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. Author manuscript; available in PMC: 2015 Aug 22.
Published in final edited form as: J Invest Dermatol. 2013 Jan 10;133(6):1512–1520. doi: 10.1038/jid.2012.478

Case–Control Study of Cutaneous Human Papillomavirus Infection in Basal Cell Carcinoma of the Skin

Michelle R Iannacone 1, Tarik Gheit 2, Tim Waterboer 3, Anna R Giuliano 1, Jane L Messina 4,5,6, Neil A Fenske 5,6, Basil S Cherpelis 6, Vernon K Sondak 4, Richard G Roetzheim 7, Sandra Ferrer-Gil 1, Kristina M Michael 3, Sandrine McKay-Chopin 2, Michael Pawlita 3, Massimo Tommasino 2, Dana E Rollison 1
PMCID: PMC4545594  NIHMSID: NIHMS714895  PMID: 23303448

Abstract

Genus-β human papillomavirus (HPV) DNA has been detected in basal cell carcinoma (BCC) tumors, but most epidemiologic studies have not observed associations between genus-β HPV seropositivity and BCC. A clinic-based case–control study was conducted to investigate cutaneous HPV infection in BCC. BCC cases (n = 224) were recruited from a dermatology clinic, and controls (n = 300) were patients who were screened negative for skin cancer. Antibodies against cutaneous HPV types in genera α, β, γ, mu, and nu were measured, and tumors from a subset of BCC cases (n = 195) were tested for HPV DNA. Overall associations were observed between BCC and seropositivity for HPV types in genus-α (odds ratio (OR) = 1.61; 95% confidence interval (CI) = 1.11–2.35), γ (OR = 1.78; 95% CI = 1.22–2.60), and mu (OR = 1.56; 95% CI = 1.06–2.30). BCC cases with β-HPV DNA in their tumors were more likely to be β-HPV seropositive than controls (OR = 1.76; 95% CI = 1.03–3.01), with type-specific associations observed for HPV8 and HPV23, whereas no association was observed between β-HPV seropositivity and β-HPV DNA–negative BCC. No concordance between seropositivity and tumor DNA status was observed for HPV types in genera α and γ. In conclusion, the combined serology and tumor DNA results suggest that β HPV types may have a role in BCC. Additional studies of BCC that assess HPV types in multiple genera are needed.

INTRODUCTION

Basal cell carcinoma (BCC) is the most common cancer in the United States (Chinem and Miot, 2011). UVR exposure is the most important environmental risk factor for BCC. Despite public awareness of the harmful effects of UVR exposure and increased use of sunscreen products, the incidence of BCC continues to rise each year.

DNA of cutaneous human papillomavirus (HPV) types has been detected in non-melanoma skin cancer (NMSC), especially in immunosuppressed individuals. Although most studies have focused on HPV DNA detection in squamous cell carcinoma (SCC) (Boxman et al., 2000; Pfister et al., 2003; Struijk et al., 2003; Forslund et al., 2007; McBride et al., 2007; Asgari et al., 2008; Cronin et al., 2008; Madan et al., 2010), genus-β HPV DNA has also been detected in BCC tumors, ranging from 8 to 55% (Shamanin et al., 1996; Harwood et al., 2000; Wieland et al., 2000; Meyer et al., 2003; Forslund et al., 2004; Andersson et al., 2008) in immunocompetent populations. In addition, no single type has been observed to predominate in skin cancers, and cutaneous HPV viral load is low, at less than one copy per cell, requiring highly sensitive techniques to detect viral DNA.

Evidence from the literature supports a potential role for cutaneous HPV seropositivity in NMSC, particularly SCC (Feltkamp et al., 2003; Struijk et al., 2006; Casabonne et al., 2007; Karagas et al., 2010). Studies that have reported on the association between seropositivity to any genus-β HPV type overall and BCC have not observed statistically significant associations (Feltkamp et al., 2003; Andersson et al., 2008; Karagas et al., 2010). However, type-specific associations with BCC have been observed for seropositivity to HPV8 and HPV20 among persons from the Netherlands (Feltkamp et al., 2003).

