A case-control study of cutaneous squamous cell carcinoma among Caucasian organ transplant recipients: The role of antibodies against human papillomavirus and other risk factors
Article first published online: 14 APR 2009
Copyright © 2009 UICC
International Journal of Cancer
Volume 125, Issue 8, pages 1935–1945, 15 October 2009
How to Cite
Casabonne, D., Lally, A., Mitchell, L., Michael, K. M., Waterboer, T., Pawlita, M., Imko-Walczuk, B., Wojnarowska, F., Proby, C., Harwood, C. and Newton, R. (2009), A case-control study of cutaneous squamous cell carcinoma among Caucasian organ transplant recipients: The role of antibodies against human papillomavirus and other risk factors. Int. J. Cancer, 125: 1935–1945. doi: 10.1002/ijc.24511
- Issue published online: 19 AUG 2009
- Article first published online: 14 APR 2009
- Accepted manuscript online: 14 APR 2009 12:00AM EST
- Manuscript Accepted: 19 MAR 2009
- Manuscript Received: 4 FEB 2009
- Barts and the London Charitable Foundation. Grant Number: RAC404
- European Commission. Grant Number: QLK2-CT-2002-0117
- Cancer Research UK
- Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik
- Jakub Hr. Potocki's Foundation
- Oxford Radcliffe Hospitals Charitable Fund
Vol. 126, Issue 5, 1282, Article first published online: 11 DEC 2009
- human papillomavirus (HPV);
- cutaneous squamous cell carcinoma (SCC);
- organ transplant recipients
A case-control study was conducted in 140 people with histology proven cutaneous squamous cell carcinoma (SCC) and 454 controls, nested within 2 cohorts of organ transplant recipients (OTR) recruited in London and Oxford between 2002 and 2006. All participants had a skin examination, completed a questionnaire and had serum tested for antibodies against the L1 antigen of 34 HPV types using Luminex technology. SCC was more common in men than women (odds ratio [OR] = 1.7, 95% confidence interval [CI]: 1.1–2.8, p = 0.02) and in people with susceptibility to burn easily (OR = 3.0, 95%CI: 1.9–4.8; p < 0.001). The risk increased with increasing age (p-trend < 0.001), increasing time since transplant (p-trend < 0.001), increasing self-reported number of sunburns as a child (p-trend < 0.001) and with the presence of viral warts (p < 0.001). As expected, antibodies against HPV 16 were associated with a self-reported history of an abnormal cervical smear among women (OR 5.1, 95%CI: 2.6–10.2) and antibodies against HPV 6 were associated with a self-reported history of genital warts (OR 4.0, 95%CI: 2.2–7.2). However, no clear associations between any of the HPV types examined (including cutaneous betaHPVs) and SCC were identified. For example, the seroprevalence of HPV 5 was 15% among cases and 9% among controls (p = 0.09) and the seroprevalence of HPV 8 was 23% among cases and 21% among controls (p = 0.6). Nor was seropositivity to multiple types associated with SCC. These serological data do not provide evidence for a role for HPV in the aetiology of cutaneous SCC among OTR in two UK-based populations. © 2009 UICC
Immunosuppressed organ transplant recipients (OTR) have a higher risk of non-melanoma skin cancer than the general population. Furthermore, cutaneous squamous cell carcinomas (SCC) occur significantly more frequently than basal cell carcinomas (BCC), reversing the ratio usually found in the general population (4:1).1, 2 Ultraviolet radiation (UVR) is the main established risk factor both for SCC and BCC.3 The strength of the association between SCC and immunosuppression, particularly after solid organ transplantation, parallels that seen for other virally associated post-transplant cancers; certain HPV types have long been proposed as potential candidates.4 However, while the oncogenic mechanism of HPV in cancers of the uterine cervix is well-understood and the causative association is now established, it remains uncertain what role, if any, HPV plays in the aetiology of skin cancer.
DNA of cutaneous HPV appears to be ubiquitous and persistent in the skin and hair follicles of healthy individuals.5, 6 Higher prevalence of cutaneous HPV-DNA has been found in the normal skin of immunosuppressed compared to immunocompetent people7 and putative novel types are often detected.5 Studies relying on HPV-DNA detection confront problems not only of contamination, a major consideration given the known ubiquity of HPV, but also the complication that HPV prevalence varies both with the type of sample (hair follicle, skin biopsy or swab) and with the section of the sample examined (e.g. skin surface or deeper within the specimen).8 To date, there are no available serological case-control data on the seroprevalence of antibodies against HPV in high-risk transplant populations. Seroprevalence studies in immunocompetent patients have not consistently found associations with specific HPV types (reviewed in Ref. 9).9–19 A direct comparison of prevalence between these studies is difficult, however, since different laboratory technologies were used and a limited number of divergent HPV types examined.
