Molecular characterization and integrative genomic analysis of a panel of newly established penile cancer cell lines.

Cell line models are essential tools to study the molecular mechanisms underlying tumor initiation and progression. There are limited treatment options for penile squamous cell carcinoma (PSCC), accounting for 1-2% of male tumors in developing countries, and limited progress in preclinical research in PSCC due to lacking available models with identified genomic characteristics. Here, biological and molecular characteristics and whole-genomic alterations were analyzed in a panel of PSCC cell lines newly established in our laboratory. These cell lines were all human papillomavirus (HPV)-negative, epithelial-like, immortalized, and tumorigenic in nude mice, whereas they displayed different proliferation, migration and invasion capacities in vitro, and tumorigenic ability in nude mice. They were all cisplatin sensitive, anti-EGFR therapy resistant, and androgen irresponsive. Whole-genomic sequecing analysis revealed that transition mutations (C:G>T:A and T:A>C:G) were the most common substitution types in these cell lines, whereas ERCC5, TP53, PTH1, CLTCL1, NOTCH2, MAP2K3, CDK11A/B, USP6, ADCH5, BCLAF1, CDKN2A, FANCD2, HRAS, and NOTCH1 were the most frequently altered genes. Amplifications of MYC, PLAG1, NCOA2, RUNX1T1, COX6C, and EGFR and losses of FBXW7, TET2, XPC, and FANCE were frequently observed in cell lines. The exomic variations between cell lines and their corresponding cancer tissues were highly consistent. Genetic variations were mainly involved in the MAPK, Jak-STAT, TGF-beta, Notch, and apoptosis signaling pathways. Conclusively, these panel of PSCC cell lines established in our laboratory harbor some common or specific biological characteristics and genomic variations, and they may serve as optimal models to investigate the molecular mechanisms underlying the progression, metastasis, relapses, and treatment resistance of PSCC and to develop effective treatment strategy.

Absence of Epstein-Barr virus in penile carcinoma. A study of 42 cases using in situ hybridization.

BACKGROUND: The relationship between Epstein-Barr virus (EBV) and carcinomas of the lower female genital tract has been the focus of some studies in recent years. However, the association between EBV and penile carcinoma never has been investigated. The aim of this study was to identify the possible role of EBV in penile carcinoma of Chinese patients. METHODS: Formalin fixed, paraffin embedded tissue of 42 cases of penile carcinoma (including 7 verrucous, 14 well differentiated, 15 moderately differentiated, and 6 poorly differentiated squamous cell carcinomas), 6 penile warts, and 9 normal penile samples were analyzed for EBV using in situ hybridization for EBV-encoded RNA (EBER). RESULTS: Epstein-Barr virus was only found in a few lymphocytes adjacent to the tumor epithelium in 12% (5 cases) of penile carcinoma. Conversely, the tumor cells, penile warts, and normal penile tissue were negative for EBV. CONCLUSIONS: The results suggest that EBV does not play a major role in the etiology of penile carcinoma.

Penile intraepithelial neoplasia. Specific clinical features correlate with histologic and virologic findings.

BACKGROUND: To evaluate the existence of the morphologic features specific for penile intraepithelial neoplasia (PIN), 1000 male sexual partners of women with genital condyloma or intraepithelial neoplasia were studied. METHODS: Ninety-two patients who presented with lesions suggesting intraepithelial neoplasia (pigmented or leukoplastic papules, keratinized condylomata, or erythroplastic macules) underwent biopsy for histologic and virologic studies. RESULTS: Histologic results showed penile intraepithelial neoplasia in 93% of the specimens. Human papillomavirus (HPV) DNA from potentially oncogenic papillomaviruses was detected in 75% of patients with Grade I PIN, in 93% of patients with Grade II PIN, and in all patients with Grade III PIN: Uncircumcised and circumcised men showed the same rate (52% vs. 45%; odds ratio [OR] = 1.3; 95% confidence interval, 0.97-1.73) of HPV-associated lesions, whereas the rate of PIN was significantly higher in uncircumcised men than in circumcised men (10% vs. 6%; OR = 1.77; 95% confidence interval, 1.02-3.07). The mean age of patients with Grade III PIN was 7 years older then the mean age of patients with Grade I PIN, which suggests a step progression similar to that of cervical intraepithelial neoplasia. CONCLUSION: Morphology seems to be a specific-enough indicator of PIN. More data are needed to determine whether treatment of PIN may contribute to preventing cervical or penile cancer. If so, the morphologic criteria here described will be clinically useful.