The aim of the current study was to investigate the association between cutaneous HPV infection and BCC, incorporating multiple biomarkers of infection. To our knowledge, this is a previously unreported case–control study in a US population to investigate the association between BCC and seroreactivity to cutaneous HPV types belonging to five different genera, as well as present serological associations between genus-α, β, or γ HPV types and BCC stratified by the presence of DNA of these HPV types in the tumor tissues.

RESULTS

As presented in Table 1, older age, male sex, ≤12 years of education, light eye and hair color, occupational sunlight exposure, cutaneous sensitivity and tanning ability to sunlight exposure, and history of smoking were associated with BCC. However, none of the risk factors presented in Table 1 were associated with cutaneous HPV serostatus among the controls, as previously described (Iannacone et al., 2012; data not shown).

Table 1.

Crude associations between demographic, skin cancer, and lifestyle risk factors and BCC

Control
(n=300)
 BCC (n=224)
Variable n (%) n (%) OR (95% CI)
Age in years (mean, SD) 55.4 (11.7) 62.5 (11.9) 1.05 (1.04–1.76)
Age
 18–39 28 (9.3) 8 (3.6) 1.00 (Reference)
 40–49 54 (18) 28 (12.5) 1.82 (0.73–4.50)
 50–59 104 (34.7) 46 (20.5) 1.55 (0.66–3.66)
 60–69 85 (28.3) 67 (29.9) 2.76 (1.18–6.45)
 70–80 29 (9.7) 75 (33.5) 9.05 (3.70–22.15)
Sex
 Female 186 (62) 89 (39.7) 1.00 (Reference)
 Male 114 (38) 135 (60.3) 2.48 (1.74–3.53)
Education
 > 12 years 267 (90.2) 157 (77.3) 1.00 (Reference)
 ≤ 12 years 29 (9.8) 46 (22.7) 2.70 (1.63 -4.47)
Eye color
 Dark brown 81 (27.3) 33 (16) 1.00 (Reference)
 Blue 85 (28.6) 85 (41.3) 2.45 (1.48–4.06)
 Green 48 (16.2) 21 (10.2) 1.07 (0.56–2.06)
 Hazel 48 (16.2) 46 (22.3) 2.35 (1.33–4.17)
 Light brown 35 (11.8) 21 (10.2) 1.47 (0.75–2.89)
Hair color
 Black/brown 234 (78.3) 144 (69.9) 1.00 (Reference)
 Blonde/red 65 (21.7) 62 (30.1) 1.55 (1.03–2.32)
Job in sun for ≥ 3 months
 No 216 (72.7) 114 (55.1) 1.00 (Reference)
 Yes 81 (27.3) 93 (44.9) 2.18 (1.50–3.16)
History of blistering sunburn
 No 95 (32) 54 (26.3) 1.00 (Reference)
 Yes 202 (68) 151 (73.7) 1.32 (0.89–1.95)
Skin reaction to season’s first sun exposure
 Tan or no change in skin color 44 (14.8) 22 (10.8) 1.00 (Reference)
 Mild sunburn turns to a tan 136 (45.6) 61 (30.1) 2.03 (1.15–3.60)
 Sunburn with or without blisters 118 (39.6) 120 (59.1) 0.9 (0.50–1.63)
Skin reaction to repeated sun exposure
 It tans easily 174 (59) 99 (49.3) 1.00 (Reference)
 It can tan if you work at it 99 (33.6) 87 (43.3) 1.55 (1.06–2.26)
 It is unable to tan 22 (7.5) 15 (7.5) 1.20 (0.59–2.42)
Alcohol consumption
 No drinks in past year 38 (12.8) 36 (17.6) 1.00 (Reference)
 ≥ 1 drink in the past year 260 (87.3) 169 (82.4) 0.69 (0.42–1.13)
Ever smoked 100 cigarettes
 No 150 (50.2) 78 (38.2) 1.00 (Reference)
 Yes 149 (49.8) 126 (61.8) 1.63 (1.13–2.34)
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Abbreviations: BCC, basal cell carcinoma; CI, confidence interval; OR, odds ratio.

