinoma. Cutaneous HPV-related disease in nongenital skin is also increased in HIV-positive patients, in the form of benign common warts, epidermodysplasia verruciformis-like skin lesions, and nonmelanoma skin cancers. This review and update addresses the above listed dermatologic manifestations of HPV disease in HIV-infected individuals, with an emphasis on new findings and published data from 2006 to 2008.     

High prevalence of high-risk oncogenic human papillomaviruses harboring atypical distribution in women of childbearing age living in Libreville, Gabon

The extent of human papillomavirus (HPV) genital shedding and type-specific diversity were evaluated in 354 consecutive women of childbearing age living in Libreville, Gabon. Detection of HPV DNA was performed by PCR using the MY09/MY11 primer set on DNA extracted from endocervical swabs. All PCR positive specimens were subjected to direct sequencing and HPV genotypes were identified on the basis of >95% sequence homology in the L1 region. Reverse line blot hybridization assay was used when a genotype could not be resolved by sequencing alone. HPV DNA was detected in 163 (46%) women, all clinically asymptomatic for HPV-related lesions. The highest prevalence of genital HPV detection (45%) was in the age group from 22 to 29 years. A total of 90 women (55%) harbored high-risk (HR) genotypes, with the most common being HPV-53 (19; 12%), HPV-58 (17; 11%), and HPV-16 (16; 10%). Low-risk genotypes were found in 36 (22%) women with HPV-54 and HPV-70 being the most frequently detected (17; 11% and 10; 6%, respectively). Finally 37 women (23%) tested positive for genotypes of unknown oncogenic risk, the most common in this category being HPV-83 (20; 12%). Multiple infections were detected in 35 (21%) women. By multivariate analysis, HPV genital shedding was significantly associated with young age (OR: 0.34; P < 0.007). The multivalent vaccine currently available against cervical carcinomas, is only active against HPV-16 and HPV-18, and will thus have a low impact in this setting.     

Prediction of 'high-risk' cervical papillomavirus infection by biopsy morphology

Certain human papillomavirus (HPV) types (such as type 16) have been linked to high-grade precancers and invasive carcinomas of the cervix. However, the accuracy with which morphologic characteristics will predict the presence and type of HPV infection is controversial. Three pathologists independently classified 102 consecutive cervical biopsies with the use of specific criteria and correlated their findings with the presence of HPV 11, 16, and 18 RNA sequences by in situ hybridization. Based on the presence and distribution of nuclear atypia, abnormal mitotic figures, and koilocytosis, biopsies were classified into borderline condyloma, condyloma, borderline cervical intraepithelial neoplasia (CIN) with koilocytotic atypia (CINK), and CIN. Two or more observers agreed on the diagnosis in 96% of cases. HPV 16-related sequences alone were detected in 0% of borderline condylomata, 17% of flat condylomata, 43% of borderline CINK, 67% of CINK, and 77% of CIN lesions. Other HPVs, including those producing signals with more than one probe, were present in 0, 50, 14, 9, and 0% of these lesions, respectively. The authors data suggest that consistent identification of HPV-related cervical disease requires the presence of specific cytologic changes. In the authors' series, when HPV-related disease is present, CIN is the most common lesion and most (71%) contain HPV 16-related nucleic acids. Thus, a high proportion (88%) of histologic abnormalities associated with HPV-16 could be distinguished as CIN by morphologic characteristics alone, and this distinction could be made by most observers.     

Detection of capsid antigen of human papillomavirus (HPV) in benign lesions of female genital tract using anti-HPV monoclonal antibody.

We established a murine monoclonal antibody (K1H8) to human papillomavirus (HPV) using alkaline-disrupted virions of HPV type 1 (HPV-1) as the immunogen. K1H8 recognized a 57 kD capsid protein of HPV-1 and detected the antigen in paraffin sections of formalin-fixed tissue. With K1H8, we examined immunohistochemically 68 biopsy specimens obtained from the female genital tract. The specimens were histologically condyloma acuminatum or koilocytotic lesions with or without dysplasia and each specimen was found to harbour a single type of genital HPV, such as types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58, by Southern blot hybridization analysis. The antigen was localized in the nuclei and occasionally in the cytoplasm of squamous cells showing koilocytotic changes. Eighty-four per cent of the specimens (57 cases) showed positivity for the antigen, indicating that K1H8 is a broadly-reactive antibody to various genital HPVs. The results suggest that benign mucosal lesions of the female genital tract are more frequently associated with viral production and are a potential source of transmission.     