Typing of human papillomaviruses in condylomata acuminata from Greece.

DNA samples from recurrent condylomata acuminata biopsies of Greek males and females were examined for the presence of human papillomavirus (HPV) DNA using high-stringency Southern blot hybridization analysis. Of the twenty-six biopsies, 25 were positive for the HPV 6/11-related DNA sequences, and when further analyzed with the polymerase chain reaction (PCR) the HPV-negative biopsy was also positive for HPV 6/11 DNA. Nineteen specimens were further characterized based on their Pstl restriction endonuclease hybridization pattern. Twelve biopsies were positive for HPV 6a, one biopsy was positive for HPV 11a, and one biopsy was positive for HPV 6c DNA. Three specimens contained HPV 6/11 related DNA that gave an unusual Pstl pattern, and one specimen appeared to represent a multiple HPV infection containing HPV 6/11- and HPV 31/35/39-related sequences. Finally, one sample contained a mixture of HPV 6a DNA and an HPV 6a-like genome. Biopsies were also taken from adjacent apparently normal tissue, 0.5 cm away from the lesion, in 19 of the patients. Only one of these was found to be positive for HPV 6a DNA by Southern blot analysis.

HPV detection and p53 alteration in squamous cell verrucous malignancies of the lower genital tract.

We examined five cases of verrucous carcinoma (VC) and two cases of giant condyloma of Buschke-Löwenstein (GCBL) associated with invasive squamous cell carcinoma (ISCC), by immunocytochemistry and molecular techniques. Neither human papillomavirus (HPV) footprints nor p53-altered expression and/or mutation were observed among the cases of VC. By contrast, both cases of GCBL with ISCC turned out to be HPV 6 or 11 positive, showed overexpression of p53 and, one of the two, a mutation in the nucleotide sequence of this tumor suppressor gene. The results point out that VC and GCBL with ISCC, in spite of some morphologic similarities, are two distinct entities, the former being unrelated to both HPV and p53 inactivation and the latter related to both. Regarding p53, immunocytochemical and molecular data on GCBL with ISCC suggest a role of mutant p53 in the progression of malignancy into invasion.

Localization of p53 protein and human papillomavirus in anogenital squamous lesions: immunohistochemical and in situ hybridization studies in benign, dysplastic, and malignant epithelia.

p53 Protein is a 53-kd nuclear phosphoprotein believed to play an important role in controlling proliferation of neoplastic and normal cells. This "natural tumor suppressor" can be rendered ineffective (or oncogenic) by mutations in the p53 gene or by interactions with proteins synthesized by DNA-transforming viruses, including specific subtypes of human papillomavirus (HPV). We describe the localization of p53 protein in association with HPV in paraffin sections of a spectrum of benign, dysplastic, and malignant anogenital squamous epithelia using immunohistochemical and in situ hybridization techniques. p53 Was detected in 81% of the 48 cases studied. Immunoreactivity for p53 was seen in 83% of the benign and low-grade squamous intraepithelial lesions (SILs), in 73% of the high-grade SILs, and in 86% of the infiltrating squamous carcinomas. In high-grade SILs p53 staining was frequently observed in individual nuclei at various levels of the abnormal epithelium and in the basal layer of the adjacent epithelium, while in squamous metaplasia and low-grade SILs immunostaining for p53 was limited to the basal layer of the epithelium. p53 Was detected in a slightly higher percentage of HPV-positive than HPV-negative epithelia as determined by in situ hybridization. No correlation was observed between p53 immunoreactivity and HPV subtypes. p53 Protein and HPV were detected in anal lesions from a small group of human immunodeficiency virus-positive individuals. Antibodies currently available mainly demonstrate mutant forms of p53 protein that are associated with longer half-lives than the wild-type protein, but demonstration of p53 protein overexpression is not necessarily indicative of malignancy.

