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Genital and cutaneous human papillomavirus (HPV) types in relation to conjunctival squamous cell neoplasia: A case-control study in Uganda

Genital and cutaneous human papillomavirus (HPV) types in relation to conjunctival squamous cell... Background: We investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia (which includes both conjunctival intraepithelial neoplasia (CIN) and carcinoma) using data and biological material collected as part of a case-control study in Uganda. Results: Among 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls (both 38%; Odds ratio [OR] 1.0, 95% confidence interval [CI] 0.4–2.7, p = 1.0). The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls (OR 8.0, 95% CI 1.0–169, p = 0.04). Conclusion: We find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases (which does not differ by histological grade of tumour) indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour. Background intraepithelial neoplasia (CIN) and carcinoma (together In the years before the HIV epidemic, corneo-conjunctival called ocular surface squamous neoplasia (OSSN)) were Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 reported to be more frequent in African countries than in vix is established. In non-melanoma skin carcinogenesis, Europe and the USA [1-3]. Using data from worldwide a role has been suggested for cutaneous HPV types from cancer registries it has been confirmed that incidence of the betapapillomavirus genus. A variety of HPV types has OSSN increases markedly with proximity to the equator, already been identified in some, but not in all, tumour presumably from increasing solar ultraviolet (UV) radia- specimens from several small case series and results from tion [3]. Exposure to UV radiation is an established cause case-control studies have, to date, been inconclusive [2]. of disease. Lesions occur in sun-exposed areas of the eye Here we present results on the association of genital and [4,5], are associated with solar elastosis [4-7] and have cutaneous (from the betapapillomavirus genus) HPV types been shown to contain classical UV-induced p53 muta- in relation to ocular surface epithelial neoplasias from a tions [8]. The incidence of the tumour increases with case-control study in Uganda, together with a review of increasing levels of ambient solar radiation and associa- published evidence. tions with sun exposure and past history of skin cancer have been identified in case-control studies [3,9-11]. Results Additional risk factors may also be important. For exam- Paraffin-embedded biopsy material was available for 81 ple, a polymorphism of TP53 codon 72 has been linked to cases (mean age 35 years) and for 29 controls (mean age an increased risk of neoplasia in one study from Uganda 30 years). Among cases, 17 people had lesions graded as including 107 cases and 115 controls [12]. Exposure to CIN (conjunctival intraepithelial neplasia) I, 18 were CIN dust and ocular trauma have also been suggested as possi- II, 22 were CIN III and 24 people had an invasive carci- ble risk factors, although evidence is scant [1,13]. noma. Fifty two cases (64%) were HIV seropositive, 22 (27%) were seronegative and the HIV serostatus was Since the 1980s there has been a marked increase in cases unknown for seven people (9%). Among controls, 15 of conjunctival neoplasia, mostly in sub-Saharan Africa people had pinguecula, 3 had chronic inflammation, two [14-19]. In Uganda for example, the reported incidence had a pyogenic granuloma, two had a cavernous angioma has more than tripled over the last decade [14,16], partic- and seven had a variety of other diagnoses. Ten controls ularly among younger people and a link with HIV infec- (34%) were HIV seropositive and 19 (66%) were seroneg- tion was suggested in case reports [20-27]. Case-control ative (Table 1). There were significantly more HIV serop- studies in several African countries [11,28-33] and cohort ositive cases than controls (70% (52/74; seven cases had studies in the USA [34,35], have confirmed a roughly 10 unknown serostatus) versus 34%; p < 0.001). fold excess risk of the tumour in HIV infected people com- pared to the uninfected; in Africa the majority of cases are Table 2 shows the prevalence of genital and cutaneous HIV infected [36]. In a recent study of 414 cases in HPV among controls, stratified by age, sex and HIV Uganda, 64% of people with conjunctival neoplasia were serostatus – none of the apparent differences was statisti- HIV infected and this applied to intraepithelial as well as cally significant (at the 5% level). Table 3 shows the pro- to invasive cases [5]. The median CD4+ T lymphocyte portion of cases with evidence of genital or cutaneous count of HIV positive cases at diagnosis has been found in HPV DNA in tumour tissue, stratified by the histological this study to be 111 cells/microL (based on results from grade of the lesion (CIN I – III and invasive carcinoma). 112 HIV infected cases) [5]. Use of antiretroviral therapy The prevalence of genital and cutaneous HPV types did has been shown to cause tumour regression in an other- not differ significantly by histological grade of the lesion, wise inoperable case [37]. A recent report from the USA but at all grades, the prevalence of genital HPV types was did not find strong associations with level of immunosup- higher than that of cutaneous types. For all tumour grades pression in HIV infected people, but the study included combined, this difference was statistically significant only 15 cases of the disease [35]. An excess risk has also (38% versus 22%; p = 0.03). been reported among immunosuppressed cancer patients and organ transplant recipients (although the number of Overall, the prevalence of genital HPV types did not differ cases remains small) [38-42]. between cases and controls (38% [31/81] in cases and 38% [11/29] among controls; OR 1.0, 95% CI 0.4–2.7, p However, the clear excess risk of ocular surface epithelial = 1.0). The genital HPV types identified were 6, 11, 16, 18, dysplasias among HIV infected people (and among 31, 33, 35, 44, 51, 52, 66 and two that were unclassifiable. immunosuppressed renal transplant recipients) suggests a High risk genital types were identified in 13 cases (types role for an underlying infection in the aetiology [43,44]. 