Detection and quantitation of human papillomavirus DNA in peripheral blood mononuclear cells from blood donors

Human papillomavirus (HPV) is the etiological agent of cervical cancer. Also, HPV has been associated with anogenital cancer, oropharyngeal cancer, genital warts, and other dermatological diseases. HPV infects epithelial cells and their replication is closely linked to epithelial differentiation. The presence of HPV DNA in peripheral blood mononuclear cells (PBMC) has been reported in some patients with head and neck cancer, cervical cancer, and other genital diseases. However, the presence of HPV DNA in blood in asymptomatic subjects is still unresolved. The objective of this study was to evaluate the presence of HPV DNA in PBMC from asymptomatic blood donors. Blood samples were collected from 207 healthy Chilean blood donors. Genomic DNA was extracted from PBMC and HPV DNA detection was performed by real-time quantitative polymerase chain reaction assays with GP5+/6+ primers. HPV typing was carried out by genetic sequencing of a 140 to 150 bp fragment of the L1 gene. HPV DNA was detected in 6.8% (14/207) of blood donors. Single HPV infections were detected in seven blood donors. High-risk HPV was found in 6.3% (13/207) of cases: nine blood donors were infected with HPV-16, five with HPV-18, two with HPV-51, and one case was infected with either 32, 33, 45, 59, 66, 70, or 82. The median viral load value was 21.3 copies/mL blood or 13.4 HPV (+) cells per 10 ⁴ PBMC. These results show that HPV DNA is present in PBMC from healthy blood donors and it suggests that blood could be a new route of HPV dissemination.     

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA

AIMS: To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions. METHODS: One hundred and seventy six cytological specimens from women with different cervical lesions were investigated. For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Semiquantitative determinations were achieved in relation to an external reference titration curve. RESULTS: HPV DNA was detected in 60.2% of the samples. HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45. HPV-11 was not detected. HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease. Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease. CONCLUSIONS: Only HPV-16 load appears to be associated with the severity of cervical disease.     

Low False-Negative Rate of PCR Analysis for Detecting Human Papillomavirus-Related Cervical Lesions

Although PCR analysis is a sensitive test for detection of human papillomavirus (HPV) in the cervix, the proportion of cases of cervical dysplasia missed, or the false-negative rate, has been unknown. We determined the accuracy of PCR analysis for HPV DNA as a predictor of HPV-related cervical lesions in a cross-sectional study of sexually active women, aged 18 to 50 years, from the University of Michigan Family Medicine HPV study. Of 133 eligible participants, 41 underwent colposcopy because of a positive result for HPV of the cervix by the PCR method and 92 underwent screening colposcopy with biopsy prior to knowing the HPV PCR results. Twenty-four of those screened were subsequently found to also be HPV DNA positive. In those found to be HPV positive, histological studies revealed the presence of condyloma or cervical intraepithelial neoplasia in 16 women (24.6%) and changes suggestive of condyloma in 5 (7.6%). No HPV-negative woman had an abnormal biopsy or cytology report (P = 0.000001). The false-negative rate (1 − sensitivity) for HPV PCR analysis for detection of the presence of a cervical HPV-related lesion was 0% (95% confidence interval, 0 to 0.047), and the specificity was 60.7%. In summary, PCR analysis for HPV DNA had a very low false-negative rate for predicting HPV-related lesions of the cervix in a community-based population. This supports the validity of using the absence of HPV at the cervix, as determined by PCR testing, as an inclusion criterion for patients in control groups in studies dealing with low-grade cervical lesions.     

High-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays

We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, -18, -31, -33, -35, -45, and -53), genital warts (HPV-6 and -11), or skin lesions (HPV-1, -2, -4, and -5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins.     

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.     

Molecular epidemiology of human papillomavirus genotype in women with high‐grade squamous intraepithelial lesion and cervical cancer: Will a quadrivalent vaccine be necessary in Thailand?

This study was designed to investigate the distribution of human papillomavirus (HPV) genotypes among a group of patients with high-grade squamous intraepithelial lesion (HSIL) or worse cytology. Consequently, the genotype-specific HPV infection in a group of HSIL and invasive cervical cancer (ICC) samples was described. Specimens were collected prospectively from 132 women referred for colposcopic examination. All the women underwent Papanicolaou (Pap) smears and colposcopies and some also underwent cervical excision procedure biopsy. The HPV genotype was determined using the INNO-LiPA assay. Among the 132 genotyped samples, 90.91% (120/132) were diagnosed HSIL, whereas 9.09% (12/132) were ICC. From the overall prevalence of HPV in the patients, 77.27% (102/132) and 22.72% (30/132) of cases had single and multiple genotype infections, respectively. The most common cases with statistical significance were high-risk HPV (HR-HPV) infections in 128 samples (96.97%), whereas, four individuals (3.03%) barely were low-risk HPV (LR-HPV) infected, P < 0.0001, χ(2). The most prevalent genotypes were frequently HPV-16 (65/167; 38.92%, followed by HPV-58 (25/167; 14.97%), HPV-18 (18/167; 10.78%), HPV-33 (13/167; 7.19%), and HPV-68 (11/167; 6.59%). In addition, HPV-11 (2/132; 1.51%) and HPV-6 (1/132; 0.76%) also were observed in this study, which confirmed the high distribution of HR-HPV among women with HSIL and ICC. HPV-58; a unique high-risk HPV, is prevalent in a group of HSIL and ICC cases. These data also contribute evidence that HPV-16, -18, -58, -33, and -68 genotypes are high-risk and high distribution among women with HSIL and ICC. Therefore, HPV-58, HPV-33, and HPV-68 should be considered for development of the next vaccine generation in Thailand.     