A case of verrucous carcinoma associated with human papillomavirus type 16 DNA.

We report here a case of verrucous carcinoma which occurred on the penis of a 75-year-old male. The nodule was first noted six months earlier and was whitish, cauliflower-like, and 17 x 19 mm in size. The histopathological examination revealed hypertrophic epidermal proliferation with pale staining keratinocytes, extending into the deep dermis. Partial penectomy and inguinal lymph node dissection were done. No lymph node metastasis was recognized. DNA was isolated from the frozen tumor tissue and examined for the presence of human papillomavirus (HPV) 16, 18, and 33 DNA by the polymerase chain reaction (PCR), using common and specific primers. A 140 base pair (bp) band was amplified and finally determined to be the HPV16 sequence by dot-blot hybridization.

[A case of verrucous carcinoma of the penis with detection of human papillomavirus using the in situ hybridization method].

A case of penile verrucous carcinoma is reported. The patient had a previous history of condyloma acuminatum of the penis. No signs of squamous cell carcinomas were observed in the histopathological findings. We also detected the histologic evidence of human papilloma virus (HPV) infection in the result of the study by in situ hybridization. The local positive reaction of HPV infection for type 6/11 was seen, and was related to the koliocytosis. However other types of HPV (16/18, 31/33, 51) were negative.

A study from south India on the association between the papilloma virus and carcinoma of the penis.

Specimens from 39 cases of squamous cell carcinoma of the penis were treated for the papilloma virus using the bovine papilloma virus antibody and the peroxidase-antiperoxidase technique. All 39 cases showed a viral antigen situated intranuclearly, intracytoplasmically or both intranuclearly and intracytoplasmically. No correlation could be seen between the presence of viral antigen and age of patient, duration of lesion, cell morphology and histological grade. The adjacent mucosa and skin were used as controls and showed no viral antigen. The inflammatory infiltrate was dense and predominantly "T" lymphocyte in type.

Human papillomavirus infection and intraepithelial, in situ, and invasive carcinoma of penis.

The role of human papillomavirus (HPV) infection and other reported cofactors in the genesis, evolution, and clinical manifestations of precancerous and cancerous squamous cell lesions of the penis were studied in 34 men. Clinically, all lesions demonstrated aceto-whitening. Histologic changes of HPV infection formed a field-of-change that involved the components of the preputial cavity in all patients. These changes were associated with minor grades of penile intraepithelial neoplasia (PIN I and II) in 19 patients, major grades of PIN/carcinoma in situ (PIN III/Tis) in 7, and invasive squamous cell carcinoma (SCCa, Stages T2 and T3) in 8. Most of the patients (79.4%) were heavy smokers; 52.9 percent had a history of HPV infection, PIN, or invasive penile SCCa; and 60 percent of 30 patients had female sexual partners who had HPV-related genital neoplasia. A pilot virologic study of specimens obtained from 20 representative patients utilizing polymerase chain reaction amplification detected HPV DNA in 80 percent. Laser therapy was aimed at the entire field-of-change in 30 patients; recurrent minor-grade PIN or SCCa developed in 2 of 23 patients (8.7%) followed for up to three years. Of the 4 remaining patients treated with local excision or partial penectomy, 3 (75%) had development of recurrent minor-grade PIN when followed for up to four years. The combination of the host of carcinogenic factors and currently rampant immunologic disorders will likely lead to an increase in the historically low incidence of SCCa of the penis in the United States.

Penile intraepithelial neoplasia: clinical presentation and an analysis of the physical state of human papillomavirus DNA.

Forty-four men with penile intraepithelial neoplasia (PIN) and a matched control group of 88 men with condyloma acuminatum were evaluated in three centers studying anogenital human papillomavirus (HPV) infections. PIN and condyloma groups could not be distinguished on the basis of historical features or clinical presentation. Although PINs were more likely than condylomata to be pigmented (31/46 [67%] vs. 33/97 [34%], P < .001), 43% of PIN III were not pigmented, suggesting that pigmentation is not a sensitive indicator of high-grade PIN. HPV-16 infection, as determined by in situ hybridization, was closely associated with PIN III (0/24 PIN I contained HPV-16 vs. 12/13 PIN III, P < .001). Southern blot analysis demonstrated only episomal viral genomes, suggesting that integration is not an early event in penile neoplasia.