16 [eight people], 51 and 66 [in one person], 18, 35, 51 Although an active search for other new oncogenic infec- and 52 [one person each]) and in three controls (types 31 tions is ongoing, no new candidate virus (if one exists) has and 33 [two people]). The most frequently detected geni- yet been identified [45]. A causal relationship between tal type was HPV 11, which was found in 22 cases and in persistent infection with several (high risk) genital human 10 controls. The prevalence of cutaneous HPV types was papillomavirus (HPV) types and cancer of the uterine cer- 22% (18/81) among cases and 3% (1/29) among controls Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 1: Distribution of cases and controls by age, sex and HIV serostatus Percentage of cases (n) n = 81 Percentage of controls (n) n = 29 Age 15–28 27% (22) 38% (11) 29–32 27% (22) 31% (9) 33–70 46% (37) 31% (9) Sex Male 44% (36) 48% (14) Female 56% (45) 52% (15) HIV serostatus Negative 27% (22) 66% (19) Positive 64% (52) 34% (10) Unknown 9% (7) 0% (0) (OR 8.0, 95%CI 1.0–168.5, p = 0.04) and did not differ ever, the prevalence of cutaneous HPV was still relatively significantly between HIV infected and uninfected cases low among cases and did not differ by histological grade and controls (Table 4). The cutaneous HPV types identi- of the lesion. fied were 5, 8, 14, 17, 19, 23, 36, 37, 80, plus 9 that were unclassifiable; HPV 14 was identified in three cases and A comprehensive review of the published literature iden- types 8, 17 and 23 were found in two people each. Evi- tified 12 case reports or case series in which the prevalence dence of infection with more than one HPV type was iden- of HPV in tumour tissue from patients with ocular surface tified in tissue from 19 cases and four controls. squamous neoplasia was investigated [46-57]. Eleven studies tested for HPV 16; seven also looked for evidence Discussion of infection with HPV 18; four studies also included HPV Our findings demonstrate that both genital and cutane- 6 and/or 11, one looked at HPV 2 and in one study the ous HPVs can be found in conjunctival tissue – the genital specific genital HPV type was not specified. Only three types were more frequently identified. However, we found studies included more than 20 cases, the largest having no evidence that genital types were associated with ocular 38. The prevalence of detectable HPV varied from 0% to surface squamous neoplasia. In relation to cutaneous 93% (summarised in Table 5) – much of this variation HPV, results reported here are broadly consistent with might be explained by the differing laboratory methodol- those from two other studies – the prevalence was signifi- ogies employed across individual studies. Sixteen case- cantly higher among cases than among controls. How- control studies were identified and are summarised in Table 6, together with results from this investigation Table 2: The proportion of controls with evidence of infection [7,11,30,58-70]. With the exception of one study, in with HPV, stratified by age, sex and HIV serostatus which HPV type was not specified, all of the studies inves- All HPV Genital HPV Cutaneous-HPV tigated HPV 16, nine also investigated HPV 18 and 5 investigated HPV 45. There is considerable heterogeneity Age in results. For example, in relation to HPV 16, four studies 15–28 27% (3/11) 27% (3/11) 9% (1/11) demonstrated a positive association and eleven showed 29–32 33% (3/9) 33% (3/9) 0% (0/9) no association with ocular surface squamous neoplasia 33–70 56% (5/9) 56% (5/9) 0% (0/9) Table 3: The proportion of cases with evidence of infection with Sex HPV, stratified by histological grade of tumour Male 29% (4/14) 29% (4/14) 0% (0/14) Female 47% (7/15) 47% (7/15) 7% (1/15) All HPV Genital HPV Cutaneous-HPV HIV serostatus CIN I 47% (8/17) 35% (6/17) 29% (5/17) Negative 32% (6/19) 32% (6/19) 0% (0/19) CIN II 56% (10/18) 50% (9/18) 28% (5/18) Positive 50% (5/10) 50% (5/10) 10% (1/10) CIN III 45% (10/22) 27% (6/22) 23% (5/22) Invasive 42% (10/24) 42% (10/24) 13% (3/24) TOTAL 38% (11/29) 38% (11/29) 3% (1/29) TOTAL 47% (38/81) 38% (31/81) 22% (18/81) None of the apparent differences in prevalence of HPV by age, sex or HIV sero-status was statistically significant. 1. χ (trend) = 1.9; p = 0.2 Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 4: The proportion of cases and controls with evidence of infection with HPV, stratified by HIV serostatus 1 2 3 All HPV Genital HPV Cutaneous-HPV 4 4 4 Case Control Odds Ratio Case Control Odds Ratio Case Control Odds Ratio (95% CI) (95% CI) (95% CI) HIV 45% (10/22) 32% (6/19) 1.8 (0.4–7.9) 36% (8/22) 32% (6/19) 1.2 (0.3–5.5) 27% (6/22) 0% (0/19) ∞ (1.8–∞) seronegativ HIV 48% (25/52) 50% (5/10) 0.9 (0.2–4.3) 40% (21/52) 50% (5/10) 0.8 (0.2–3.8) 21% (11/52) 10% (1/10) 2.2 (0.2–52) seropositiv TOTAL 47% (38/81) 38% (11/29) 1.5 (0.6–3.8) 38% (31/81) 38% (11/29) 1.0 (0.4–2.7) 22% (18/81) 3% (1/29) 8.0 (1.0–169) 1. More than one HPV type was identified in tissue from 19 cases and four controls 2. Genital HPV types investigated: 6, 11, 16, 18, 31, 33–35, 39, 40, 42–45, 51–54, 56, 58, 59, 66, 68, 70, 74. Genital HPV types identified: 6, 11, 16, 18, 31, 33, 35, 44, 51, 52, 66, plus two unclassifiable; high risk genital types were identified in 13 cases (types 16 [eight people], 51 and 66 [in one person] 18, 35, 51 and 52 [one person each]) and in three controls (types 31 and 33 [two people]); HPV 11 was most frequently detected (22 cases and 10 controls) 3. Cutaneous HPV types investigated: 5, 8, 9, 12, 14, 15, 17, 19–25, 36–38, 47, 49, 75, 76, 80, 92, 93, 96. Cutaneous HPV types identified: 5, 8, 14, 17, 19, 23, 36, 37, 80, plus 9 unclassifiable; HPV 14 was identified in three cases and types 8, 17 and 23 were found in two people each 4. Among cases, 7 had unknown HIV serostatus (five studies failed to identify HPV 16 in either the cases There is substantial variation in HPV prevalence rates or controls). In most studies, type-specific methods of between different studies, which may have arisen, in part, HPV detection were used and so the types shown in the because of differences in patient selection, sample taking, tables were the only ones that were tested for. preparation and storage and detection method. Even for PCR as a detection system, there are many variables that Only three studies (including this one [68,69]) investi- influence the sensitivity and specificity and so could gated cutaneous HPV types – each demonstrated a signif- impact on the reported prevalence. These include PCR icantly higher prevalence of cutaneous HPV in cases as design (nested, broad spectrum or type-specific), the size compared to controls (summarised in Table 7). Two of of the amplified product and the choice of the polymerase the three studies examined the prevalence according