Genus-mu (odds ratio (OR) = 1.56; 95% confidence interval (CI) = 1.06–2.30; P = 0.03) and genus-α (OR = 1.61; 95% CI = 1.11–2.35; P = 0.01) HPV seropositivity was significantly associated with BCC, with a greater risk observed among individuals seropositive for ≥2 types in genus-α (OR = 1.75; 95% CI = 1.08–2.85; Ptrend=0.01; Table 2). However, for both genera, statistical significance was lost after correction for multiple comparisons. A positive association between BCC and genus-γ HPV seropositivity was observed (OR = 1.78; 95% CI = 1.22–2.60; P=0.003), and remained statistically significant after correction for multiple comparisons. No statistically significant associations were observed for genus-β seropositivity or the polyomaviruses JC virus and KI virus (Table 2). Analyses restricted to those aged 40–69 years revealed only minor differences in the associations between genus-specific HPV seropositivity and BCC compared with the total study population (Table 2).

Table 2.

Associations between genus-specific HPV seroreactivity and BCC cases and controls

Control
(n=300)
 BCC (n=224)
 BCC (n=141)
n (%) n (%) OR (95% CI)1 OR (95% CI)2
 
Alpha
 Seronegative 194 (64.7) 121 (54.0) 1.00 (Reference) 1.00 (Reference)
 Seropositive 106 (35.3) 103 (46.0) 1.61 (1.11–2.35) 1.68 (1.09–2.59)
  1 type 59 (19.7) 52 (23.2) 1.50 (0.94–2.38) 1.55 (0.91–2.65)
  ≥ 2 types 92 (30.7) 80 (35.7) 1.59 (1.03–2.45) 1.38 (0.84–2.29)
ptrend=0.01 ptrend=0.02
Beta
 Seronegative 119 (39.7) 72 (32.1) 1.00 (Reference) 1.00 (Reference)
 Seropositive 181 (60.3) 152 (67.9) 1.32 (0.90–1.93) 1.07 (0.69–1.66)
  1 type 47 (15.7) 42 (18.8) 1.35 (0.79–2.32) 0.93 (0.70–1.73)
  2-3 types 51 (17.0) 40 (17.8) 1.30 (0.76–2.21) 1.30 (0.70–2.42)
  4-8 types 36 (12.0) 36 (15.2) 1.75 (0.97–3.15) 1.01 (0.51–2.01)
  > 8 types 47 (15.7) 36 (16.1) 1.02 (0.59–1.78) 1.08 (0.57–2.02)
ptrend=0.43 ptrend=0.72
Gamma
 Seronegative 145 (48.3) 73 (32.6) 1.00 (Reference) 1.00 (Reference)
 Seropositive 155 (51.7) 151 (67.4) 1.78 (1.22–2.60)3 1.58 (1.02–2.44)
  1 type 63 (21.0) 71 (31.7) 2.06 (1.29–3.27)3 1.86 (1.09–3.17)
  ≥2 types 47 (15.7) 51 (22.8) 1.75 (1.08–2.85) 1.84 (1.04–3.24)
ptrend=0.03 ptrend=0.17
Mu
 Seronegative 204 (68.0) 138 (61.6) 1.00 (Reference) 1.00 (Reference)
 Seropositive 96 (32.0) 86 (38.4) 1.56 (1.06–2.30) 1.38 (0.89–2.13)
Nu
 Seronegative 266 (88.7) 198 (88.4) 1.00 (Reference) 1.00 (Reference)
 Seropositive 34 (11.3) 26 (11.6) 1.00 (0.56–1.77) 1.25 (0.64–2.42)
Control antigens
 JCV
  Seronegative 108 (36.0) 62 (27.7) 1.00 (Reference) 1.00 (Reference)
  Seropositive 192 (64.0) 162 (72.3) 1.32 (0.89–1.96) 1.34 (0.84–2.11)
KIV
  Seronegative 24 (8.0) 14 (6.3) 1.00 (Reference) 1.00 (Reference)
  Seropositive 276 (92.0) 210 (93.8) 0.88 (0.42–1.83) 0.81 (0.35–1.88)
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Abbreviations: BCC, basal cell carcinoma; CI, confidence interval; HPV, human papillomavirus; JCV, JC virus; KIV, KI virus; OR, odds ratio.

1

OR and 95% CI adjusted for age and sex.