Here, we describe the results of a case-control study of cutaneous SCC, nested within 2 cohorts of OTR recruited in the UK at 2 regional transplant centres 100 km apart (London and Oxford). We assess the role of possible risk factors for SCC, including the relationship between the tumour and the prevalence of antibodies against the major capsid protein L1 of 34 HPV types, measured using Luminex technology.
Material and methods
We conducted a case-control study of 140 people with histology proven cutaneous squamous cell carcinoma (SCC) and 454 controls, nested within two cohorts of OTR (570 patients [96%] with renal transplant, 12 patients [2%] with kidney-pancreas transplant, 7 patients [1%] with heart transplant and 5 patients [1%] with lung, lung-kidney, liver, heart-lung-kidney and heart-lung transplants) recruited in London and in Oxford. All transplant recipients from Oxford Radcliffe Hospitals and from the Barts and London NHS Trust were invited to participate in the study and were recruited between October 2002 and August 2006. Patients underwent transplantation between 1964 and 2005 in Oxford and between 1972 and 2006 in London. In London, all patients have access to a dedicated dermatology clinic after their usual visit to the transplant centre, are seen routinely within 6–12 months of transplant and undergo routine dermatological examinations thereafter at which all benign and malignant lesions are recorded and treated if necessary. For the present study, patients were recruited at routine clinic visits and completed a questionnaire delivered by a specialist nurse and were examined by a dermatologist. In Oxford, patients are referred to a dermatologist if a suspicious skin lesion is present, but are not otherwise under routine surveillance. Therefore, OTR attending the Oxford Transplant Centre were invited by mail to take part in the study and to complete a questionnaire. At the next clinic visit, this questionnaire was checked and finalised by a dermatologist who also conducted an examination of the participants' skin, recording all benign and malignantcutaneous lesions. Treatments were initiated where indicated and educational information relating to the risks of skin cancer in OTR was also provided. In both centres, a blood sample was taken and serum, buffy coat and red blood cells were separated, aliquoted and frozen at −80°C.
The same questionnaire was used in both centres to collect information on (i) social and demographic details (age, sex, height, weight, ethnicity, marital status, educational level, area of residence, country of birth); (ii) smoking and alcohol history; (iii) medical history (skin and/or other cancers, psoriasis); (iv) exposure to ultraviolet (UV) radiation (outdoor occupation and hobbies, sun exposure before and after transplantation, sun exposure currently, number of moles and freckles before and after transplantation, history of sunburn in childhood, protective measures against UV radiation, time spent abroad); (v) history of HPV-related viral infection (cutaneous and genital warts and history of abnormal smear in women); (vi) transplantation and dialysis (number of transplantations, dates, type of dialysis, time spent on dialysis before and after transplantation, primary diagnosis); (vii) gynaecological and reproductive history for women (age at menopause, number of pregnancies, use of hormonal contraception, hormone replacement therapy, surgical removal of uterus). As it is not clear how cutaneous HPV are transmitted, the questionnaire also included some questions on possible risk factors for infection (e.g. shared bedroom or bed as a child, number of siblings and number in household, as surrogates for crowding and proximity). No information on HLA or other infections was collected. Current immunosuppressive treatment at recruitment was asked but we did not have full history on specific or combined immunosuppressive treatments over time and therefore felt that we could not address this issue based on our study design. All information on transplantation, medications and skin cancers was cross-checked against information held in the renal-centre database and medical records.
Patients: Case and control status
A flow chart of the recruitment process is shown in Figure 1. Only malignant lesions with confirmed pathology verification of diagnosis were included. Patients were classified as cases if review of histology records revealed evidence of SCC with or without other skin cancers. Of the 145 patients with SCC, 70 (48%) had SCC with or without in situ carcinoma of the skin (CIS), 66 (46%) had SCC with a history of BCC and 9 (6%) had SCC with a history of other non-melanoma skin cancers (porocarcinoma, Merkel cell carcinoma, eccrine nodular carcinoma or tricholemmal carcinoma) and with or without BCC. Controls were patients without confirmed diagnosis of skin cancer. In the course of the study, 20 patients from the London group developed their first SCC and were excluded from the control group (prospective data will be examined after a sufficient period of follow-up).
Overall, we relied on results on the unmatched analyses as none of the main factors (age at recruitment, time since transplantation or sex) was associated with HPV seropositivity for cutaneous types (Table III). However, due to discrepancies between cases and controls in terms of age at recruitment, time since transplantation and sex distributions we randomly selected a subgroup of controls after matching cases on centre, sex, age at recruitment (±5 years) and time since transplantation (±5 years). Final number of cases and controls by centre, ethnicity, availability of blood specimen and/or completed questionnaire are summarised in Figure 1.
