Journal of Medical Virology 86:1556–1559 (2014)

Presence of Betapapillomavirus in Kaposi Sarcoma Lesions Alltalents T. Murahwa,1,2 Faith C. Muchemwa,1* Kerina Duri,1 Margaret Z. Borok,3 Russell B. Kanyera,1 Monalisa T. Manhanzva,2 Munyaradzi P. Mapingure,4 and Babill Stray-Pedersen5 1

Department of Immunology, College of Health Sciences, University of Zimbabwe, Avondale, Harare, Zimbabwe Department of Medical Laboratory Sciences, College of Health Sciences, University of Zimbabwe, Avondale, Harare, Zimbabwe 3 Department of Medicine, College of Health Sciences, University of Zimbabwe, Avondale, Harare, Zimbabwe 4 Research Support Centre, College of Health Sciences, University of Zimbabwe, Avondale, Harare, Zimbabwe 5 Division of Obstetrics and Gynecology, Institute of Clinical Medicine, Rikshospitalet, Oslo University Hospital, Oslo, Norway 2

Human herpes virus 8 (HHV 8) is recognized as the necessary cause of Kaposi sarcoma (KS) and in the recent past the human papillomavirus (HPV) has been linked to the development of cutaneous basal cell and squamous cell carcinomas. In a cross sectional study investigating Beta-HPV infections in skin lesions, an unexpected occurrence of HPV DNA was found in KS lesions of HIV infected individuals. Of the 18 KS cases included in the study 16 (89%) had HPV DNA detected. The most common Betapapillomavirus types were HPV14 [15 cases (83.3%)], HPV12 [8 cases (44.4%)], and HPV24 [7 cases (39%)]. Multiple Beta-HPV types were detected in 10 (62.5%) of the participants with HPV DNA positive lesions; of these 7 had a CD4þ count below 350 cells/ml and 3 had CD4þ counts above 350 cells/ml. The presence of Beta-HPV DNA in KS lesions is a newly described phenomenon. Further studies to elucidate the role of Beta-HPV in KS need to be conducted as it is possible that HHV 8 may not be the solitary viral carcinogen in KS tumorigenesis. J. Med. Virol. 86:1556–1559, 2014.

occurrence of KS is significantly associated with HIV infection, several studies have shown increased incidence of KS among HIV infected individuals and the synergistic interactions between these two viruses [Cattelan et al., 2001; Newton et al., 2006; Sullivan et al., 2008]. HPV is the most commonly implicated virus in many human malignancies, 5.2% of all cancers are attributable to HPV infection [Parkin and Bray, 2006]. The involvement of HPV in cervical, penile, oral, genital, and laryngeal cancers and cutaneous lesions such as skin warts, squamous cell carcinomas (SCC), and basal cell carcinomas (BCC) has been documented extensively [Alba and Cararach, 2009]. There is an increasing body of evidence linking HPV to non-melanoma skin cancers [Plasmeijer et al., 2011; Viarisio et al., 2011]. Several molecular epidemiological and sero-epidemiological surveys done in various populations have verified the association between HPV and non-melanoma skin cancers (NMSC) [Bouwes Bavinck et al., 2010; Zakrzewska et al., 2012; Iannacone et al., 2013]. The possibility of HHV 8 and HPV co-existence and synergy has hardly been explored. This study reports

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INTRODUCTION Human herpes virus 8 (HHV 8) formerly named Kaposi sarcoma-associated herpesvirus (KSHV) was discovered in 1994 as a cause for Kaposi’s sarcoma (KS) [Kemeny et al., 1997]. HHV 8 has been firmly established as the viral etiology of KS [Li et al., 1998; Renwick et al., 1998; Nsubuga et al., 2008]. The C 2014 WILEY PERIODICALS, INC. 

Abbreviations: HHV 8, human herpesvirus 8; HPV, human papillomavirus; KS, Kaposi sarcoma Grant sponsor: Letten Foundation (Oslo, Norway); Grant sponsor: International Clinical Operational and Health Services Research Training Award (ICOHRTA) The authors state no conflict of interest.  Correspondence to: Faith C. Muchemwa, Department of Immunology, College of Health Sciences, University of Zimbabwe, A178 Avondale, Harare, Zimbabwe. E-mail: [email protected] Accepted 14 March 2014 DOI 10.1002/jmv.23944 Published online 7 April 2014 in Wiley Online Library (wileyonlinelibrary.com).