2

OR and 95% CI adjusted for age and sex, restricted to participants aged 40–69 years. The age-restricted analysis included 141 BCC cases and 243 controls.

3

Statistical significance retained after correction for multiple comparisons (β-coefficient P<0.01).

Sex-stratified analyses demonstrated a statistically significant greater association between BCC and seropositivity to the single type in genus-mu among women (OR = 2.46; 95% CI = 1.40–4.30) compared with men (OR = 1.00; 95% CI = 0.58–1.72; Pinteraction=0.02; Table 3) within the total study population, as well among individuals aged 40–69 years (P=0.007; data not shown). No significant differences were observed by sex between BCC and seropositivity to HPV type(s) in genera α, β, γ, and nu (Table 3).

Table 3.

Associations between (a) genus-specific HPV seroreactivity and BCC stratified by sex; (b) genus-specific seropositivity and BCC stratified by gender, restricted to population aged 40–69 years

Male (n=249)
 Female (n=275)
HPV genus n (%) OR (95% CI)1 n (%) OR (95% CI)1 P-value2
 
(a)
Alpha
 Seronegative 145 (58.2) 1.00 (Reference) 170 (61.8) 1.00 (Reference)
 Seropositive 104 (41.8) 1.92 (1.13-3.27) 105 (38.2) 1.35 (0.79-2.29) 0.36
Beta
 Seronegative 83 (33.3) 1.00 (Reference) 108 (39.3) 1.00 (Reference)
 Seropositive 166 (66.7) 1.24 (0.72-2.13) 167 (60.7) 1.39 (0.81-2.39) 0.76
Gamma
 Seronegative 87 (34.9) 1.00 (Reference) 131 (47.6) 1.00 (Reference)
 Seropositive 162 (65.1) 1.64 (0.95-2.81) 144 (52.4) 1.93 (1.13-3.28) 0.67
Mu
 Seronegative 162 (65.1) 1.00 (Reference) 180 (65.5) 1.00 (Reference)
 Seropositive 87 (34.9) 1.00 (0.58-1.72) 95 (34.5) 2.46 (1.40-4.30) 0.02
Nu
 Seronegative 216 (86.8) 1.00 (Reference) 248 (90.2) 1.00 (Reference)
 Seropositive 33 (13.2) 1.00 (0.47-2.12) 27 (9.8) 0.99 (0.40-2.42) 0.99
(b)
Male (age 40–69, n=177)
Female (age 40–69, n=207)
HPV Genus OR (95% CI)1 OR (95% CI)1
 Alpha 2.05 (1.11-3.79) 1.37 (0.73-2.55)
 Beta 1.13 (0.61-2.09) 1.01 (0.55-1.88)
 Gamma 1.43 (0.77-2.66) 1.74 (0.94-3.22)
 Mu 0.83 (0.45-1.52) 2.37 (1.27-4.43)
 Nu 1.30 (0.54-3.12) 1.19 (0.43-3.31)
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Abbreviations: BCC, basal cell carcinoma; CI, confidence interval; HPV, human papillomavirus; OR, odds ratio.

1

Adjusted for age.

2

P-value for β-coefficient corresponding to the genus-specific HPV×sex interaction term in logistic regression model.

Among BCC cases, the greatest type-specific seroprevalence was observed for HPV1, and it was statistically significantly associated with BCC (OR = 1.56; 95% CI = 1.06–2.30; Table 4). In genus-α, compared with controls, seropositivity to HPV types 3 (OR = 1.70; 95% CI = 1.03–2.82) and 27 (OR = 2.18; 95% CI = 1.28–3.71) were significantly associated with BCC; however, none of these associations remained statistically significant after correcting for multiple comparisons. No associations were observed between BCC and genera β, γ, or nu seropositivity (Table 4).

Table 4.