The study in London has been approved by the East London and City Health Authority Research Ethics Committee and in Oxford by the Mid and South Buckinghamshire Local Research Ethics Committee. Informed consent was obtained for all participants.
HPV antibody detection was by multiplex serology, an antibody detection method that is based on a glutathione S-transferase (GST) capture enzyme-linked immunosorbent assay, as previously described20, 21 in combination with fluorescent bead technology.22, 23 All antigens were expressed in E. coli as double fusion of full-length viral proteins with a N-terminal GST domain and a C-terminal peptide consisting of the last 11 amino acids from the large T antigen of simian virus 40.20 The expression constructs for the full-length L1 proteins of all HPV types analyzed here (1, 2, 3, 4, 5, 6, 7, 8, 9, 13, 15, 16, 17, 20, 23, 24, 27, 36, 38, 41, 48, 49, 50, 60, 63, 65, 75, 76, 92, 93, 95, 96, 101 and 103) are described in detail elsewhere.9, 21, 24 Glutathione-casein was coupled to internally fluorescence-labelled polystyrene beads (Luminex, Austin, TX), and fusion proteins were affinity-purified on the beads directly in a one-step procedure. Beads with GST and the C-terminal peptide alone were prepared for background determination. Binding of the antigens (i.e. the GST fusion proteins) to various bead sets was verified with a monoclonal antibody against the common C-terminal peptide.20 The differently labelled bead sets carrying different antigens were mixed and incubated in 96-well plates with human plasma diluted 1:100 in blocking buffer, as described previously.23 The analyses were performed blinded with respect to the case or control status of the samples. Antibodies bound to the beads via the viral antigens were then stained with biotinylated anti-human immunoglobulin and fluorescent reporter conjugate streptavidin-R-phycoerythrin. Antibodies bound to antigens on beads were quantified via the reporter fluorescence in the Luminex analyzer, which also identified the internal bead colour and thus the antigen carried by the bead. Antibody quantity was determined as the median R-phycoerythrin fluorescence intensity (MFI) from at least 100 beads of the same internal colour after subtraction of background reactivity (GST and C-terminal peptide alone). For all HPV types but HPV6 analyzed here, MFI cut-offs to define seropositivity for all antigens were set to 200 MFI as described and discussed previously.9, 24 To reduce the influence of borderline seropositive sera, a stringent (doubled) cut-off of 400 MFI was applied to HPV6. In our previous analysis,9 data analysis using geometric mean MFI values instead of cut-off values did not materially change the results.
As non-melanoma skin cancers occur mainly in Caucasian populations, analyses looking at SCC were restricted to Caucasian patients. Analyses looking at the association between seropositivity to HPV16 and a self-reported history of abnormal smear test and between seropositivity to HPV6 and self-reported history of genital warts were not restricted to Caucasian patients. Odds ratios (OR) were estimated to measure the association between SCC and risk factors from the questionnaire using conditional on centre logistic regression adjusted for sex, age at recruitment (<45, 45–59, ≥60 years), time since transplantation (<5, 5–9 years, ≥10 years) and skin type (I and II, III and IV). Skin type was defined using Fitzpatrick classification scale as follows: (I) never tans, always burns, (II) rarely tans, usually burns, (III) usually tans, can burn and (IV) always tans, rarely burns. As the number of patients who answered the questionnaire differed from the ones that gave specimens (Fig. 1), the figures in tables do not always compare directly between analyses involving HPV results and those based on questionnaire only. Where results are presented in the form of plots, black circles indicate the odds ratios and horizontal lines represent 95% confidence intervals (CI).
To examine the relationship between seropositivity to a single HPV type or multiple HPV seropositivity (seronegative to all, seropositive to 1 type, seropositive to 2 or more) and SCC, odds ratios were estimated using conditional logistic regression on centre and adjusted for sex, age at recruitment (<45, 45–59, ≥ 60 years), time since transplantation (<5, 5 to 9, ≥10 years) and skin type (I and II, III and IV). Multiple HPV seropositivity was also examined by species within genus. All analyses were also performed for each centre separately using unconditional logistic regression adjusted for the same factors. Conditional logistic regression on centre, sex, age at recruitment (±5 years) and time since transplantation (±5 years) and adjusted for skin type (I and II, III and IV) was used to estimate odds ratio of the matched analysis.