Betapapillomavirus in Kaposi Sarcoma Lesions

the findings from a set of KS skin biopsies, which were analyzed for the presence of Beta-HPV DNA. MATERIALS AND METHODS Patient Recruitment and Sample Collection Study participants were part of a study to determine the prevalence of cutaneous Beta papillomavirus infections among Black Zimbabweans in which 98 non-melanoma skin cancer, 34 cutaneous wart, and 18 KS biopsies were collected from participants for HPV DNA analysis. The current study included the 18 patients who had skin lesions confirmed to be KS from previous histological records and who were on treatment for KS. Blood samples for an HIV rapid test were collected from all participants willing to participate in the study and a CD4þ count was done for the samples that were HIV positive. Samples were collected between June 2011 and December 2012. Tissue biopsies were formalin fixed and embedded in paraffin wax on the same day to reduce the genotoxic effects of formalin [Tan and Ding, 2006]. The wax-embedded biopsies were archived until all samples were collected. Ethical Approval Permission to carry out the study was sought from the Joint Research Ethics Committee of the University of Zimbabwe College of Health Sciences and Parirenyatwa Hospital (JREC). The Medical Research Council of Zimbabwe (MRCZ) granted ethical approval. Informed consent was obtained from all participants. Laboratory Tests Tissue biopsy preparation. The procedure for preparation of the tissue biopsies was adopted as described elsewhere [Shi et al., 2002; Jung et al., 2004; Keohavong et al., 2004]. The formalin fixed paraffin embedded (FFPE) tissue blocks were melted on a hot plate and cut in to small pieces of the tissue, 1–2 mm. The tissues were deparaffinized in xylene thrice in a 65˚C water bath for 15 min each time. To remove the xylene, the tissue was washed in decreasing concentrations of ethanol, 90%, 70%, and 50% and finally dried in an incubator at 40˚C to remove residual ethanol. DNA extraction from tissue. DNA extraction was done using the Invisorb kit (Stratec Molecular, Berlin, Germany). The dried tissue pieces were then treated in proteinase K overnight at 60˚C in a shaking water bath. The resulting cell lysate was subjected to DNA extraction according to manufacturer’s instructions. HPV DNA genotyping. Genotyping was done using the Diassay kit (Diassay B.V, Rijswijk, Netherlands). The assay detects 25 known Betapapillomaviruses (5, 8, 9, 12, 14, 15, 17, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96). The

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procedure involves an amplification step and a reverse hybridization step, described elsewhere [de Koning et al., 2009]. HPV plasmids 5, 8, and 38 obtained from the HPV Reference Laboratory (DKFZ, Germany) courtesy of J. Dillner and E.M. De Villiers were used as positive controls in the assays. The PCR amplicons were run on a 3% agarose gel to check for an 117 bp amplicon and then genotyped. Statistical Analysis Stata version 10 (StataCorp LP, TX) was used for analyzing the data. Baseline description of study participants was presented using percentages for categorical variables such as sex, and mean (standard deviation) for continuous variables, or median and range where the variable was not normally distributed. Prevalence of HPV genotypes found in KS lesions was presented as percentages. Multiple infections were determined with a HPV multiple infection being defined as infection with two or more HPV subtypes. RESULTS AND DISCUSSION Demographics and Characteristics of Study Participants The mean age (SD) of the participants was 42 (9.4) years ranging from 21 to 59 years. Most of the patients were in the 30–44 years age group. All the KS patients were HIV infected. The mean CD4þ cell count was 298 cells/ml ranging from 0 to 734 cells/ml. The majority of the participants were male (Table I). HPV DNA in KS Lesions The prevalence of Beta-HPVs in KS lesions from HIV positive patients in this study is very high (89%). However, the prevalence of HPV in normal healthy skin of HIV positive patients is not known in the Black Zimbabwean population and needs to be explored. It has been demonstrated elsewhere that the frequency of Beta-HPVs in normal skin from immunocompromised patients such as organ transplant recipients is very high [de Koning et al., 2009]. The occurrence of Beta-HPV DNA in KS lesions has

TABLE I. Baseline Demographic Characteristics of Study Participants Variable Sex Male Female Age 15–29 30–44 45–59 HIV infected CD4 count >350

Presence of Betapapillomavirus in Kaposi sarcoma lesions.

Human herpes virus 8 (HHV 8) is recognized as the necessary cause of Kaposi sarcoma (KS) and in the recent past the human papillomavirus (HPV) has bee...
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