to used. This review was not done to draw attention to these histological grade of tumour (this study and reference 69) differences, but rather to show that there is no consistent and no association was demonstrated in either. evidence for a causal association between HPV and OSSN. In addition, however, it should be noted that the total Table 5: Summary of case series investigating the prevalence of HPV DNA in tumour tissue from patients with ocular surface squamous neoplasias. Study [Reference] Detection method Number HPV positive/total (%) HPV type McDonnell et al, 1987 [46] In situ hybridisation (ISH) 0/28 (0%) HPV 2, 6, 16, 18 McDonnell et al, 1989 [47] PCR 1/1 (100%) HPV 16 Lauer et al, 1990 [48] PCR 4/5 (80%) HPV 16 2/5 (40%) HPV18 Odrich et al, 1991 [49] PCR 2/2 (100%) HPV 16 McDonnell et al, 1992 [50] PCR 33/38 (87%) HPV 16 Tuppurainen et al, 1992 [51] ISH and PCR 0/4 (0%) HPV 6, 11, 16 and 18 Serna et al, 1995 [52] PCR 1/9 (11%) HPV 16 Nakamura et al, 1997 [53] ISH and PCR 2/8 (25%) HPV 16 2/8 (25%) HPV 18 Toth et al, 2000 [54] PCR 5/23 (9%) HPV types not specified Eng et al, 2002 [55] PCR 0/20 (0%) HPV 6, 11, 16, 18 Moubayed et al, 2004 [56] ISH 12/14 (86%) HPV 16 13/14 (93%) HPV 18 12/14 (86%) HPV 6 and 11 Reszec and Sulkowski, 2005 [57] PCR 1/11 (9%) HPV 16 1/11 (9%) HPV 18 Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 6: Summary of case-control studies investigating various HPV types in the aetiology of ocular surface squamous neoplasias Study Detection method Number HPV positive/total (%) HPV type Case Control McDonnell et al, 1986 [58] In situ HPV antigen detection 5/61 (8%) 0/6 (0%) Unknown genital HPV type McDonnell et al, 1989 [59] PCR 6/6 (100%) 0/6 (0%) HPV 16 Saegusa et al, 1995 [60] ISH and PCR 3/8 (38%) 0/12 (0%) HPV 16 Adachi et al, 1995 [61] PCR 1/5 (20%) 0/9 (0%) HPV 16 Waddell et al, 1996 [30] PCR 7/20 (35%) 2/15 (13%) HPV 16 Karcioglu and Issa, 1997 [62] PCR 4/45 (9%) 8/70 (11%) HPV 16 10/45 (22%) 10/70 (14%) HPV 18 Tabrizi et al, 1997 [63] PCR 20/88 (23%) 5/66 (8%) HPV 16 or 18 Dushku et al, 1999 [64] PCR 0/8 (0%) 0/16 (0%) L1 (all types) Palazzi et al, 2000 [65] PCR 2/30 (7%) 1/30 (3%) HPV 16 Scott et al, 2002 [66] ISH and in situ reverse transcriptase PCR 5/10 (50%) 0/5 (0%) HPV 16 5/10 (50%) 0/5 (0%) HPV 18 Newton et al, 2002 [11] Serological analysis 8/39 (21%) 43/418 (10%) HPV 16 4/39 (10%) 16/418 (4%) HPV 18 2/39 (5%) 24/418 (6%) HPV 45 Tulvatana et al, 2003 [7] PCR 0/28 (0%) 0/23 (0%) Multiple types Waddell et al, 2003 [67] Serological analysis 37/253 (15%) 6/37 (16) HPV 16 Ateenyi-Agaba et al, 2004 [68] PCR 0/21 (0%) 0/22 (0%) HPV 16, 18 and 45 0/22 (0%) 2/22 (9%) HPV 11 18/21 (86%) 7/20 (35%) Multiple cutaneous HPV types Tornesello et al, 2006 [69] PCR 0/86 (0%) 1/63 (2%) HPV 6 2/86 (2%) 0/63 (0%) HPV 18 15/86 (17%) 0/63 (0%) Multiple cutaneous HPV types Sen et al, 2007 [70] In situ HPV antigen detection 0/30 (0%) 0/30 (0%) Multiple genital HPV types de Koning et al [this study] PCR 31/81 (38%) 11/29 (38%) Multiple genital HPV types 18/81 (22%) 1/29 (3%) Multiple cutaneous HPV types number of cases and controls studied in this and in other HPV types among 39 cases of cutaneous squamous cell studies, remains relatively small. carcinoma (SCC) for whom plasma was collected prior to diagnosis (incident) and 80 controls was examined [72]. Results reported here are also broadly similar to those Fifteen cases having already developed SCC at blood col- from case-control studies investigating the role of HPV in lection (prevalent) were also tested. There were no statis- the aetiology of cutaneous squamous cell carcinoma tically significant differences in the seroprevalence of (SCC) [71,72]. Moderate associations between cutaneous antibodies against any of the HPV types examined HPV types and cutaneous SCC have been identified, but between incident cases and controls, nor was there a dif- doubt remains about whether this is causal. It has been ference in the seroprevalence of multiple infections. How- suggested that the increased serorecognition of HPV ever, consistent with results from published case-control among cases as compared to controls may arise as a result studies, the seroprevalence against many cutaneous HPV of tumour formation [73]. Some support for this view types was higher among prevalent cases than among comes from a recent small prospective study, in which the either incident cases or controls. This might suggest that if seroprevalence of antibodies against the L1 antigen of 38 HPV is involved in the aetiology of cutaneous squamous Table 7: Summary of case-control studies investigating cutaneous HPV types in the aetiology of ocular surface squamous neoplasias Study [Reference] Prevalence of cutaneous HPV (number/total) Odds Ratio (95% Confidence Interval) and p value Cases Controls Ateenyi-Agaba et al, 2004 [68] 86% (18/21) 35% (7/20) 12.0 (1.7–84.9), p = 0.002 Tornesello et al, 2006 [69] 17% (15/86) 0% (0/63) ∞ (2.5–∞), p = 0.001 de Koning [this study] 22% (18/81) 3% (1/29) 8.0 (1.0–168.5), p = 0.04 1. For comparative purposes, the unadjusted odds ratio is shown 2. Estimated using Fisher exact test Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 carcinoma, the process occurs close to the time of diagno- Conclusion sis, or that the antibody response observed in people with We find no evidence of an association between genital the tumour is a consequence of tumour formation. HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher The possibility that the presence of a tumour facilitates among cases as compared to controls. Although consist- detection of antibodies against HPV is supported by the ent with results from two other case-control studies, the findings of Favre et al (2000), who reported a higher sero- relatively low prevalence of cutaneous HPV types among prevalence of HPV-5 among patients with burns or with cases (which does not differ by histological grade of proliferative cutaneous autoimmune diseases than among tumour) indicates that there remains considerable uncer- controls [74]. Patients with psoriasis, involving abnormal tainty about a role for cutaneous HPV in the aetiology of keratinocyte differentiation and proliferation, have also this tumour. shown a high HPV-5 seroprevalence [75]. This is thought to arise as a consequence of cell proliferation in the skin Methods providing an environment that favours viral replication, Participants From November 1995 to May 2001 in country-wide clin- resulting in a rise in antibodies against the relevant HPV type. Similarly, there is debate concerning the results