Association between type-specific HPV seropositivity and BCC

Controls (n=300)
 BCC (n=224)
HPV genus n (%) n (%) OR (95% CI)1
 
Any HPV type 256 (85.3) 203 (90.6) 1.72 (0.96–3.05)
α
 2 29 (9.7) 29 (12.9) 1.55 (0.87–2.77)
 3 36 (12) 43 (19.2) 1.70 (1.03–2.82)
 7 22 (7.3) 14 (6.3) 0.87 (0.42–1.82)
 10 17 (5.7) 22 (9.8) 1.70 (0.84–3.44)
 27 33 (11) 42 (18.8) 2.18 (1.28–3.71)
 57 22 (7.3) 21 (9.4) 1.39 (0.71–2.72)
 77 47 (15.7) 40 (17.9) 1.18 (0.72–1.92)
β
 Any β1 100 (33.3) 94 (42) 1.30 (0.89–1.90)
  5 38 (12.7) 30 (13.4) 1.01 (0.59–1.75)
  8 69 (23) 69 (30.8) 1.32 (0.87–2.00)
  20 63 (21) 54 (24.1) 1.11 (0.72–1.73)
  24 37 (12.3) 42 (18.8) 1.37 (0.82–2.28)
  36 52 (17.3) 40 (17.9) 0.90 (0.55–1.46)
 Any β2 143 (47.7) 114 (50.9) 1.14 (0.79–1.64)
  9 75 (25) 57 (25.4) 0.88 (0.58–1.35)
  15 76 (25.3) 67 (29.9) 1.18 (0.78–1.77)
  17 77 (25.7) 59 (26.3) 1.06 (0.70–1.61)
  23 61 (20.3) 52 (23.2) 1.28 (0.82–1.99)
  38 74 (24.7) 58 (25.9) 1.05 (0.69–1.59)
  107 48 (16) 33 (14.7) 0.83 (0.50–1.38)
 Any β3 101 (33.7) 76 (33.9) 0.96 (0.65–1.42)
  49 89 (29.7) 67 (29.9) 0.96 (0.65–1.44)
  75 46 (15.3) 35 (15.6) 1.01 (0.61–1.68)
  76 54 (18) 45 (20.1) 1.07 (0.67–1.70)
β 4
  (92) 50 (16.7) 30 (13.4) 0.78 (0.46–1.31)
β 5
  (96)5 45 (15) 33 (14.7) 0.86 (0.52–1.44)
γ
 4 103 (34.3) 84 (37.5) 1.11 (0.76–1.62)
 48 64 (21.3) 56 (25) 1.23 (0.80–1.91)
 50 47 (15.7) 34 (15.2) 1.01 (0.61–1.67)
 65 78 (26) 62 (27.7) 0.98 (0.65–1.48)
 88 20 (6.7) 13 (5.8) 1.05 (0.49–2.24)
 95 67 (22.3) 55 (24.6) 1.00 (0.65–1.54)
 101 33 (11) 24 (10.7) 0.97 (0.54–1.74)
 103 13 (4.3) 15 (6.7) 1.37 (0.61–3.05)
Mu (1) 96 (32) 86 (38.4) 1.56 (1.06–2.30)
Nu (41) 34 (11.3) 26 (11.6) 1.00 (0.56–1.77)
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Abbreviations: BCC, basal cell carcinoma; CI, confidence interval; HPV, human papillomavirus; OR, odds ratio.

1

ORs and 95% CIs adjusted for age and sex.

Of the 238 BCC tumor tissues (98.3%) that tested positive for the β-globin gene, 120 (50.4%) tested positive for DNA to at least one genus-β HPV type, including 43 tumors (18.1%) with a single-type infection and 77 (32.4%) with multiple-type infections (Table 5). Individuals with genus-β HPV DNA–positive tumors were significantly older (mean age (SD) = 65.1 (12.2) compared with those with genus-β HPV DNA–negative tumors (mean age (SD) = 60.1 (11.7); Pwilcoxon=0.001). No additional factors listed in Table 1 were statistically significantly associated with genus-β HPV DNA status in the tumor (data not shown). In contrast to the high DNA prevalence observed for genus-β HPV types, the prevalence for HPV type(s) in genera α, γ, and mu was much lower. Twenty (8.4%) BCC tumors tested DNA positive to at least one genus-γ HPV type, seven (2.9%) tumors tested positive for genus-α HPV DNA, although none were DNA positive for HPV2 or HPV3, and only one (0.4%) BCC tumor tested HPV DNA positive for the single type in genus-mu (Table 5). In addition, within genus, only single-type infections were present in the tumors for α and γ HPV types, and a single tumor was DNA positive for HPV27 in genus-α and HPV50 in genus-γ.