Four sensitivity analyses were performed to explore further the association between HPV and SCC (i) with CIS included as cases; (ii) with cases restricted to SCC only; (iii) restricted to cases diagnosed 4 years before recruitment; (iv) fully adjusted model for sex, age at recruitment [<44, 45–59, ≥60 years], time since transplantation [<5, 5–9, ≥10 years], skin type [I and II, III and IV], number of sunburns before the age of 18 [none, 1–2, ≥3], birth order [1, 2, ≥3] and currently living with partner or being married [yes, no]. To deal with multiple significance tests, agreement between results of the 2 centres was used to detect genuine associations and level of statistical significance was set to 1%. Missing value categories were added to adjustment variables with incomplete information in order to retain all the observations in the analysis. Likelihood ratio tests were used to assess heterogeneity and trend tests (obtained by treating the categorical variable as a continuous variable in the model). All p values were two-sided. Data not shown are available upon request. Statistical analyses were carried out using STATA 9 (StataCorp, 2005, College Station, TX).
The characteristics of the study populations are shown in Table I. There was no statistically significant difference between London and Oxford in terms of sex, age at recruitment, time since transplantation and skin type distributions of cases and controls. In relation to the matched analysis, the male to female ratio was 2 (61/30), the mean age at recruitment was 58.5 (standard deviation [SD]: 9.7) and 58.9 (SD: 9.8) years, and the mean time since transplantation was 14.5 (SD: 6.7) and 14.7 (SD: 6.8) years for controls and cases, respectively.
|Controls N=182||Cases N=50||Controls N=243||Cases N=89||Controls N=425||Cases N=139||Controls N=91||Cases N=91|
|Sex [ratio (number), M/F]||1.5 (110/72)||2.1 (34/16)||1.6 (150/93)||2.2 (61/28)||1.5 (260/165)||2.2 (95/44)||2.0 (61/30)||2.0 (61/30)|
|Age at recruitment [mean (SD), in years]||48 (13)||61 (10)||47 (13)||58 (10)||47 (13)||59 (10)||58.5 (9.7)||58.9 (9.8)|
|Time since transplantation [mean (SD), In years]||9 (7)||17 (9)||10 (7)||15 (7)||9 (7)||16 (7)||14.5 (6.7)||14.7 (6.8)|
|Skin type [number with type I or II (%)]1||46 (26)||21 (43)||87 (38)||58 (66)||133 (33)||79 (58)||–||–|
The mean time between transplantation and the development of the first skin cancer was 11.9 years [SD: 7.3 years] for SCC and 10.0 years [SD: 6.7 years] for BCC. The median time between diagnosis of SCC and recruitment was −4 years [IQ25= −2 years and IQ75= −7 years]. Patients with SCC had an average of 5.2 lesions [SD: 8.5] and those with BCC had 3.5 lesions [SD: 4.2] (data not shown). As expected, BCC were more often detected on non sun-exposed skin only (25%) than SCC (12%) (p = 0.01).25, 26 For SCC, the main body sites involved were the head (39%), hand/wrist (28%), arms (12%) whereas for BCC, lesions tended to occur on the head (58%), the back (19%) and on the chest/shoulder (8%). In contrast to SCC, BCC were uncommon on hands (2%) (data not shown).
Risk factors from the questionnaire in relation to SCC
Figures 2 and 3 show the odds ratios for OTR with SCC (140 patients) compared to controls (454 patients) for various risk factors examined using data from the questionnaire. Men were more likely to develop SCC than women (p = 0.02) and odds ratios for SCC also increased with increasing age at recruitment (p-trend < 0.001) and with time since transplantation (p-trend < 0.001). Cases were 13 times (95% CI: 6.3–28.1) more likely than controls to have been transplanted more than 10 years prior to recruitment. Patients with skin type I and II were also more likely to develop SCC than those with skin type III and IV (p < 0.001). A history of actinic keratoses (AKs) or cutaneous warts was positively associated with the development of SCC. Transplant data variables and self-reported history of non-cutaneous cancer, psoriasis, genital warts or herpes zoster was not associated with the development of SCC; nor were any of the data for women relating to sexual and reproductive factors. Being born first compared to being born second or third or higher order was associated with increased odds of developing SCC (p-value for trend = 0.02). A self-report of being married or currently living with a partner was associated with an increased risk of SCC (OR: 2.1, 95% CI: 1.2–3.6: p = 0.01). Current smokers appeared less likely to develop SCC than controls but this finding was driven by data from only 1 centre (London). There was no significant association between SCC and types of academic qualification, eye or hair colour, birth country, body mass index, physical activity or markers of crowding and proximity. Regarding UVR exposure, the number of sunburns before the age of 18 years was positively associated with SCC (p-value for trend < 0.002; 3 or more burns vs. never: OR: 3.3, 95% CI: 1.8–6.2). Cases with SCC were more likely to have had holidays in sunny countries after transplantation but no clear dose-relationship was detected (p-trend = 0.08). Patients with SCC and controls did not differ in terms of other markers of UV exposure. Conditional on centre logistic regression with all potential confounding variables in the model (sex, age at recruitment [less than 44, 45–59,60 or more], time since transplantation [less than 5 years, 5–9 years, 10 or more years], skin type [I and II, III and IV], number of sunburns before the age of 18 [none, 1–2, 3 or more], birth order [1, 2, 3 or more] and currently living with partner or being married [yes, no]) did not produce any material difference for any of the factors involved (data not shown). Risk factors (age at recruitment, sex, time since transplantation, skin type, birth order, number of sunburns before the age of 18, currently living with partner or being married) that were found to be linked with SCC in the pooled analyses were also found to be associated with SCC when each centre was examined separately (data not shown).
