This article has Open Peer Review reports available.
Implication of human papillomavirus-66 in vulvar carcinoma: a case report
© Kotsopoulos et al; licensee BioMed Central Ltd. 2011
Received: 18 June 2010
Accepted: 25 June 2011
Published: 25 June 2011
Vulvar cancer in older women is seldom associated with human papillomavirus infection.
We present the case of an 80-year-old Greek Caucasian woman with an undetermined obstetric and gynecologic history. The patient underwent radical vulvectomy and bilateral inguinal lymphadenectomy for a vulvar carcinoma. A human papillomavirus infection was suggested on the basis of histological and cytological examinations followed by human papillomavirus DNA typing, which revealed the presence of human papillomavirus-66.
Even though human papillomavirus-16 and human papillomavirus-18 are most frequently implicated in the pathogenesis of vulvar carcinoma, human papillomavirus-66 can also be regarded as a causative factor. Suspicious lesions should be biopsied, and in the presence of carcinoma, vulvectomy with bilateral lymphadenectomy, if necessary, must be performed. Furthermore, polymerase chain reaction assay analysis with clinical arrays in cytological samples is an accurate test for the detection of a wide range of human papillomavirus genotypes and can be used to verify the infection and specify the human papillomavirus type implicated.
Vulvar carcinoma in older women is seldom associated with any type of human papillomavirus (HPV) infection, representing less than 15% of reported cases . Atypia of the squamous epithelium of the vulva is considered to be a co-factor in the carcinogenesis of vulvar cancer and usually a non-neoplastic lesion, such as vulvar inflammation, lichen sclerosus or hyperplasia of squamous cells, pre-exists . Two models have been suggested for the development of vulvar cancer . Type 1 occurs in relatively young women and is associated with warty or basaloid vulvar intra-epithelial neoplasia. According to its definition, type 2 is represented by keratinizing squamous cell carcinoma and mainly presents in post-menopausal women, and its association with HPV infection is quite rare (< 15%). Although smoking and sexually transmitted diseases are considered to be co-factors in type 1 vulvar cancer, there is a low correlation with type 2 . The most common HPV genotypes in vulvar carcinoma are types 16 and 18, while genotypes 31, 33, 45, 52, 53 and 62 have also been considered as etiological factors [2–5]. HPV-66 is a rare type of α-papillomavirus. Even though the prevalence and distribution of HPV-66 in most studies have depended highly upon the origin of the population involved, in a meta-analysis of carcinomas and intra-epithelial neoplasia of the vulva, vagina and anus, HPV-66, among other rare types, was found in no more than 0.5% of any anogenital carcinomas that were tested . This type has also been associated with cervical intra-epithelial neoplasia 1 lesions and Verruca vulgaris[7, 8]. On the basis of a review of the latest international literature, we found only one other case in which the co-existence of HPV-66 and vulvar carcinoma was reported; however, it was reported in combination with HPV-52 infection .
In this report, we describe the case of an 80-year-old woman of Greek Caucasian origin, gravida 2 para 2, with an undetermined obstetric and gynecologic history. After her second delivery, no data referring to the clinical history of the patient was available because the patient deferred any preventive or diagnostic medical examination until she presented to our hospital. Her last menstrual period had occurred 28 years ago. Her medical history included hypertension and angina pectoris.
Initially, multiple biopsies from the center of the lesion and from the lateral margins were obtained, the histological examination of which revealed the presence of squamous cell carcinoma. At the periphery of the lesion, the squamous stratified epithelium exhibited abnormalities consistent with vulvar intra-epithelial neoplasia (vulvar intra-epithelial neoplasia (VIN) grade I/II). Numerous lesional cells showed koilocytic atypia, which is representative of HPV-related infection. However, initial hybrid capture 2 testing for HPV was negative in all the samples tested, which were obtained from either the center or the periphery of the lesion. A pre-operative computed tomographic scan of the abdomen and inguinal areas showed bilaterally enlarged inguinal lymph nodes with central fusion.
The patient underwent radical vulvectomy extending centrally to the level of the perineal membrane, as well as bilateral inguinal lymphadenectomy. Post-operatively, the woman had no major complications and was discharged 14 days after the procedure.