Table 5.

Type-specific HPV DNA prevalence among 238 BCC tumor tissues

HPV DNA+
HPV genus/type n (%)
Any β type 120 (50.4)
β Species
  β 1
  5 31 (13.0)
  8 31 (13.0)
  12 9 (3.8)
  14 7 (2.9)
  19 3 (1.3)
  20 2 (0.8)
  21 0 (0.0)
  24 35 (14.7)
  25 3 (1.3)
  36 21 (8.8)
  47 10 (4.2)
  93 9 (3.8)
  β 2
  9 10 (4.2)
  15 6 (2.5)
  17 9 (3.8)
  22 17 (7.1)
  23 7 (2.9)
  37 17 (7.1)
  38 32 (13.5)
  80 3 (1.3)
  β 3
  49 0 (0.0)
  75 4 (1.7)
  76 18 (7.6)
  β 4
  92 6 (2.5)
  β 5
  96 7 (2.9)
Number of β HPV infections
 1 Type 43 (18.1)
 2 Types 31 (13.0)
 3 Types 22 (9.2)
 4 Types 9 (3.8)
 ≥5 Types 15 (6.3)
Any α type 1 7 (3.0)
 2 0 (0.0)
 3 0 (0.0)
 10 4 (1.7)
 27 2 (0.8)
 57 1 (0.4)
IAny γ type 20 (8.4)
 4 1 (0.4)
 50 11 (4.6)
 60 1 (0.4)
 65 3 (1.3)
 88 1 (0.4)
 103 1 (0.4)
 121 2 (0.8)
Mu1 (1) 1 (0.4)
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Abbreviations: BCC, basal cell carcinoma; HPV, human papillomavirus.

1

DNA prevalence estimation based on a sample size of 236 BCC tumor tissues.

Roughly half of the BCC cases (49.7%) were HPV DNA positive, corresponding to at least one type in genus-β. When comparing seropositivity between controls and BCC cases stratified by HPV DNA status, a statistically significantly greater seroprevalence of genus-β antibodies was observed among BCC cases with HPV DNA–positive tumors compared with controls (OR = 1.76; 95% CI=1.03–3.01), whereas no association was observed between genus-β seropositivity and BCC cases negative for HPV DNA (OR = 1.06; 95% CI = 0.65–1.72; Table 6). Of the 97 BCC cases with genus-β HPV DNA–positive tumors, 36.1% tested seropositive for the same HPV type detected in the tumor tissue (data not shown). Among cases with HPV8 and HPV23 DNA–positive tumors, statistically significant associations were observed between BCC and seropositivity for HPV types 8 (OR = 2.77; 95% CI = 1.11–6.94) and 23 (OR = 15.8; 95% CI = 1.60–155.0; Table 6). No differences in seroprevalence were observed between HPV DNA–negative BCC cases and controls for any single HPV type in genus-β (Table 6).

Table 6.

Association between genus-specific HPV seroposivity and BCC, stratified by HPV DNA status of the same HPV type in the tumor

