1679 Squamous Papilloma of the Urinary Tract Is Unrelated to Condyloma Acuminata Liang Cheng, M.D.1,2 Bradley C. Leibovich, M.D.3 John C. Cheville, M.D.4 Dharamdas M. Ramnani, M.D.4 Thomas J. Sebo, M.D.4 Ajay Nehra, M.D.3 Reza S. Malek, M.D.3 Horst Zincke, M.D., Ph.D.3 David G. Bostwick, M.D.3,4 1 Department of Pathology, Indiana University School of Medicine, Indianapolis, Indiana. 2 Department of Urology, Indiana University School of Medicine, Indianapolis, Indiana. 3 Department of Urology, Mayo Clinic, Rochester, Minnesota. 4 Department of Pathology, Mayo Clinic, Rochester, Minnesota. BACKGROUND. To the authors’ knowledge, there is no previous report of squamous papilloma of the urinary tract. It is uncertain whether there is a correlation between squamous papilloma, condyloma acuminatum, and verrucous carcinoma. METHODS. The authors evaluated the clinical and pathologic features of squamous papilloma (5 of the bladder, 2 of the urethra), condyloma acuminatum (3 cases), and verrucous carcinoma (3 cases) of the urinary bladder and performed human papillomavirus (HPV) DNA in situ hybridization studies to determine whether HPV was a common feature shared by these lesions. In addition, DNA ploidy evaluation by image cytometry and p53 immunohistochemical staining were performed. RESULTS. Squamous papilloma of the urinary tract occurred in elderly women and followed a benign clinical course with infrequent recurrence. All squamous papillomas were HPV DNA negative and DNA diploid with no or minimal p53 nuclear accumulation. Condyloma acuminata of the bladder contained HPV DNA, increased p53 protein expression, and aneuploid DNA content. All three cases of condyloma acuminata were associated with coexistent condylomata of the external genitalia, and two required pelvic exenteration for uncontrolled expansile growth. Verrucous carcinoma of the bladder occurred in elderly patients. All three cases of verrucous carcinoma were negative for HPV DNA and DNA aneuploid, and they exhibited consistent p53 expression. CONCLUSIONS. These data indicate that squamous papilloma is a distinct entity not related to condyloma or verrucous carcinoma. These lesions are benign, HPV DNA negative, DNA diploid, and they lack p53 overaccumulation. Cancer 2000;88: 1679 – 86. © 2000 American Cancer Society. KEYWORDS: bladder, neoplasms, squamous lesion, precursor, koilocyte, condylomata acuminata, verrucous carcinoma, papilloma, human papillomavirus, p53, DNA ploidy. S The authors thank Dr. Ricardo V. Lloyd for excellent technical assistance. Dr. Bostwick’s current address: Bostwick Laboratories, 6722 Patterson Ave., Richmond, VA 23226. Address for reprints: Liang Cheng, M.D., Department of Pathology, University Hospital 3465, Indiana University School of Medicine, 550 North University Blvd., Indianapolis, IN 46202. Email: Icheng@iupui.edu. Received October 8, 1999; revision received December 28, 1999; accepted December 28, 1999. © 2000 American Cancer Society quamous papilloma, condylomata acuminatum, and verrucous carcinoma of the urinary bladder comprise the spectrum of rare proliferative squamous lesions. There is morphologic overlap between these lesions, and some authors indicate that squamous papilloma and condyloma acuminata of the urinary bladder are histologically indistinguishable,1 although formal evaluation is lacking. Mostofi et al. stated that some of squmaous papilloma “may be condyloma acuminata that have extended into the bladder from the urethra.”2 Some investigators suggest that there is a relation between squamous papilloma and low grade squamous cell carcinoma, and that human papillomavirus (HPV) may play an important role in the development of these squamous proliferations.1–3 However, the correlations among squamous papilloma, condyloma, and verrucous carcinoma remain largely undefined, and, because of the rarity of the 1680 CANCER April 1, 2000 / Volume 88 / Number 7 TABLE 1 Clinical and Pathologic Findings in 13 Squamous Proliferative Lesions of the Urinary Tract Case no. Age/ gender Diagnosis Symptomsa Cystoscopic findings Location Treatment length of follow-up (yrs) 1 82 F Squamous papilloma