Childhood condyloma acuminatum: association with genital and cutaneous human papillomaviruses.

We studied 25 children, age 7 months to 12 years 6 months, with anogenital warts, and their parents. In most children the warts were localized in the anal area, in 3 of 18 girls perianally and on the vulva, and in 4 girls exclusively on the vulva. Southern blot hybridization studies disclosed an association of condylomata with human papillomaviruses (HPV) 6 and 11 in 74% and HPV 2 in 17.4% of patients. The clinical features were similar in warts induced by genital and cutaneous HPVs. Even the HPV 2-associated warts in the vulva of two girls were typical of condyloma acuminatum. In all children with HPV 2-induced condylomata, cutaneous common warts coexisted, also induced by HPV 2. However, three mothers had cutaneous warts, and the children's condylomata were associated with HPV 6. Thus, the mere presence of skin warts in family members does not rule out other sources of infection. Sexual abuse was suspected in four girls and two boys, but was not confirmed in any. Nonsexual transmission could occur by persons with the lesions taking care of children. Perinatal transmission also appears to be an important route of infection in small babies. Infection in utero was probable in one girl in whom anal warts appeared in the first week of life and whose mother had cervical condylomata during pregnancy. This study provides further confirmation of possible nonsexual transmission of genital HPVs and the not infrequent association of childhood condylomata with HPV 2.

Human papillomavirus DNA in the urogenital tracts of men with gonorrhoea, penile warts or genital dermatoses.

OBJECTIVE: To assess the presence of human papillomavirus (HPV) DNA in urethral and urine specimens from men with and without sexually transmitted diseases. DESIGN: Prospective study. SETTING: Two London departments of genitourinary medicine PATIENTS: 100 men with urethral gonorrhoea, 31 men with penile warts and 37 men with genital dermatoses. METHODS: Urethral and urine specimens were taken, HPV DNA extracted and then amplified using the polymerase chain reaction. HPV types 6, 11, 16, 18, 31 and 33 were identified using Southern blotting followed by hybridisation. RESULTS: HPV DNA was detected in 18-31% of urethral swab specimens and in 0-14% of urine specimens. Men with penile warts had HPV detected in urethral swabs more often than did men in the other two clinical groups. "High risk" HPV types were found in 71-83% of swab specimens and in 73-80% of urine specimens containing HPV DNA. CONCLUSIONS: HPV is present in the urogenital tracts of men with gonorrhoea, penile warts and with genital dermatoses. In men with urethral gonorrhoea, detection of HPV in urethral specimens is not related to the number of sexual partners, condom usage, racial origin or past history of genital warts. HPV DNA in the urethral swab and urine specimens may represent different aspects of the epidemiology of HPV in the male genital tract. The preponderance of HPV types 16 and 18 in all three groups of men may be relevant to the concept of the "high risk male".

Detection and typing of genital papillomaviruses in men with a single polymerase chain reaction and type-specific DNA probes.

BACKGROUND: Human papillomavirus (HPV) infection of the genitalia can produce both visible and subclinical lesions. Because different genotypes are preferentially associated with benign or malignant lesions, HPV detection and typing is clinically important. OBJECTIVE: Our purpose was to assess a polymerase chain reaction (PCR)-based, noninvasive procedure for HPV diagnosis and evaluate the reliability of the acetic acid test for revealing subclinical HPV lesions. METHODS: Mucosal samples were collected by gentle scraping, and PCR-positive samples were typed by hybridization with specific DNA probes. RESULTS: Seventy-eight men were assessed. The PCR procedure was reliable and easy to perform. No significant difference was found in the prevalence of HPV infection between patients whose results were acetowhite-positive and those whose results were acetowhite-negative. CONCLUSION: Detection of acetowhite epithelium, although useful for clinical examination, is not sufficiently specific and should not be used as a sole criterion for the diagnosis of HPV infection.

The prevalence of "high-risk" HPV types in penile condyloma-like lesions: correlation between HPV type and morphology.