BCC DNA status
Positive
 Negative
HPV type Antibody status Controls
n (%)		 n (%) OR (95% CI)1 n (%) OR (95% CI)1
Genus-β
N 300 97 98
Any β Negative 121 (40.3) 26 (26.8) 1.00 (Reference) 31 (37.8) 1.00 (Reference)
Positive 179 (59.7) 71 (73.2) 1.76 (1.03–3.01) 61 (62.2) 1.06 (0.65–1.72)
β 1
 5 Negative 262 (87.3) 21 (84) 1.00 (Reference) 86 (87.8) 1.00 (Reference)
Positive 38 (12.7) 4 (16) 1.17 (0.36–3.87) 12 (12.2) 0.91 (0.45–1.86)
 8 Negative 231 (77) 14 (53.8) 1.00 (Reference) 73 (74.5) 1.00 (Reference)
Positive 69 (23) 12 (46.2) 2.77 (1.11–6.94) 25 (25.5) 1.02 (0.59–1.77)
 20 Negative 237 (79) 0 (0) 1.00 (Reference) 74 (75.5) 1.00 (Reference)
Positive 63 (21) 2 (100) NA (NA) 24 (24.5) 1.13 (0.65–1.96)
 24 Negative 263 (87.7) 22 (73.3) 1.00 (Reference) 82 (83.7) 1.00 (Reference)
Positive 37 (12.3) 8 (26.7) 1.80 (0.69–4.70) 16 (16.3) 1.15 (0.59–2.22)
 36 Negative 248 (82.7) 14 (70) 1.00 (Reference) 81 (82.7) 1.00 (Reference)
Positive 52 (17.3) 6 (30) 1.12 (0.37–3.39) 17 (17.3) 0.85 (0.46–1.59)
β 2
 9 Negative 225 (75) 6 (66.7) 1.00 (Reference) 74 (75.5) 1.00 (Reference)
Positive 75 (25) 3 (33.3) 1.02 (0.22–4.63) 24 (24.5) 0.93 (0.54–1.60)
 15 Negative 224 (74.7) 3 (60) 1.00 (Reference) 72 (73.5) 1.00 (Reference)
Positive 76 (25.3) 2 (40) 1.48 (0.23–9.48) 26 (26.5) 1.00 (0.59–1.71)
 17 Negative 223 (74.3) 4 (57.1) 1.00 (Reference) 78 (79.6) 1.00 (Reference)
Positive 77 (25.7) 3 (42.9) 2.40 (0.49–11.70) 20 (20.4) 0.81 (0.46–1.43)
 23 Negative 239 (79.7) 1 (20) 1.00 (Reference) 81 (82.7) 1.00 (Reference)
Positive 61 (20.3) 4 (80) 15.80 (1.60–155.00) 17 (17.3) 0.85 (0.46–1.56)
 38 Negative 226 (75.3) 21 (75) 1.00 (Reference) 78 (79.6) 1.00 (Reference)
Positive 74 (24.7) 7 (25) 0.97 (0.37–2.56) 20 (20.4) 0.80 (0.45–1.43)
β 3
 75 Negative 254 (84.7) 3 (75) 1.00 (Reference) 87 (88.8) 1.00 (Reference)
Positive 46 (15.3) 1 (25) 2.14 (0.19–24.40) 11 (11.2) 0.67 (0.32–1.37)
β 4
 92 Negative 250 (83.3) 5 (100) 1.00 (Reference) 86 (87.8) 1.00 (Reference)
Positive 50 (16.7) 0 (0) 0.00 (NA) 12 (12.2) 0.64 (0.32–1.28)
β 5
 96 Negative 255 (85) 6 (85.7) 1.00 (Reference) 82 (83.7) 1.00 (Reference)
Positive 45 (15) 1 (14.3) 0.70 (0.08–6.42) 16 (16.3) 0.96 (0.50–1.82)
Genus-α2
N 300 7 186
Negative 194 (64.7) 3 (42.9) 1.00 (Reference) 98 (52.7) 1.00 (Reference)
Positive 106 (35.3) 4 (57.1) 2.53 (0.55–11.72) 88 (47.3) 1.64 (1.11–2.44)
Genus-γ
N 300 17 178
Negative 145 (48.3) 6 (35.3) 1.00 (Reference) 55 (30.9) 1.00 (Reference)
Positive 155 (51.7) 11 (64.7) 1.40 (0.49–4.00) 123 (69.1) 1.97 (1.31–2.97)
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Abbreviations: BCC, basal cell carcinoma; CI, confidence interval; HPV, human papillomavirus; NA, not applicable; OR, odds ratio.

1

ORs and 95% CIs adjusted for age and sex.

2

OR and 95% CI estimations based on a sample size of 193 BCC cases and 300 controls.