Irritative symptomsb Whitish plaque Dome TURB 10.0 2 3 46 F 79 M Squamous papilloma Squamous papilloma Polypoid lesion Erythematous Bladder Lateral wall None None 2.0 1.5 4 62 F Squamous papilloma Whitish plaque Vesical neck TURB 0.3 NED 5 6 7 70 F 32 F 63 F Squamous papilloma Squamous papilloma Squamous papilloma Not documented Detected at surveillance H/O invasive urothelial carcinoma Irritative symptoms and Hematuria Not documented Not documented Irritative symptoms H/O urothelial carcinoma Recurrence of squamous papilloma at 5 yrs NED Died of bladder cancer — Papillary lesion Sessile tumor Urethra Urethra Posterior wall None None TURB 4.3 2.5 8.8 8 45 F Condyloma acuminatum Extensive condyloma Entire bladder TURB 15 NED NED Nephroureterectomy for ureter urothelial carcinoma 7 yrs later Died of urothelial carcinoma Multiple recurrences Pelvic exenteration due to perianal vesical fistula — Pelvic exenteration at 4 yrs Lost to follow-up 9 51 M Condyloma acuminatum 10 27 M Condyloma acuminatum 11 81 F 12 13 Irritative symptoms Extensive vulval condyloma Extensive perineal condyloma Intravesical chemotherapy Lateral wall vesical neck Condyloma Outcome Condyloma Entire bladder — — — Verrucous carcinoma Extensive urethral condyloma Irritative symptoms Erythematous Dome TURB 5.3 62 M Verrucous carcinoma H/O bladder calculi Whitish plaque Radical cystectomy 5.0 54 F Verrucous carcinoma H/O diabetes Whitish plaque and erythematous Trigone, lateral wall and dome Bladder Recurrence at 1 yr after diagnosis Died of cerebrovascular accident NED Unknown 6.0 Reccurence at last follow-up H/O: history of; TURB: transurethral resection of the bladder; NED: no evidence of disease. a Other than 3 patients with condyloma acuminatum of the bladder (Cases 8, 9, and 10), none of the other patients had history of genital, perineal or perianal condylomas. b This patient also had history of interstial cystitis. lesions, the comparative clinical pathologic features are not well documented. The objective of this study was to examine the clinical and histologic features of squamous papilloma, condyloma acuminatum, and verrucous carcinoma of the urinary tract. In addition, HPV DNA in situ hybridization, DNA ploidy determination by image cytometry, and p53 immunostaining were performed to define the relation between these squamous proliferative lesions. MATERIALS AND METHODS We reviewed cases of squamous papilloma, condyloma acuminata, and verrucous carcinoma of the uri- nary tract from the Mayo Clinic surgical pathology files between 1971 and 1998. All patients underwent transurethral resection. All histologic slides were evaluated by the authors without the knowledge of clinical information. The 1973 World Health Organization histologic criteria2 were used for the diagnosis of squamous papilloma, which was defined as a papillary lesion with a delicate fibrovascular stroma lined by squamous epithelium. The diagnostic criteria for condyloma acuminata and verrucous carcinoma were the same as those used for other organ sites.3–5 Patient follow-up was obtained from the medical records. Routine 5-m sections from formalin fixed, par- Squamous Cell Proliferation of the Bladder/Cheng et al. 1681 FIGURE 1. Squamous papilloma of the bladder is shown (A and B). The papillary configuration of the lesion resembles configurations seen in condyloma. affin embedded tissue were used for in situ hybridization for HPV detection, immunohistochemical studies, and DNA ploidy analysis. In situ hybridization was performed using microwave pretreatment with digoxigenin-labeled probes.6 Briefly, oligonucleotide probes specific for HPV 6 and 11, HPV 16 and 18, and HPV 31 and 33 (Enzo Diagnostics, Farmingdale, NY) were diluted 1:5 in hybridization solution containing 10% dextran sulphate, 3⫻ standard saline solution (SCC) buffer, 1⫻ Denhardt 100 g/mL salmon sperm DNA, 125 g/mL yeast tRNA, 10 g/mL polyadenylic-cytidylic acid, 0.1% sodium pyrophosphate–inorganic, and 50% deionized formamide. cDNA probes were applied to pretreated slides, denatured for 5 minutes at 95 °C, and hybridized for 60 minutes at 37 °C. Slides were subsequently incubated with an