HPV16 and HPV6 in relation to history of cytological abnormalities and genital warts
Table II shows the odds ratio for HPV16 seropositivity associated with a self-reported history of abnormal smear tests and for HPV6 seropositivity associated with self-reported history of genital warts using data from all study participants (irrespective of ethnic group) and for each centre separately. As expected, highly statistically significant associations (p ≤ 0.003) were observed after adjustment for confounding variables when data were pooled and when each centre was examined separately.
|Risk factor||Centre (No. never/ever)||Never No. POS (%)||Ever No. POS (%)||adjusted OR 1 (95%CI)||p-value|
|Self-reported abnormal smear test2,3|
|HPV16||Both (215/56)||32 (15)||28 (50)||5.1 (2.6-10.2)||<0.001|
|Oxford (83/27)||9 (11)||13 (48)||8.6 (2.5-29.4)||<0.001|
|London (132/29)||23 (17)||15 (52)||4.3 (1.8-10.5)||0.001|
|Self-reported history of genital warts3|
|HPV6||Both (688/53)||172 (25)||31 (58)||4.0 (2.2-7.2)||<0.001|
|Oxford (270/26)||51 (19)||15 (58)||4.6 (1.9-11.2)||<0.001|
|London (418/27)||121 (29)||16 (59)||3.4 (1.5-7.6)||0.003|
Association between HPV seropositivity and age at recruitment, time since transplantation, sex and skin type among Caucasian control OTR
Table III shows seroprevalence of the 34 HPV types by sex, age at recruitment, time since transplantation and skin type among Caucasian controls (N = 425). As expected, higher HPV seroprevalence was observed in women for HPV16 (25% in female vs. 10% in male) and seroprevalence for mucosal HPV types (16, 6 and 13) decreased with increasing age (p-trend < 0.01). Apart from an increase in seroprevalence of HPV4 with time since transplantation (p-trend = 0.01) and a decrease in seroprevalence of HPV65 with increasing age (p-trend < 0.001), no clear association was found with time since transplantation for any of the other HPV types examined. Similarly, no association was found between HPV seroprevalence and skin phototype.
Seropositivity to a single HPV type in relation to SCC
In Table IV, the seroprevalence of HPV types is examined among cases and controls. There was no statistically significant difference at the 1% level between the prevalence of antibodies against any of the HPV types between cases with SCC and controls. The seroprevalence of antibodies against mucosal HPV types in all patients with prevalent SCC were similar than in the control group but age was a strong confounder of the association producing a 2-fold increased risk of SCC in patients seropositive to any of the 3 mucosal HPV types examined. The seroprevalence of antibody to HPV5 was statistically significantly higher in patients with SCC from Oxford (p = 0.02) but not in cases from London (p = 0.5) (data not shown). The seroprevalence of antibodies against HPV17 was consistently higher among patients with SCC (all: 37/119 [31%]; Oxford: 14/50 [28%]; London: 23/69 [33%]) compared to controls (all: 100/425 [24%]; Oxford: 39/182 [21%]; London: 61/243 [25%]), but this result was not significant at the 5% level (data not shown). Matching did not produce any material differences in results.
Seropositivity to multiple HPV types in relation to SCC
In Table V, the relationship between the presence of antibodies against multiple HPV types (seronegative, positive to 1 or positive to 2 or more) and the risk of SCC is shown for cases and for controls among all patients and by centre. Overall, there was no statistically significant difference in multiple HPV seroprevalence for any of the examined genera between cases and controls at the 1% level except for mucosal HPV types due to the strong confounding effect of age. Further matched analyses did not substantially alter the results.