In the tissue specimen obtained pre-operatively for the performance of liquid-based Cytology (The Thin prep - pap test, Cytyc Corp., Marborough, Massachusetts, USA), we used a polymerase chain reaction (PCR) assay with CLINICAL ARRAYS Human Papillomavirus Kit (Genomica, Madrid, Spain) and a Hybrid Capture 2 HPV DNA test (Digene, Madrid, Spain). The latter test is an accurate qualitative and quantitative method used to detect five low-risk types of HPV (6, 11, 42, 43 and 44) and 13 high-risk types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). It can also determine the viral load. This technology is based on hybrid analysis to identify indentifying a signal enhancement in a microplate using chemiluminescence. Samples containing the target DNA are hybridized with a specific detector HPV RNA. Hybrid-produced RNA-DNA binds to the surface of a microplate coated with specific antibodies for RNA-DNA hybrids. The immobilized hybrids react with alkaline phosphatase (ALK)-conjugated antibodies and are detected with a chemiluminescence substrate. While the binding ALK cleaves the substrate, it attracts light, which is measured by chemiluminescence in relative light units .
In our case, none of the above-mentioned HPV types was detected with the use of hybrid capture 2 testing. The DNA of HPV-66 was the only one detected using the CLINICAL ARRAYS Human Papillomavirus Kit.
On the basis of these results, a standard PCR assay was also performed. Four histological specimens from four different levels of the lesion, as well as from a lymph node with metastasis, were examined. Extraction of total DNA from formalin fixed, paraffin-embedded sections was performed using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The quality and integrity of extracted DNA were assessed by PCR amplification of an amplicon of the interferon (IFN)-γ gene and by agarose gel (0.6%) electrophoresis, respectively. A nested, multiplex, highly sensitive PCR method (approximately 1 fg/103 viral copies) was used for HPV detection and genotyping. In this assay, consensus primers for first-run amplification of a broad spectrum of mucosal HPV genotypes, including all high-risk HPV genotypes, were combined with nested PCR amplifications of type-specific primers. Despite robust detection of the IFN-γ gene product amplification, no positive signal for HPV presence was found in any of the examined samples, even after substantially increasing the PCR cell cycles. Considering that there were clear indications of HPV infection in the microscopic examination of the specimens, the PCR assay was unable to detect the DNA of the virus, most probably because of low viral load in the specimens examined, which could have been caused and amplified by inadequate superficial lesional tissue. Furthermore, tissue fixation techniques could have a negative impact on the preservation of the viral genome.
The presence, coexistence and possible cause of HPV infection in women's anogenital squamous neoplasia have been studied extensively over the past decade. It must be emphasized that the presence of condylomatous lesions does not exclude the possibility of a coexisting invasive malignancy. However, the correlation appears to be stronger as far as intra-epithelial lesions (VIN grade III) are concerned.
HPV-66 is an α-papillomavirus considered to belong among the potentially high-risk mucosal HPV types. Nevertheless, it can also be encountered in benign lesions. Even though this HPV type is reported to be associated with cervical squamous carcinomas, little is known concerning vulvar squamous cell carcinomas. On the other hand, vulvar carcinomas are mainly correlated with HPV-16 and 18 genotypes. In our case report, we describe the case of a woman with a HPV-66-related vulvar carcinoma. This diagnosis was made on the basis of the use of PCR with the CLINICAL ARRAYS Human Papillomavirus Kit.
In conclusion, in patients with HPV-66 infection the possibility of a coexisting invasive malignancy, albeit rare, should be considered even in the presence of benign lesions. Caution should be taken, especially in older women with cancer, as the majority of these cancers are HPV-negative. In cases that raise clinical suspicions of HPV, examination of tissue using PCR with the CLINICAL ARRAYS Human Papillomavirus Kit should be considered in patients with histological features of HPV infection and negative Hybrid Capture 2 assay testing, as it detects a wider range of HPV genotypes than other types of testing, including HPV-66. A standard PCR assay of formalin-fixed samples seemed to be less effective, as it appeared to be affected by sampling, tissue fixation and/or viral load. Patients should be followed up meticulously at short time intervals.
Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
We recognize Dr Kartsiounis Christos for his help in using data from gynecological oncology department archives, Mr Mousidis Ioannis for photographing the histopathological images and Mr Kotsinas Athanassios and Dr Gorgoulis Vasilios of the University of Athens for performing PCR assays in formalin-fixed samples, as well as Dr Destouni Chariklia for her consultation in interpreting the cytological and PCR results.
- Schorge JO, Schaffer JI, Halvorson LM, Hoffman BL, Brandshaw KD, Cunningham FG: Invasive cancer of the vulva. Williams Gynecology. 2008, New York: McGraw-Hill Medical, 665-668.Google Scholar
- Smith JS, Backes DM, Hoots BE, Kurman RJ, Pimenta JM: Human papillomavirus type-distribution in vulvar and vaginal cancers and their associated precursors. Obstet Gynecol. 2009, 113: 917-924.View ArticlePubMedGoogle Scholar
- Sutton BC, Allen RA, Moore WE, Dunn ST: Distribution of human papillomavirus genotypes in invasive squamous carcinoma of the vulva. Mod Pathol. 2008, 21: 345-354. 10.1038/modpathol.3801010.View ArticlePubMedGoogle Scholar
- Venuti A, Marcante ML: Presence of human papillomavirus type 18 DNA in vulvar carcinomas and its integration into the cell genome. J Gen Virol. 1989, 70: 1587-1592. 10.1099/0022-1317-70-6-1587.View ArticlePubMedGoogle Scholar
- Insinga RP, Liaw KL, Johnson LG, Madeleine MM: A systematic review of the prevalence and attribution of human papillomavirus types among cervical, vaginal, and vulvar precancers and cancers in the United States. Cancer Epidemiol Biomarkers Prev. 2008, 17: 1611-1622. 10.1158/1055-9965.EPI-07-2922.View ArticlePubMedPubMed CentralGoogle Scholar
- De Vuyst H, Clifford GM, Nascimento MC, Madeleine MM, Franceschi S: Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer. 2009, 124: 1626-1636. 10.1002/ijc.24116.View ArticlePubMedGoogle Scholar
- Wu D, Cai L, Huang M, Zheng Y, Yu J: Prevalence of genital human papillomavirus infection and genotypes among women from Fujian province, PR China. Eur J Obstet Gynecol Reprod Biol. 2010, 151: 86-90. 10.1016/j.ejogrb.2010.02.040.View ArticlePubMedGoogle Scholar
- Davis MD, Gostout BS, McGovern RM, Persing DH, Schut RL, Pittelkow MR: Large plantar wart caused by human papillomavirus-66 and resolution by topical cidofovir therapy. J Am Acad Dermatol. 2000, 43: 340-343. 10.1016/S0190-9622(00)70287-8.View ArticlePubMedGoogle Scholar
- van de Nieuwenhof HP, van Kempen LC, de Hullu JA, Bekkers RL, Bulten J, Melchers WJ, Massuger LF: The etiologic role of HPV in vulvar squamous cell carcinoma fine tuned. Cancer Epidemiol Biomarkers Prev. 2009, 18: 2061-2067. 10.1158/1055-9965.EPI-09-0209.View ArticlePubMedGoogle Scholar
- Dutra I, Santos MR, Soares M, Couto AR, Bruges-Armas M, Teixeira F, Monjardino L, Hodgson S, Bruges-Armas J: Characterisation of human papillomavirus (HPV) genotypes in the Azorean population, Terceira island. Infect Agent Cancer. 2008, 3: 6-10.1186/1750-9378-3-6.View ArticlePubMedPubMed CentralGoogle Scholar
- García-Sierra N, Martró E, Castellà E, Llatjós M, Tarrats A, Bascuñana E, Díaz R, Carrasco M, Sirera G, Matas L, Ausina V: Evaluation of an array-based method for human papillomavirus detection and genotyping in comparison with conventional methods used in cervical cancer screening. J Clin Microbiol. 2009, 47: 2165-2169. 10.1128/JCM.00402-09.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.