antidigoxigenin antibody linked to alkaline phosphatase (BoehringerMannheim Biochemicals, Indianapolis, IN) and developed in nitroblue tetrazolium and 5-bromo-chloro-3indolylphosphate. Positive controls consisted of formalin fixed, paraffin embedded sections of cases from other organs, such as the cervix, known to be infected with HPV 6, 11, 16, 18, and 31, 33, 51. Negative control consisted of substituting another cDNA probe 1682 CANCER April 1, 2000 / Volume 88 / Number 7 TABLE 2 Human Papillomavirus Typing, p53 Alteration, and DNA Ploidy in Squamoproliferative Lesions of the Bladder HPV type Case no. Diagnosis 6/11 16/18 31/33 p53a DNA ploidy 1 2 3 4 5 6 7 8 9 10 11 12 13 Squamous papilloma Squamous papilloma Squamous papilloma Squamous papilloma Squamous papilloma Squamous papilloma Squamous papilloma Condyloma Condyloma Condyloma Verrucous carcinoma Verrucous carcinoma Verrucous carcinoma ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ND ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ND ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ND ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ 10 5 5 0 0 0 ND 40 20 20 60 90 95 Diploid Diploid Diploid Diploid Diploid Diploid ND Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid HPV: human papillomavirus; ND: not done. a % of positive staining cells. (adenovirus) for the HPV probe. All negative slides were examined for DNA integrity by using an alu repeat probe (Blur 8, Diagnostic Enzo, Farmingdale, NY) and were positive for alu repeat DNA sequence. Immunohistochemical stains were performed on a Ventana ES autostainer (Tucson, AZ) using the avidinbiotin-complex method. Primary monoclonal antibodies were used for evaluation of p53 (DO-7, Dako, dilution 1:100). The percentage of immunoreactive cells was recorded for each case. Positive and negative controls were run in parallel and gave appropriate results. Digital image analysis for DNA ploidy was performed with the CAS 200 digital image analyzer (Becton Dickinson, Cellular Imaging Systems). Quantification of Feulgen staining was performed with the quantitative DNA analysis program (version 3.0). None of these ancillary studies were used in the original file classification and none of the original file diagnoses were changed after performance of ancillary studies. RESULTS Table 1 summarizes the patient characteristics and clinical findings. None had positive urine culture. Of 7 cases of squamous papilloma (5 in the urinary bladder and 2 in the urethra), the male-to-female ratio was 1:6 and the mean age was 62 years (range, 32– 82 years). The mean follow-up was 4.2 years (range, 0.3–10 years). One patient had history of interstitial cystitis and had recurrence at 5 years after diagnosis (Case 1). None had history of genital, perineal, or perianal condyloma. Squamous papilloma is characterized by pro- liferation of squamous epithelium surrounding a central fibrovascular core (Fig. 1). In situ hybridization for HPV was negative in all cases (Table 2). No or minimal p53 nuclear accumulation was observed (Table 2). All squamous papillomas were diploid by digital image analysis. Condyloma acuminata of the bladder is indistinguishable from that seen in other organ sites. It shows papillary growth of squamous epithelium displaying koilocytosis and nuclear changes characteristic of HPV infection (Fig. 2). The underlying stroma is well vascularized with scattered lymphocytic infiltration. No stromal invasion is seen. The adjacent epithelium showed squamous change with acanthosis and hyperkeratosis. The involvement of bladder by condyloma acuminata is usually extensive. All three cases of condyloma acuminata of the bladder were associated with coexistent extensive condyloma acuminata of external genitalia and were suspected during cystoscopic examinations. All three cases were positive for HPV types 6 and 11 (Fig. 2). Subsequently, two patients underwent pelvic exenteration for uncontrolled condylomas. DNA ploidy analysis revealed aneuploidy pattern in both cases. Abnormal p53 expression was seen in all three cases. Verrucous carcinoma of the bladder is morphologically identical to