ics, anyone with a suspect corneo-conjunctival lesion was obtained from studies using tests for cutaneous HPV offered removal and histology, and enrolment in a follow- DNA. The prevalence of HPV DNA was significantly lower up study with home visits. HIV serology was also offered in tumour biopsies than in swabs of the tested lesion [76]. after pre-test counselling. Lesions were photographed and Furthermore, evidence of cutaneous HPV DNA has been details of the eyes and general health were recorded and found to be both highly prevalent and persistent in the analysed in EPI INFO version 6. Those who subsequently healthy population [77]. It is possible that the results turned out to have lesions other than ocular surface squa- reported here reflect a similar situation. However, there is mous neoplasia were used as a control group in the anal- now some preliminary evidence from studies of molecu- yses of HPV. lar mechanisms, suggesting that HPV might interact with ultra-violet radiation disturbing apoptotic pathways and Consent and ethical approval Information about the disease, its treatment and HIV test- leading to cell immortalization [78]. Transforming prop- erties of E6 and E7 proteins of some cutaneous HPV types ing was given in private in vernacular by counsellors, and have also been described (reviewed in reference [71]). It consent confirmed by signature or thumbprint. The study remains to be established what role, if any, HPV plays in was approved by the Science and Ethics Committee of the the pathological processes that lead to the development of Uganda Virus Research Institute, and by the Uganda both conjunctival and cutaneous squamous cell neopla- National Council for Science and Technology. sia. Serology and histopathology It should be noted that the relatively high percentage of Venous blood was taken and screening tests for HIV anti- samples with unclassified cutaneous HPV types could rep- bodies done, with confirmation at the Uganda Virus resent infections with novel types of which only subge- Research Institute (two enzyme immunoassay tests in par- nomic amplicons have been sequenced [79]. However, allel, with Western blot if required). Biopsies went to St the other possibility is that these were infections with low Thomas' Hospital London for histopathology. CIN was copy numbers of one of the 25 tested cutaneous HPV classified (by SBL) into 3 stages according to one, two or types allowing only for general detection and not the three thirds thickness being dysplastic; invasive tumours identification of specific types. With the broad spectrum were diagnosed when the epithelial basement membrane SPF PCR – DEIA (see Methods section) more than 50 was breached. HPV types can be detected. It cannot, therefore, be excluded that the two cases with an indeterminate genital HPV typing HPV result actually represent a cutaneous HPV type. The HPV analyses were performed on DNA isolated from for- SPF -LiPA system amplifies a small fragment from 65 malin-fixed, paraffin-embedded specimens. Chances of 10 25 base pairs and is therefore very suitable for the testing of contamination during the cutting of the sections were paraffin-embedded, formalin-fixed samples. Although the minimised by discarding the initial section that was cut to conjunctiva represent mucosal tissue, the detection of remove any environmental contamination which had genital HPV types in 40% of the HIV seronegative cases occurred while blocks were stored and by changing cryo- and in 32% of the HIV seronegative controls was unex- stat blades in between sections. DNA was extracted from pected. This finding indicates that the natural history of the sections in a cabinet which had been UV-treated to HPV and their tissue tropism is not fully understood. remove any contaminating DNA. Additionally, 15 nega- tive DNA isolation controls were included. For both the Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 genital HPV test and the beta HPV test, 10 μl of a 20 ng/μl used in this study, with assistance from KP, CP and CH. SL DNA solution per specimen was used as input for the PCR conducted the histopathology. RN conducted the statisti- analyses. Genital HPV genotyping was carried out using cal analyses. The manuscript was drafted by RN and MK. the SPF -LiPA system (SPF HPV LiPA, version 1; man- All authors read, contributed to and approved the manu- 10 25 10 ufactured by Labo Bio-Medical Products, Rijswijk, The script. Netherlands) as described previously [80,81]. 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Br J Ophthalmol 2007, Authors' contributions 91:120-1. 19. Waddell KM, Newton R: The aetiology and associations of con- KW, JM, RD and RN conducted the original study and col- junctival intraepithelial neoplasia – further evidence. Br J Oph- lected all the biological material used for work described thalmol 2007, 91(1):120-121. here. MK and WQ developed the HPV assays, which MK Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 20. Winward KE, Curtin VT: Conjunctival squamous cell carcinoma 42. Vajdic CM, van Leeuwen MT, McDonald SP, McCredie MR, Law M, in a patient with human immunodeficiency virus infection. Chapman JR, Webster AC, Kaldor JM, Grulich AE: Increased inci- Am J Ophthalmol 1989, 107(5):554-555. dence of squamous cell carcinoma of the eye after kidney 21. Kim RY, Seiff SR, Howes EL Jr, O'Donnell JJ: Necrotizing scleritis transplantation. 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J "BioMed Central will be the most significant development for Pathol 2006, 208:165-75. disseminating the results of biomedical researc h in our lifetime." 79. Forslund O, Iftner T, Andersson K, Lindelof B, Hradil E, Nordin P, Sir Paul Nurse, Cancer Research UK Stenquist B, Kirnbauer R, Dillner J, de Villiers EM, Viraskin Study Group: Cutaneous human papillomaviruses found in sun- Your research papers will be: exposed skin: beta-papillomavirus species 2 predominates in available free of charge to the entire biomedical community squamous cell carcinoma. J Infect Dis 2007, 196:876-83. 80. Kleter B, van Doorn LJ, Ter Schegget J, et al.: Novel short-fragment peer reviewed and published immediately upon acceptance PCR assay for highly sensitive broad-spectrum detection of cited in PubMed and archived on PubMed Central anogenital human papillomaviruses. Am J Pathol 1998, 153:1731-9. yours — you keep the copyright 81. 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Genital and cutaneous human papillomavirus (HPV) types in relation to conjunctival squamous cell neoplasia: A case-control study in Uganda