人乳头状瘤病毒感染与宫颈癌前病变的关系

目的 探讨人乳头状瘤毒（ＨＰＶ）感染与宫颈湿疣、癌前病变的相关性。方法 对１７９例宫颈细胞涂片异常患者行宫颈多点活检,病理形这观察,其中１２８例同时采用ＰＣＲ方法检测ＨＰＶ－ＤＮＡ,１０例行原位杂交分析。结果（１）形态学观察：宫颈湿疣多为扁平型（９７．３％）;除表现经典的诊断性控空细胞（３９．７％）外,国可见异型性明显的非典型性控空细胞（６０．３％）;宫颈湿疣常伴宫颈上皮内肿瘤（ＣＩＮ,４２．５％     

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

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

Detection of human papillomavirus in skin and genital lesions of renal allograft recipients by in situ hybridization

Renal allograft recipients have an increased incidence of malignancy including squamous carcinoma of cervix and skin. There is growing evidence that human papillomavirus (HPV) has a part to play in malignant transformation at these sites. We have previously identified HPV DNA in the skin and genital lesions of such patients by dot and Southern blotting. In situ hybridization studies, using biotinylated DNA probes for HPV 4, 5 and 8 in skin lesions and 6, 11. 16 and 18 in genital lesions, were performed on tissues derived from the same group of patients. In the cutaneous lesions, only 25% of the specimens probed were found to contain virus by in situ hybridization; 60% of these specimens were found to harbour virus by dot and Southern blotting. In situ hybridization revealed HPV 16 and/or 18 in 86% of the genital lesions probed.     

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.     

Cervical mucus antibodies against human papillomavirus type 16, 18, and 33 capsids in relation to presence of viral DNA.

To investigate whether cervical mucus antibodies against human papillomavirus (HPV) capsids are associated with the detection of HPV DNA or HPV-related cytological diagnoses, 611 samples of cervical secretions from 359 women referred to a colposcopy clinic were tested by an enzyme-linked immunosorbent assay for the presence of immunoglobulin A (IgA) antibodies against HPV capsids of HPV type 16, 18, or 33 and for the presence of cervical HPV DNA by PCR. Among subjects with at least one cervical sample positive for HPV type 16 (HPV-16) DNA, 28.1% also had at least one HPV-16 IgA-positive cervical sample (odds ratio [OR] = 2.9; P = 0.0003). IgA to HPV-18 was also more common among HPV-18 DNA-positive subjects (OR = 3.1; P = 0.0325) and IgA to HPV-33 was more common among HPV-33 DNA-positive subjects (OR = 4.2; P = 0.0023). Cervical IgA antibodies to HPV-16 were also more common among patients with cervical intraepithelial neoplasia, particularly among patients with cervical intraepithelial neoplasia grade I (P < 0.0005). The data indicate that an HPV type-restricted IgA antibody response against HPV capsids is detectable in cervical mucus and is associated with a concomitant cervical HPV infection.     

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.     

The molecular-based differentiation of Heck's disease from its mimics including oral condyloma and white sponge nevus

Heck's disease (focal or multifocal epithelial hyperplasia) is a benign, rare condition of the skin and mucous membranes induced by human papillomavirus (HPV) infection. Other entities that can induce large papillomatous lesions that involve the mucous membranes and skin include condyloma acuminatum, which is sexually transmitted, and white sponge nevus, often due to a mutation of cytokeratin 4 or 13. Six cases diagnosed as either Heck's disease (n = 2) or white sponge nevus (n = 4) and 6 oral condyloma were compared on histologic grounds and analyzed in situ for HPV DNA, including HPVs 6,11, and 13, as well as cytokeratins 4 and 13. Each case showed marked acanthosis, and para/hyperkeratosis. More variable histologic findings included rete ridge elongation, keratinocyte degeneration, and perinuclear halos. High copy HPV 13 DNA was evident in the squamous cells towards the surface in the two cases diagnosed as Heck's disease and in two cases diagnosed as white sponge nevus on clinical grounds. HPV 6/11 was found in each of the six condyloma. Marked decrease in either cytokeratin 4 or 13 was evident in the two cases diagnosed as white sponge nevus that were HPV DNA negative. It is concluded that in situ hybridization analyses including HPVs 6, 11, and 13 as well as immunohistochemistry for cytokeratins 4 and 13 can differentiate Heck's disease from condyloma and white sponge nevus, which can be difficult to differentiate on clinical and histologic grounds.