|Genus Seropositivity to:||BOTH CENTRES|
|Controls N=425 no (%)||no (%)||Cases1N=119 OR2 (95%CI)||p-value trend||Controls N=91 no (%)||no (%)||Cases1N=91 OR3 (95%CI)||p-value trend|
|negative||250 (59)||67 (56)||ref||64 (70)||52 (57)||ref|
|1||127 (30)||37 (31)||1.7 (1.0 to 3.0)||22 (24)||30 (33)||1.5 (0.7 to 3.0)|
|2 or more||48 (11)||15 (13)||2.8 (1.2 to 6.6)||0.007||5 (6)||9 (10)||2.7 (0.8 to 9.6)||0.09|
|negative||285 (67)||90 (76)||ref||63 (69)||69 (76)||ref|
|1||92 (22)||12 (10)||0.4 (0.2 to 0.9)||20 (22)||10 (11)||0.3 (0.1 to 1.0)|
|2 or more||48 (11)||17 (14)||1.6 (0.7 to 3.4)||0.6||8 (9)||12 (13)||1.9 (0.6 to 6.1)||0.9|
|any alpha types||241 (57)||64 (54)||1.1 (0.9 to 1.4)||0.4||45 (49)||50 (55)||1.1 (0.8 to 1.5)||0.5|
|any alpha cutaneous||140 (33)||29 (24)||0.8 (0.4 to 1.3)||0.3||28 (31)||22 (24)||0.7 (0.3 to 1.5)||0.3|
|any alpha mucosal||175 (41)||52 (44)||1.9 (1.1 to 3.2)||0.02||27 (30)||39 (43)||1.7 (0.8 to 3.3)||0.1|
|negative||188 (44)||51 (43)||ref||34 (37)||39 (43)||ref|
|1||76 (18)||18 (15)||0.9 (0.4 to 1.9)||0.5||21 (23)||13 (14)||0.5 (0.2 to 1.3)|
|2||161 (38)||50 (42)||1.2 (0.7 to 2.1)||36 (40)||39 (43)||1.0 (0.5 to 2.0)||1.0|
|negative||292 (69)||80 (67)||ref||63 (69)||63 (69)||ref|
|1||58 (14)||18 (15)||1.2 (0.6 to 2.5)||0.7||15 (16)||13 (14)||0.9 (0.4 to 2.0)|
|2||75 (18)||21 (18)||1.1 (0.6 to 2.1)||13 (14)||15 (16)||1.2 (0.5 to 2.8)||0.7|
|negative||248 (58)||64 (54)||ref||45 (50)||47 (51)||ref|
|1||73 (17)||21 (18)||1.1 (0.6 to 2.3)||0.4||23 (25)||18 (20)||0.9 (0.4 to 2.0)|
|2||104 (24)||34 (29)||1.3 (0.7 to 2.3)||23 (25)||26 (29)||1.2 (0.5 to 2.8)||0.6|
|negative||328 (77)||85 (71)||ref||71 (78)||65 (71)||ref|
|1||48 (11)||14 (12)||1.1 (0.5 to 2.3)||8 (9)||10 (11)||1.8 (0.2 to 1.3)|
|2||49 (12)||20 (17)||1.3 (0.6 to 2.6)||0.5||12 (13)||16 (18)||1.0 (0.5 to 2.0)||0.2|
|any beta types||237 (56)||68 (57)||1.1 (0.7 to 1.9)||0.6||57 (62)||52 (57)||0.8 (0.4 to 1.5)||0.5|
|any beta species 1||133 (31)||39 (33)||1.2 (0.7 to 2.0)||0.6||28 (31)||28 (31)||1.0 (0.6 to 1.9)||0.9|
|any beta species 2||177 (42)||55 (46)||1.2 (0.7to 2.0)||0.4||46 (51)||44 (48)||1.0 (0.5 to 1.9)||0.9|
|negative||255 (53)||57 (48)||ref||50 (55)||47 (52)||ref|
|1||92 (22)||29 (24)||1.2 (0.6 to 2.2)||20 (22)||19 (21)||1.0 (0.4 to 2.1)|
|2 or more||108 (25)||33 (28)||1.3 (0.7 to 2.4)||0.3||21 (23)||25 (27)||1.3 (0.7 to 2.8)||0.5|
|negative||247 (58)||65 (55)||ref||52 (57)||51 (56)||ref|
|1||86 (20)||30 (25)||1.2 (0.7 to 2.2)||20 (22)||21 (23)||1.0 (0.5 to 2.2)|
|2 or more||92 (22)||24 (20)||1.0 (0.5 to 1.9)||0.9||19 (21)||19 (21)||1.0 (0.5 to 2.1)||0.9|
|any gamma types||200 (47)||62 (52)||1.3 (0.8 to 2.1)||0.4||41 (45)||44 (48)||1.2 (0.6 to 2.1)||0.6|
|any gamma species 1||178 (42)||55 (46)||1.1 (0.7 to 1.6)||0.7||39 (43)||40 (44)||1.0 (0.6 to 1.9)||0.9|
|negative||379 (89)||103 (87)||ref||80 (88)||78 (86)||ref|
|positive||46 (11)||16 (13)||1.5 (0.7 to 3.2)||0.3||11 (12)||13 (14)||1.4 (0.6 to 3.5)||0.4|
|negative||264 (62)||74 (62)||ref||65 (71)||55 (60)||ref|
|1||100 (24)||30 (25)||1.1 (0.6 to 2.0)||10 (11)||24 (26)||3.9 (1.4 to 10.8)|
|2 or more||61 (14)||15 (13)||0.8 (0.4 to 1.8)||0.8||16 (18)||12 (13)||1.1 (0.5 to 2.8)||0.3|
|negative||378 (89)||102 (85)||ref||77 (85)||77 (85)||ref|
|1||38 (9)||13 (11)||1.2 (0.5 to 2.7)||12 (13)||11 (12)||1.1 (0.4 to 2.6)|
|2 or more||9 (2)||4 (3)||1.1 (0.3 to 4.8)||0.7||2 (2)||3 (3)||2.1 (0.3 to 13.6)||0.5|
|Any HPV types|
|negative||59 (14)||12 (10)||ref||11 (12)||8 (9)||ref|
|1 or 2||136 (32)||39 (33)||2.0 (0.8 to 4.7)||33 (36)||31 (34)||1.8 (0.5 to 6.4)|
|3 to 6||123 (29)||33 (28)||1.7 (0.7 to 4.0)||27 (30)||27 (30)||1.8 (0.5 to 6.0)|
|7 or more||107 (25)||35 (29)||2.4 (1.0 to 5.8)||0.1||20 (22)||25 (27)||2.5 (0.7 to 9.3)||0.2|
|any HPV types||366 (86)||108 (91)||2.1 (0.9 to 4.8)||0.07||80 (88)||83 (91)||1.9 (0.6 to 5.9)||0.3|
Fully adjusted and sensitivity analyses