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Springer Journals
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Copyright © 2008 by de Koning et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-3-12
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18783604
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Abstract

Background: We investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia (which includes both conjunctival intraepithelial neoplasia (CIN) and carcinoma) using data and biological material collected as part of a case-control study in Uganda. Results: Among 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls (both 38%; Odds ratio [OR] 1.0, 95% confidence interval [CI] 0.4–2.7, p = 1.0). The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls (OR 8.0, 95% CI 1.0–169, p = 0.04). Conclusion: We find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases (which does not differ by histological grade of tumour) indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour. Background intraepithelial neoplasia (CIN) and carcinoma (together In the years before the HIV epidemic, corneo-conjunctival called ocular surface squamous neoplasia (OSSN)) were Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 reported to be more frequent in African countries than in vix is established. In non-melanoma skin carcinogenesis, Europe and the USA [1-3]. Using data from worldwide a role has been suggested for cutaneous HPV types from cancer registries it has been confirmed that incidence of the betapapillomavirus genus. A variety of HPV types has OSSN increases markedly with proximity to the equator, already been identified in some, but not in all, tumour presumably from increasing solar ultraviolet (UV) radia- specimens from several small case series and results from tion [3]. Exposure to UV radiation is an established cause case-control studies have, to date, been inconclusive [2]. of disease. Lesions occur in sun-exposed areas of the eye Here we present results on the association of genital and [4,5], are associated with solar elastosis [4-7] and have cutaneous (from the betapapillomavirus genus) HPV types been shown to contain classical UV-induced p53 muta- in relation to ocular surface epithelial neoplasias from a tions [8]. The incidence of the tumour increases with case-control study in Uganda, together with a review of increasing levels of ambient solar radiation and associa- published evidence. tions with sun exposure and past history of skin cancer have been identified in case-control studies [3,9-11]. Results Additional risk factors may also be important. For exam- Paraffin-embedded biopsy material was available for 81 ple, a polymorphism of TP53 codon 72 has been linked to cases (mean age 35 years) and for 29 controls (mean age an increased risk of neoplasia in one study from Uganda 30 years). Among cases, 17 people had lesions graded as including 107 cases and 115 controls [12]. Exposure to CIN (conjunctival intraepithelial neplasia) I, 18 were CIN dust and ocular trauma have also been suggested as possi- II, 22 were CIN III and 24 people had an invasive carci- ble risk factors, although evidence is scant [1,13]. noma. Fifty two cases (64%) were HIV seropositive, 22 (27%) were seronegative and the HIV serostatus was Since the 1980s there has been a marked increase in cases unknown for seven people (9%). Among controls, 15 of conjunctival neoplasia, mostly in sub-Saharan Africa people had pinguecula, 3 had chronic inflammation, two [14-19]. In Uganda for example, the reported incidence had a pyogenic granuloma, two had a cavernous angioma has more than tripled over the last decade [14,16], partic- and seven had a variety of other diagnoses. Ten controls ularly among younger people and a link with HIV infec- (34%) were HIV seropositive and 19 (66%) were seroneg- tion was suggested in case reports [20-27]. Case-control ative (Table 1). There were significantly more HIV serop- studies in several African countries [11,28-33] and cohort ositive cases than controls (70% (52/74; seven cases had studies in the USA [34,35], have confirmed a roughly 10 unknown serostatus) versus 34%; p < 0.001). fold excess risk of the tumour in HIV infected people com- pared to the uninfected; in Africa the majority of cases are Table 2 shows the prevalence of genital and cutaneous HIV infected [36]. In a recent study of 414 cases in HPV among controls, stratified by age, sex and HIV Uganda, 64% of people with conjunctival neoplasia were serostatus – none of the apparent differences was statisti- HIV infected and this applied to intraepithelial as well as cally significant (at the 5% level). Table 3 shows the pro- to invasive cases [5]. The median CD4+ T lymphocyte portion of cases with evidence of genital or cutaneous count of HIV positive cases at diagnosis has been found in HPV DNA in tumour tissue, stratified by the histological this study to be 111 cells/microL (based on results from grade of the lesion (CIN I – III and invasive carcinoma). 112 HIV infected cases) [5]. Use of antiretroviral therapy The prevalence of genital and cutaneous HPV types did has been shown to cause tumour regression in an other- not differ significantly by histological grade of the lesion, wise inoperable case [37]. A recent report from the USA but at all grades, the prevalence of genital HPV types was did not find strong associations with level of immunosup- higher than that of cutaneous types. For all tumour grades pression in HIV infected people, but the study included combined, this difference was statistically significant only 15 cases of the disease [35]. An excess risk has also (38% versus 22%; p = 0.03). been reported among immunosuppressed cancer patients and organ transplant recipients (although the number of Overall, the prevalence of genital HPV types did not differ cases remains small) [38-42]. between cases and controls (38% [31/81] in cases and 38% [11/29] among controls; OR 1.0, 95% CI 0.4–2.7, p However, the clear excess risk of ocular surface epithelial = 1.0). The genital HPV types identified were 6, 11, 16, 18, dysplasias among HIV infected people (and among 31, 33, 35, 44, 51, 52, 66 and two that were unclassifiable. immunosuppressed renal transplant recipients) suggests a High risk genital types were identified in 13 cases (types role for an underlying infection in the aetiology [43,44]. 