its counterpart in the oral cavity, the lower female genital tract, the digestive tract, and other locations. It is composed of well-differentiated keratinizing squamous epithelium that invades the stroma in a pushing fashion. The degree of cytologic atypia is mild (Fig. 3). None had history of genital, Squamous Cell Proliferation of the Bladder/Cheng et al. 1683 FIGURE 2. Condyloma acuminata of the bladder is shown. Koilocytotic change can be subtle (A), and human papillomavirus in situ hybridization is positive in the deeper section (B). perineal, or perianal condyloma. HPV was not detected by in situ hybridization. Strong p53 nuclear accumulation and aneuploid tumor population were observed in all 3 cases (Table 2). The adjacent epithelium was squamous in nature, showing acanthosis and hyperkeratosis. Two cases (Cases 11 and 13) showed features that resembled squamous papilloma, including proliferation of benign-appearing squamous epithelium with underlying dilated vessels; subsequently, both patients developed recurrence of verrucous carcinoma (Table 1). One patient underwent radical cystectomy and there was no evidence of cancer recurrence during 5 years of follow-up (Case 12). DISCUSSION Squamous lesions of the urinary bladder are rarely encountered, and the distinctions among squamous papilloma, condyloma acuminata, and verrucous carcinoma are difficult to make in small biopsies.1,3,4,7–9 Because of the morphologic similarity to condyloma, some investigators have suggested that squamous papilloma of the bladder is associated with HPV infec- 1684 CANCER April 1, 2000 / Volume 88 / Number 7 FIGURE 3. Verrucous carcinoma of the bladder is shown. The pushing margins are characteristic of verrucous carcinoma (A). p53 immunostaining is strongly positive (B). tion,1–3 although this relation has not been confirmed. In this study, we found that all squamous papillomas were negative for HPV DNA, were DNA diploid, and displayed no or minimal p53 protein nuclear accumulation. These lesions were benign and infrequently recurred. Our study indicates that squamous papilloma is a distinct entity not related to condyloma or verrucous carcinoma. Some investigators have considered that condyloma acuminatum of the bladder morphologically indistinguishable from squamous papilloma1 and postulated that some squamous papillomas may represent extensions of condylomas from the urethra.2 Clinical information and ancillary studies, such as HPV DNA in situ hybridization studies, p53 immunostaining, and DNA ploidy analysis, may be useful in the differential diagnosis (Table 3). Less than 2 dozen cases of condyloma acuminata3,10 –19 of the bladder have been reported. The majority of patients had a history of long-standing uncontrolled condyloma of the external genitalia, similar to our cases. Isolated condyloma of the bladder is rare and almost always presents in immunosuppressed patients.1,11 Del Mistro et al. reported three cases of condyloma, two of Squamous Cell Proliferation of the Bladder/Cheng et al. 1685 TABLE 3 Differential Diagnosis of Squamous Papilloma, Condyloma Acuminata, and Verrucous Carcinoma of the Urinary Bladder Age (yrs) Gender (male:female) Clinical history Clinical presentation Biologic behavior Location Extent Histologic changes Architecture Pushing margin Cytologic atypia Stromal invasion p53 alteration HPV detection DNA ploidy a b Squamous papilloma Condyloma acuminataa Verrucous carcinomab 62 (range, 32–82) 1:6 Nonspecific Irritative symptoms Rarely recurs No predilection Small, solitary 40 (range, 17–76) 1:1.6 External genitalia condyloma or history of immunosuppression Irritative symptoms Aggressive No predilection Multiple, extensive 66 (range, 43–83) 1.2:1 Nonspecific Irritative symptoms Aggressive No predilection Diffuse, extensive Papillary Absent Usually not seen or mild Absent ⫺/⫹ ⫺ Diploid Papillary Absent Usually not seen or mild Absent ⫹ ⫹ Aneuploid Expansive and endophytic Present May be present Present ⫹⫹ ⫺ Aneuploid References 3, 10–19. References 22–28. which occurred in immunocompromised patients and one in a patient with extensive treatment-resistant external genitalia