Fully adjusted (for sex, age at recruitment [less than 44, 45–59,60 or more], time since transplantation [less than 5 years, 5 to 9 years, 10 or more years], skin type [I and II, III and IV], number of sunburns before the age of 18 [none, 1–2, 3 or more], birth order [1, 2, 3 or more] and currently living with partner or being married [yes, no]) conditional on centre logistic regression did not produce any material difference (data not shown). Analyses looking at the association between HPV and SCC with different case status or restricted to cases with SCC diagnosed within 4 years of recruitment did also not produce any material differences in results (data not shown).
This is the first nested case-control study conducted in a population of OTR that has examined the relationship between antibodies against HPV-L1 proteins and the risk of cutaneous SCC. Seropositivity to any HPV type was high in both cases and controls with only 10% of cases and 14% of controls being seronegative to all of the 34 HPV types tested. Despite the fact that OTR are at a greatly increased risk of SCC compared to the general population, there remains limited power to examine associations with all the different HPV types and to detect small or moderate effect size, in part because the prevalence of some is low. Consequently, some associations might not have reached statistical significance. However, the study was conducted in 2 separate centres that were nonetheless in close geographical proximity, both to increase numbers and to compare and validate findings across centres.
In accordance with all of the published literature, patients with SCC were more likely to be older, to have fairer skin, to have been transplanted for a longer period of time and to have had a history of AKs than controls. We also found that men were more likely to have SCC than women, a finding that has also been identified in some other studies,27 but not in all.28 Although the risk of SCC is thought to increase with lifetime cumulative sun exposure,29 a higher number of sunburns before the age of 18 has also been associated with SCC in Australia30 and a moderate association was also observed in a recent multi-centre study.31 The increased odds of having SCC in patients being married or living with a partner is likely to be a screening effect—a partner may identify lesions not seen by the patient themselves.32, 33 To our knowledge, the association between birth order and the presence of cutaneous SCC has not been reported elsewhere. The possibility of a chance finding is reduced by the fact that the trend was observed in both centres. Birth order has also been inversely associated with allergic disease or eczema34, 35 in some studies. In this context, it is hypothesised that individuals born first lack early exposure to infectious agents and are therefore more susceptible to develop certain conditions than their siblings.36 However, its significance in this context remains highly speculative however.
As expected, we found a statistically significant (and well-established) association between self-reported history of cervical cytological abnormalities and/or genital warts and HPV16 or HPV6, respectively. This was present in both centres and validated the methodology used for HPV serological detection. In contrast, there was no consistent relationship between the presence of antibodies against any of the HPV types examined and SCC even, after adjustment for multiple confounding factors. In addition, it is also notable that the prevalence of specific HPV types was not as high among cases in our study as would be expected on the basis of studies of other oncogenic viruses (such as HPV16 in relation to cervical cancer). Younger patients tend to have higher seropositivity to mucosal HPV types, as a group, than those older. Moreover, independently of HPV, younger people have a lower risk of SCC than older people. Hence, age is a strong positive confounder of the association between HPV seroprevalence of mucosal types and SCC suggesting that the association observed can probably be removed by a strong unmeasured confounder. As none of the main potential confounding factors (i.e., time since transplantation, age and sex) was associated with the presence of antibodies against cutaneous HPV types among Caucasian controls, matched analysisfor these factors did not produce, as expected, any materialdifferences in results for cutaneous HPV types.