16 [eight people], 51 and 66 [in one person], 18, 35, 51 Although an active search for other new oncogenic infec- and 52 [one person each]) and in three controls (types 31 tions is ongoing, no new candidate virus (if one exists) has and 33 [two people]). The most frequently detected geni- yet been identified [45]. A causal relationship between tal type was HPV 11, which was found in 22 cases and in persistent infection with several (high risk) genital human 10 controls. The prevalence of cutaneous HPV types was papillomavirus (HPV) types and cancer of the uterine cer- 22% (18/81) among cases and 3% (1/29) among controls Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 1: Distribution of cases and controls by age, sex and HIV serostatus Percentage of cases (n) n = 81 Percentage of controls (n) n = 29 Age 15–28 27% (22) 38% (11) 29–32 27% (22) 31% (9) 33–70 46% (37) 31% (9) Sex Male 44% (36) 48% (14) Female 56% (45) 52% (15) HIV serostatus Negative 27% (22) 66% (19) Positive 64% (52) 34% (10) Unknown 9% (7) 0% (0) (OR 8.0, 95%CI 1.0–168.5, p = 0.04) and did not differ ever, the prevalence of cutaneous HPV was still relatively significantly between HIV infected and uninfected cases low among cases and did not differ by histological grade and controls (Table 4). The cutaneous HPV types identi- of the lesion. fied were 5, 8, 14, 17, 19, 23, 36, 37, 80, plus 9 that were unclassifiable; HPV 14 was identified in three cases and A comprehensive review of the published literature iden- types 8, 17 and 23 were found in two people each. Evi- tified 12 case reports or case series in which the prevalence dence of infection with more than one HPV type was iden- of HPV in tumour tissue from patients with ocular surface tified in tissue from 19 cases and four controls. squamous neoplasia was investigated [46-57]. Eleven studies tested for HPV 16; seven also looked for evidence Discussion of infection with HPV 18; four studies also included HPV Our findings demonstrate that both genital and cutane- 6 and/or 11, one looked at HPV 2 and in one study the ous HPVs can be found in conjunctival tissue – the genital specific genital HPV type was not specified. Only three types were more frequently identified. However, we found studies included more than 20 cases, the largest having no evidence that genital types were associated with ocular 38. The prevalence of detectable HPV varied from 0% to surface squamous neoplasia. In relation to cutaneous 93% (summarised in Table 5) – much of this variation HPV, results reported here are broadly consistent with might be explained by the differing laboratory methodol- those from two other studies – the prevalence was signifi- ogies employed across individual studies. Sixteen case- cantly higher among cases than among controls. How- control studies were identified and are summarised in Table 6, together with results from this investigation Table 2: The proportion of controls with evidence of infection [7,11,30,58-70]. With the exception of one study, in with HPV, stratified by age, sex and HIV serostatus which HPV type was not specified, all of the studies inves- All HPV Genital HPV Cutaneous-HPV tigated HPV 16, nine also investigated HPV 18 and 5 investigated HPV 45. There is considerable heterogeneity Age in results. For example, in relation to HPV 16, four studies 15–28 27% (3/11) 27% (3/11) 9% (1/11) demonstrated a positive association and eleven showed 29–32 33% (3/9) 33% (3/9) 0% (0/9) no association with ocular surface squamous neoplasia 33–70 56% (5/9) 56% (5/9) 0% (0/9) Table 3: The proportion of cases with evidence of infection with Sex HPV, stratified by histological grade of tumour Male 29% (4/14) 29% (4/14) 0% (0/14) Female 47% (7/15) 47% (7/15) 7% (1/15) All HPV Genital HPV Cutaneous-HPV HIV serostatus CIN I 47% (8/17) 35% (6/17) 29% (5/17) Negative 32% (6/19) 32% (6/19) 0% (0/19) CIN II 56% (10/18) 50% (9/18) 28% (5/18) Positive 50% (5/10) 50% (5/10) 10% (1/10) CIN III 45% (10/22) 27% (6/22) 23% (5/22) Invasive 42% (10/24) 42% (10/24) 13% (3/24) TOTAL 38% (11/29) 38% (11/29) 3% (1/29) TOTAL 47% (38/81) 38% (31/81) 22% (18/81) None of the apparent differences in prevalence of HPV by age, sex or HIV sero-status was statistically significant. 1. χ (trend) = 1.9; p = 0.2 Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 4: The proportion of cases and controls with evidence of infection with HPV, stratified by HIV serostatus 1 2 3 All HPV Genital HPV Cutaneous-HPV 4 4 4 Case Control Odds Ratio Case Control Odds Ratio Case Control Odds Ratio (95% CI) (95% CI) (95% CI) HIV 45% (10/22) 32% (6/19) 1.8 (0.4–7.9) 36% (8/22) 32% (6/19) 1.2 (0.3–5.5) 27% (6/22) 0% (0/19) ∞ (1.8–∞) seronegativ HIV 48% (25/52) 50% (5/10) 0.9 (0.2–4.3) 40% (21/52) 50% (5/10) 0.8 (0.2–3.8) 21% (11/52) 10% (1/10) 2.2 (0.2–52) seropositiv TOTAL 47% (38/81) 38% (11/29) 1.5 (0.6–3.8) 38% (31/81) 38% (11/29) 1.0 (0.4–2.7) 22% (18/81) 3% (1/29) 8.0 (1.0–169) 1. More than one HPV type was identified in tissue from 19 cases and four controls 2. Genital HPV types investigated: 6, 11, 16, 18, 31, 33–35, 39, 40, 42–45, 51–54, 56, 58, 59, 66, 68, 70, 74. Genital HPV types identified: 6, 11, 16, 18, 31, 33, 35, 44, 51, 52, 66, plus two unclassifiable; high risk genital types were identified in 13 cases (types 16 [eight people], 51 and 66 [in one person] 18, 35, 51 and 52 [one person each]) and in three controls (types 31 and 33 [two people]); HPV 11 was most frequently detected (22 cases and 10 controls) 3. Cutaneous HPV types investigated: 5, 8, 9, 12, 14, 15, 17, 19–25, 36–38, 47, 49, 75, 76, 80, 92, 93, 96. Cutaneous HPV types identified: 5, 8, 14, 17, 19, 23, 36, 37, 80, plus 9 unclassifiable; HPV 14 was identified in three cases and types 8, 17 and 23 were found in two people each 4. Among cases, 7 had unknown HIV serostatus (five studies failed to identify HPV 16 in either the cases There is substantial variation in HPV prevalence rates or controls). In most studies, type-specific methods of between different studies, which may have arisen, in part, HPV detection were used and so the types shown in the because of differences in patient selection, sample taking, tables were the only ones that were tested for. preparation and storage and detection method. Even for PCR as a detection system, there are many variables that Only three studies (including this one [68,69]) investi- influence the sensitivity and specificity and so could gated cutaneous HPV types – each demonstrated a signif- impact on the reported prevalence. These include PCR icantly higher prevalence of cutaneous HPV in cases as design (nested, broad spectrum or type-specific), the size compared to controls (summarised in Table 7). Two of of the amplified product and the choice of the polymerase