OBJECTIVE: To evaluate the prevalence of "high-risk" human papilloma virus (HPV) types in penile condyloma-like lesions and to correlate HPV types with clinical and histological features. DESIGN: The study included 94 male patients with signs of penile HPV infection. From acuminate, papular and macular lesions, specimens were collected for HPV DNA hybridisation, using the dot blot and Southern blot techniques. Biopsy specimens from 51 cases were examined by light microscopy for signs of koilocytosis and dysplasia. SETTING: The STD outpatient clinic of the Department of Dermatovenereology of Sahlgrenska Hospital, Göteborg, Sweden. RESULTS: In 79 (90%) of 88 patients HPV DNA was detected by dot blot. Of 51 cases examined by histology 88% disclosed an evident koilocytosis. "High-risk" HPV types (16, 18, 31, 33, 35) were demonstrated in 8% of acuminate, 24% of papular and 56% of macular lesions. In 29% of 51 lesions examined histologically moderate to severe dysplasia was observed. There was a significant correlation between "high-risk" HPV types and dysplasia. CONCLUSION: "High-risk" HPV types are prevalent in papular and especially macular penile condyloma-like lesions. The histological finding of koilocytosis concomitant with dysplasia strongly indicates infection with a "high-risk" HPV type. Although the risk of penile cancer is low, it is from an epidemiological point of view important to diagnose these lesions. Until simple tests for HPV typing are available, biopsy for light microscopy (histology) should be obtained liberally from papular and macular condyloma-like lesions. In atypical cases of balanoposthitis HPV aetiology should also be considered.

Human papillomavirus in penile squamous cell lesions. A comparison of an isotopic RNA and two commercial nonisotopic DNA in situ hybridization methods.

Sensitive isotopic in situ hybridization analysis of 34 paraffin-embedded penile squamous cell lesions for human papillomavirus (HPV) types 6, 11, 16, 18, and 31 revealed similar HPV detection rates, HPV-type specificity, and viral distribution patterns to those described in analogous cervical and vulvar lesions. Human papillomavirus was detected in six of six cases of condylomata acuminata (HPV type 6 [n = 4], HPV type 11 [n = 2]), in six of eight cases of squamous cell carcinoma in situ (HPV type 16 [n = 5], HPV type 31 [n = 1]), and in four of 10 cases of squamous cell carcinoma (all were HPV type 16). Interestingly, all 10 cases of penile verrucous carcinoma analyzed were HPV negative. Reevaluation of the HPV-positive penile lesions with two commercial nonisotopic HPV-typing in situ hybridization kits (Pathogene, Enzo, New York, NY; Viratype, Life Technologies, Gaithersburg, Md), revealed positive results for HPV type 6 and/or type 11 in all six cases of condylomata acuminata studied. However, only the Viratype assay detected HPV genome in the high-grade squamous cell lesions, with a relative sensitivity of 70% compared with that of the isotopic assay.

Squamous cell carcinoma of the scrotum associated with human papillomaviruses.

A 53-year-old man presented with recurrent squamous cell carcinoma, and dysplasia of the scrotum and penis. Risk factors included psoralen and ultraviolet radiation therapy for psoriasis, and x-ray therapy for primary lymphoma of the groin. Three different human papillomavirus types were documented using the polymerase chain reaction in distinct anatomical areas. The scrotal carcinoma was associated with human papillomavirus type 18, while regions of dysplasia contained either type 18, 16 or 6/11. Diagnosis of squamous dysplasia and carcinoma of the scrotum in men with psoriasis is complicated by chronic active inflammation, and molecular biological methods may be necessary to detect human papillomavirus infected cells.

Polymerase chain reaction in detection of human papillomavirus DNA and types of condyloma acuminata.

Sections from 88 paraffin-embedded tissues identified histopathologically as condyloma acuminata were assayed for the presence of human papillomavirus (HPV) types 6, 11, 16, 18 and 33 using polymerase chain reaction followed by specific hybridization. HPV DNA sequences were found in all tissues of condyloma acuminata. HPV type 6 was present in 71 tissues, type 11 in 16, and type 16 in 2. One man was doubly infected with HPV types 6 and 16. No HPV types 18 and 33 were found. We conclude that condyloma acuminata is mainly produced by HPV types 6 and 11.