condylomata with secondary involvement of the urinary bladder.3 All 3 patients had multiple recurrences, and their lesions were aneuploid and positive for HPV DNA types 6 and 11.3 Similarly, we found that our 3 patients with condylomata had aneuploid tumors that were HPV DNA positive for types 6 and 11. All three patients had extensive external genitalia involvement by condylomata, and two required pelvic exenteration for uncontrolled condylomata. In most reported cases, condyloma of the bladder is refractory to conservative therapy, necessitating cystectomy in more than half the reported cases. The relation between condyloma acuminata and verrucous carcinoma is not well understood. Most cases of vesical verrucous carcinoma have occurred in association with schistosomiasis infection,20 and, to the best of our knowledge, only eight cases have been reported without such infection.21–28 Of these eight unique cases, two had extensive external genitalia condyloma with secondary involvement of the bladder,21,22 suggesting the possibility of misinterpretation of the bladder tumor, although HPV typing was not performed. Alternatively, there may be a relation between verrucous carcinoma and condyloma. We could not detect HPV DNA in our cases of verrucous carcinoma or any association with schistosomiasis infection. Clearly, additional cases of verrucous carcinoma are needed to study HPV DNA status. Together with findings from other studies, squamous papilloma does not appear to be associated with schistosomiasis or condyloma, and some verrucous carcinomas apparently are not associated with schistosomiasis or condyloma and have no recognizable connection with infectious agents or any other predisposing condition. The diagnosis of verrucous carcinoma relies on the findings of expansive growth, pushing margins, lack of well-formed papillae, and cytologic atypia, although the atypia is mild and usually presents in the deeper portions of the lesion (Table 3). Condyloma acuminata is almost always associated with extensive external genitalia condyloma or immunocompromising conditions, lacks stromal invasion, and is HPV DNA positive by in situ hybridization. Several limitations of this study should be considered. The current study includes only a small number of patients with relatively short follow-up. Nonetheless, this is the first report of squamous papilloma of the bladder with follow-up, HPV analysis, DNA ploidy determination, and p53 immunostaining. We were not able to demonstrate HPV infection in squamous papilloma, although the possibility of some unusual types of HPV as etiologic agents cannot be ruled out. The sensitivity of our HPV analysis may also be limiting, because approximately 50 or more copies of HPV DNA are necessary for in situ hybrization detection. If an infected sample has less than 50 copies of HPV DNA, this could lead to false-negative results.6 More sensitive methods, such as in situ polymerase chain reaction (PCR), in situ reverse PCR, and self-sustained sequence replication, may yield a higher detection rate.29 The possible link between squamous papilloma and condyloma could not be established by the current investigation. Furthermore, the value of ancillary 1686 CANCER April 1, 2000 / Volume 88 / Number 7 studies may be limited in small cystoscopic biopsies because intralesional heterogeneity of DNA content and expression of HPV and p53 potentially influences interpretation. In addition, the cystoscopic examinations may be nonspecific, and the findings of whitish, plaquelike lesions should alert the urologists and pathologists to consider these unusual squamous lesions. Squamous papilloma is a benign lesion that is HPV DNA negative and DNA diploid and lacks p53 overaccumulation. In contrast, condyloma acuminatum is clinically aggressive, frequently recurrent, usually associated with extensive external genitalia involvement, HPV DNA positive, and DNA aneuploid, and exhibits abnormal p53 protein accumulation. Verrucous carcinoma is an indolent neoplasm that is HPV negative and DNA aneuploid and exhibits abnormal p53 accumulation. 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