the three studies examined the prevalence according to used. This review was not done to draw attention to these histological grade of tumour (this study and reference 69) differences, but rather to show that there is no consistent and no association was demonstrated in either. evidence for a causal association between HPV and OSSN. In addition, however, it should be noted that the total Table 5: Summary of case series investigating the prevalence of HPV DNA in tumour tissue from patients with ocular surface squamous neoplasias. Study [Reference] Detection method Number HPV positive/total (%) HPV type McDonnell et al, 1987 [46] In situ hybridisation (ISH) 0/28 (0%) HPV 2, 6, 16, 18 McDonnell et al, 1989 [47] PCR 1/1 (100%) HPV 16 Lauer et al, 1990 [48] PCR 4/5 (80%) HPV 16 2/5 (40%) HPV18 Odrich et al, 1991 [49] PCR 2/2 (100%) HPV 16 McDonnell et al, 1992 [50] PCR 33/38 (87%) HPV 16 Tuppurainen et al, 1992 [51] ISH and PCR 0/4 (0%) HPV 6, 11, 16 and 18 Serna et al, 1995 [52] PCR 1/9 (11%) HPV 16 Nakamura et al, 1997 [53] ISH and PCR 2/8 (25%) HPV 16 2/8 (25%) HPV 18 Toth et al, 2000 [54] PCR 5/23 (9%) HPV types not specified Eng et al, 2002 [55] PCR 0/20 (0%) HPV 6, 11, 16, 18 Moubayed et al, 2004 [56] ISH 12/14 (86%) HPV 16 13/14 (93%) HPV 18 12/14 (86%) HPV 6 and 11 Reszec and Sulkowski, 2005 [57] PCR 1/11 (9%) HPV 16 1/11 (9%) HPV 18 Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 Table 6: Summary of case-control studies investigating various HPV types in the aetiology of ocular surface squamous neoplasias Study Detection method Number HPV positive/total (%) HPV type Case Control McDonnell et al, 1986 [58] In situ HPV antigen detection 5/61 (8%) 0/6 (0%) Unknown genital HPV type McDonnell et al, 1989 [59] PCR 6/6 (100%) 0/6 (0%) HPV 16 Saegusa et al, 1995 [60] ISH and PCR 3/8 (38%) 0/12 (0%) HPV 16 Adachi et al, 1995 [61] PCR 1/5 (20%) 0/9 (0%) HPV 16 Waddell et al, 1996 [30] PCR 7/20 (35%) 2/15 (13%) HPV 16 Karcioglu and Issa, 1997 [62] PCR 4/45 (9%) 8/70 (11%) HPV 16 10/45 (22%) 10/70 (14%) HPV 18 Tabrizi et al, 1997 [63] PCR 20/88 (23%) 5/66 (8%) HPV 16 or 18 Dushku et al, 1999 [64] PCR 0/8 (0%) 0/16 (0%) L1 (all types) Palazzi et al, 2000 [65] PCR 2/30 (7%) 1/30 (3%) HPV 16 Scott et al, 2002 [66] ISH and in situ reverse transcriptase PCR 5/10 (50%) 0/5 (0%) HPV 16 5/10 (50%) 0/5 (0%) HPV 18 Newton et al, 2002 [11] Serological analysis 8/39 (21%) 43/418 (10%) HPV 16 4/39 (10%) 16/418 (4%) HPV 18 2/39 (5%) 24/418 (6%) HPV 45 Tulvatana et al, 2003 [7] PCR 0/28 (0%) 0/23 (0%) Multiple types Waddell et al, 2003 [67] Serological analysis 37/253 (15%) 6/37 (16) HPV 16 Ateenyi-Agaba et al, 2004 [68] PCR 0/21 (0%) 0/22 (0%) HPV 16, 18 and 45 0/22 (0%) 2/22 (9%) HPV 11 18/21 (86%) 7/20 (35%) Multiple cutaneous HPV types Tornesello et al, 2006 [69] PCR 0/86 (0%) 1/63 (2%) HPV 6 2/86 (2%) 0/63 (0%) HPV 18 15/86 (17%) 0/63 (0%) Multiple cutaneous HPV types Sen et al, 2007 [70] In situ HPV antigen detection 0/30 (0%) 0/30 (0%) Multiple genital HPV types de Koning et al [this study] PCR 31/81 (38%) 11/29 (38%) Multiple genital HPV types 18/81 (22%) 1/29 (3%) Multiple cutaneous HPV types number of cases and controls studied in this and in other HPV types among 39 cases of cutaneous squamous cell studies, remains relatively small. carcinoma (SCC) for whom plasma was collected prior to diagnosis (incident) and 80 controls was examined [72]. Results reported here are also broadly similar to those Fifteen cases having already developed SCC at blood col- from case-control studies investigating the role of HPV in lection (prevalent) were also tested. There were no statis- the aetiology of cutaneous squamous cell carcinoma tically significant differences in the seroprevalence of (SCC) [71,72]. Moderate associations between cutaneous antibodies against any of the HPV types examined HPV types and cutaneous SCC have been identified, but between incident cases and controls, nor was there a dif- doubt remains about whether this is causal. It has been ference in the seroprevalence of multiple infections. How- suggested that the increased serorecognition of HPV ever, consistent with results from published case-control among cases as compared to controls may arise as a result studies, the seroprevalence against many cutaneous HPV of tumour formation [73]. Some support for this view types was higher among prevalent cases than among comes from a recent small prospective study, in which the either incident cases or controls. This might suggest that if seroprevalence of antibodies against the L1 antigen of 38 HPV is involved in the aetiology of cutaneous squamous Table 7: Summary of case-control studies investigating cutaneous HPV types in the aetiology of ocular surface squamous neoplasias Study [Reference] Prevalence of cutaneous HPV (number/total) Odds Ratio (95% Confidence Interval) and p value Cases Controls Ateenyi-Agaba et al, 2004 [68] 86% (18/21) 35% (7/20) 12.0 (1.7–84.9), p = 0.002 Tornesello et al, 2006 [69] 17% (15/86) 0% (0/63) ∞ (2.5–∞), p = 0.001 de Koning [this study] 22% (18/81) 3% (1/29) 8.0 (1.0–168.5), p = 0.04 1. For comparative purposes, the unadjusted odds ratio is shown 2. Estimated using Fisher exact test Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 carcinoma, the process occurs close to the time of diagno- Conclusion sis, or that the antibody response observed in people with We find no evidence of an association between genital the tumour is a consequence of tumour formation. HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher The possibility that the presence of a tumour facilitates among cases as compared to controls. Although consist- detection of antibodies against HPV is supported by the ent with results from two other case-control studies, the findings of Favre et al (2000), who reported a higher sero- relatively low prevalence of cutaneous HPV types among prevalence of HPV-5 among patients with burns or with cases (which does not differ by histological grade of proliferative cutaneous autoimmune diseases than among tumour) indicates that there remains considerable uncer- controls [74]. Patients with psoriasis, involving abnormal tainty about a role for cutaneous HPV in the aetiology of keratinocyte differentiation and proliferation, have also this tumour. shown a high HPV-5 seroprevalence [75]. This is thought to arise as a consequence of cell proliferation in the skin Methods providing an environment that favours viral replication, Participants From November 1995 to May 2001 in country-wide clin- resulting in a rise in antibodies against the relevant