Until recently, only antibodies against HPV-L1 types 5, 8, 9, 15, 20, 23, 24, 36 and 38 of the beta genus, HPV 16 of the alpha genus and HPV1 of the mu genus have been examined in the context of skin cancer (reviewed in Ref. 9).9–19 the introduction of Luminex technology, antibodies against up to 100 HPV types can now be tested simultaneously.9, 16, 18, 19 Andersson et al. did not report any differences in seropositivity between 72 immunocompetent individuals with SCC and 121 controls for any of the 14 HPV types they examined (HPV types: 1, 5, 6, 8, 9, 10, 15, 16, 20, 24, 32, 36, 38 and 57)18 whereas Karagas et al. examined 16 HPV types (HPV types: 1, 2, 3, 5, 6, 8, 9, 10, 15, 16, 20, 24, 32, 36, 38, 57) in 252 immunocompetent patients with SCC and 461 controls and found a 2-fold increase risk of SCC in patients who were seropositive to HPV5 compared to those who were seronegative for all investigated beta types.16 In a previous prospective pilot study, we did not find an association between any of the 38 HPV types examined or multiple seropositivity and SCC.9 In a small study also using Luminex technology, Waterboer et al. (2008) re-tested sera from 43 immunocompetent patients with SCC from Italy and from 77 controls for antibodies against 31 HPV types and found an association between prevalence of antibodies against L1 of HPV 15, 17 and 38 (beta genus species 2) and also with l1 of HPV 50 (gamma genus) and the presence of SCC.19 These findings were consistent with a study in which HPV-DNA from the beta genus of species 2 predominated in SCC compared to healthy skin samples.37
The natural history of cutaneous HPV types is not well understood24 and the concordance between HPV-DNA in skin biopsies18 or in plucked hairs17 and antibody detection has been low. In comparison to the clear results relating HPV 16 and 6 to self-reported histories of cervical cytological abnormalities or genital warts, the role of any of the 34 HPV types examined in relation to SCC remains unclear. HPV might only be latently present in the skin.38 The prevalence of antibodies against different HPV-L1 antigens varies from study to study and any association might be a consequence of increased viral replication after cell proliferation in patients with cutaneous SCC (this may also explain associations identified for other proliferative skin lesions, such as psoriasis or burns).13 Low HPV-DNA copy numbers in tumour cells39 and the lack of HPV integration to the host-DNA40 would also support this theory. It is possible that the increased risk of SCC observed in OTR is simply a result of immunosuppression impairing the normal capacity to repair UV-damaged DNA. On the other hand, inhibition of UV-induced apoptosis leading to increased capacity for cells to accumulate UV-induced mutations has been shown to be a general effect of multiple HPV types,41 such that diverse HPV types may play a contributing role, rather than there existing specific ‘high-risk’ HPV types as shown for cervical cancer. Equally, few sero-epidemiological studies have used the recently developed multiplexed and high throughput technologies such as Luminex and new HPV types or combination of types19 might still be found to be associated with SCC development in the future.
In conclusion, our serological data do not support a role for any of the 34 HPV types examined in the aetiology of transplant associated SCC. Further research is needed to clarify the association between SCC and HPV and to allow direct comparison between sero-epidemiological, HPV DNA detection42 and functional studies. In particular, large prospective studies, with recording of possible confounding variables, are necessary to elucidate any genuine associations.
We thank all the participants from Oxford Radcliffe Hospitals and from the Barts and the London NHS Trust for their contribution to the study, Ms. Krys Baker for data handling, Ms. Sarah Tipper, Dr. Karin Purdie and Ms. Sally Lambert for support in the laboratory, Dr. Paul Harden for facilitating the study and Dr. Andrew Roddam for statistical advice. Dr. C.P. and Dr. C.H. are supported by Cancer Research UK, Ms. L.M. was supported by funding from Barts and the London Charitable Foundation (grant RAC404) and the European Commission (grant QLK2-CT-2002-0117). Dr. T.W. was supported by the Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik. Dr. B.I.-W. was supported by Jakub Hr. Potocki's Foundation and Dr. A.L. by Oxford Radcliffe Hospitals Charitable Fund. The authors declare no conflict of interest.
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