HPV type. Similarly, there is debate concerning the results ics, anyone with a suspect corneo-conjunctival lesion was obtained from studies using tests for cutaneous HPV offered removal and histology, and enrolment in a follow- DNA. The prevalence of HPV DNA was significantly lower up study with home visits. HIV serology was also offered in tumour biopsies than in swabs of the tested lesion [76]. after pre-test counselling. Lesions were photographed and Furthermore, evidence of cutaneous HPV DNA has been details of the eyes and general health were recorded and found to be both highly prevalent and persistent in the analysed in EPI INFO version 6. Those who subsequently healthy population [77]. It is possible that the results turned out to have lesions other than ocular surface squa- reported here reflect a similar situation. However, there is mous neoplasia were used as a control group in the anal- now some preliminary evidence from studies of molecu- yses of HPV. lar mechanisms, suggesting that HPV might interact with ultra-violet radiation disturbing apoptotic pathways and Consent and ethical approval Information about the disease, its treatment and HIV test- leading to cell immortalization [78]. Transforming prop- erties of E6 and E7 proteins of some cutaneous HPV types ing was given in private in vernacular by counsellors, and have also been described (reviewed in reference [71]). It consent confirmed by signature or thumbprint. The study remains to be established what role, if any, HPV plays in was approved by the Science and Ethics Committee of the the pathological processes that lead to the development of Uganda Virus Research Institute, and by the Uganda both conjunctival and cutaneous squamous cell neopla- National Council for Science and Technology. sia. Serology and histopathology It should be noted that the relatively high percentage of Venous blood was taken and screening tests for HIV anti- samples with unclassified cutaneous HPV types could rep- bodies done, with confirmation at the Uganda Virus resent infections with novel types of which only subge- Research Institute (two enzyme immunoassay tests in par- nomic amplicons have been sequenced [79]. However, allel, with Western blot if required). Biopsies went to St the other possibility is that these were infections with low Thomas' Hospital London for histopathology. CIN was copy numbers of one of the 25 tested cutaneous HPV classified (by SBL) into 3 stages according to one, two or types allowing only for general detection and not the three thirds thickness being dysplastic; invasive tumours identification of specific types. With the broad spectrum were diagnosed when the epithelial basement membrane SPF PCR – DEIA (see Methods section) more than 50 was breached. HPV types can be detected. It cannot, therefore, be excluded that the two cases with an indeterminate genital HPV typing HPV result actually represent a cutaneous HPV type. The HPV analyses were performed on DNA isolated from for- SPF -LiPA system amplifies a small fragment from 65 malin-fixed, paraffin-embedded specimens. Chances of 10 25 base pairs and is therefore very suitable for the testing of contamination during the cutting of the sections were paraffin-embedded, formalin-fixed samples. Although the minimised by discarding the initial section that was cut to conjunctiva represent mucosal tissue, the detection of remove any environmental contamination which had genital HPV types in 40% of the HIV seronegative cases occurred while blocks were stored and by changing cryo- and in 32% of the HIV seronegative controls was unex- stat blades in between sections. DNA was extracted from pected. This finding indicates that the natural history of the sections in a cabinet which had been UV-treated to HPV and their tissue tropism is not fully understood. remove any contaminating DNA. Additionally, 15 nega- tive DNA isolation controls were included. For both the Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:12 http://www.infectagentscancer.com/content/3/1/12 genital HPV test and the beta HPV test, 10 μl of a 20 ng/μl used in this study, with assistance from KP, CP and CH. SL DNA solution per specimen was used as input for the PCR conducted the histopathology. RN conducted the statisti- analyses. Genital HPV genotyping was carried out using cal analyses. The manuscript was drafted by RN and MK. the SPF -LiPA system (SPF HPV LiPA, version 1; man- All authors read, contributed to and approved the manu- 10 25 10 ufactured by Labo Bio-Medical Products, Rijswijk, The script. Netherlands) as described previously [80,81]. Briefly, the broad spectrum SPF PCR amplifies a 65-base pair frag- References 1. Templeton AC: Tumours of the eye and adnexa. Tumours of a ment from the L1 region of the HPV genome. By using Tropical Country: A survey of Uganda 1964–1968. Recent Result Cancer biotinylated reverse primers the amplimers could be cap- Research 1973, 41:203-214. tured onto streptavidin-coated microtiter plates. After 2. Newton R: A review of the aetiology of squamous cell carci- noma of the conjunctiva. Br J Cancer 1996, 74:1511-1513. denaturation of the PCR products by alkaline treatment, a 3. Newton R, Ferlay J, Reeves G, Beral V, Parkin DM: Incidence of defined cocktail of digoxigenin-labeled probes was used squamous cell carcinoma of the eye increases with increas- to detect HPV positive samples. This method that is desig- ing levels of ambient solar ultraviolet radiation. Lancet 1996, i:1450-1. nated the HPV DNA Enzyme Immunoassay (DEIA) pro- 4. 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J "BioMed Central will be the most significant development for Pathol 2006, 208:165-75. disseminating the results of biomedical researc h in our lifetime." 79. Forslund O, Iftner T, Andersson K, Lindelof B, Hradil E, Nordin P, Sir Paul Nurse, Cancer Research UK Stenquist B, Kirnbauer R, Dillner J, de Villiers EM, Viraskin Study Group: Cutaneous human papillomaviruses found in sun- Your research papers will be: exposed skin: beta-papillomavirus species 2 predominates in available free of charge to the entire biomedical community squamous cell carcinoma. J Infect Dis 2007, 196:876-83. 80. Kleter B, van Doorn LJ, Ter Schegget J, et al.: Novel short-fragment peer reviewed and published immediately upon acceptance PCR assay for highly sensitive broad-spectrum detection of cited in PubMed and archived on PubMed Central anogenital human papillomaviruses. Am J Pathol 1998, 153:1731-9. yours — you keep the copyright 81. 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