Among the seven BCC cases with genus-α HPV DNA–positive tumors, none was seropositive for the same genus-α HPV type. In addition, compared with controls, the seroprevalence for genus-α HPV types was greater among cases with genus-α HPV DNA–positive (OR = 2.53; 95% CI = 0.55–11.72) or DNA–negative tumor tissues (OR = 1.64; 95% CI = 1.11–2.44; Table 6). Only 17 (8.7%) BCC cases had genus-γ HPV DNA–positive tumors (Table 6), of which one tested seropositive for the same type (HPV50). Using the controls as a common reference group, no association was observed between genus-γ seropositivity and BCC cases with tumors that were HPV DNA positive (OR = 1.40; 95% CI = 0.49–4.00), whereas a statistically significant positive association was observed between genus-γ HPV seropositivity and BCC cases with tumors that were negative for γ HPV DNA (OR = 1.97; 95% CI = 1.31–2.97; Table 6).

DISCUSSION

To our knowledge, this is the first study to report seroprevalence for cutaneous HPV types from five different genera and to include both serological and DNA-based markers of infection for cutaneous HPV types in relation to BCC. Similar associations with SCC have been previously reported (Iannacone et al., 2012).

The current study did not observe an association between overall genus-β seropositivity and BCC, which is consistent with previous studies (Feltkamp et al., 2003; Andersson et al., 2008; Karagas et al., 2010). However, stratified analyses revealed that BCC cases with genus-β HPV DNA–positive tumors were significantly more likely to have genus-β HPV antibodies compared with controls. In addition, among BCC cases DNA positive for HPV types 8 and 23, a higher seroprevalence for the same type was observed in the cases compared with the controls. These results may indicate that HPV infection is involved in some but not all skin cancers, and as a result the association between genus-β HPV seropositivity and BCC would be obscured when HPV DNA status of the tumors is not taken into account.

In the current study, BCC was associated with genus-α seropositivity, with type-specific associations observed for HPV3 and HPV27. The single published study reporting results for BCC and seroreactivity to cutaneous HPV types outside of genus-β (Andersson et al., 2008) reported a null association with genus-α seropositivity. Differences across study findings may be due to differences in the HPV types and number of types investigated. Only seven BCC tumors were DNA positive for genus-α HPV types; however, no tumors were positive for HPV3 and only two were positive for HPV27. Regardless of HPV DNA status in the tumors, seroprevalence was greater among BCC cases compared with controls. These findings may indicate that seroresponse for genus-α HPV types is not related to the presence of DNA for these types in the tumor. In addition, if in fact genus-α HPV types are involved in BCC development, the low detection of these HPV types in the tumor tissues may indicate that HPV has a role in the earlier stages of skin cancer tumorigenesis. A similar phenomenon may hold true for the single HPV type in genus-mu, where 38% of BCC cases were observed to be seropositive while DNA for HPV1 was detected in a single tumor tissue only.

A statistically significant association was observed between genus-γ HPV seropositivity and BCC; however, only 8.4% of BCC tumors were DNA positive for genus-γ HPV types. Furthermore, when stratified by the presence or absence of DNA in the tumor tissue, an association with genus-β HPV seropositivity was observed among BCC cases with HPV DNA–negative tumors but not among BCC cases with DNA-positive tumors. This trend is contrary to that observed for genus-β HPV seropositivity in relation to DNA status in the tumor. No other studies have presented information on genus-γ HPV seroreactivity or detection of DNA in tumor tissue among BCC cases. Furthermore, there is no information available on the functionality of genus-γ HPV types. Thus, other than chance, the explanation for the observed association between genus-γ seropositivity and γ-HPV DNA–negative BCC is unknown.

The associations with BCC observed in the present analysis differ from those previously reported for SCC from the same underlying clinic population (Iannacone et al., 2012), with seroprevalence being significantly elevated for genus-β HPV types in SCC but for genus-γ types in BCC. In addition, in genus-α, HPV10 seropositivity was associated with SCC, whereas HPV types 3 and 27 were associated with BCC. The observed differences in risk of cutaneous HPV infection by NMSC type within the same study population support the hypothesis that these malignancies have distinct etiologies. Recent evidence in vitro demonstrated functional differences in the E6 and E7 oncoproteins encoded for by different genus-β HPV types, as they relate to the life span and immortalization of primary foreskin keratinocytes (Cornet et al., 2012). For example, Cornet and colleagues demonstrated the ability of HPV38 and HPV49 E6 to inactivate p53, although the mechanisms for each HPV type may not be the same. These functional differences may translate to their differential involvement in BCC versus SCC tumorigenesis by targeting signaling proteins implicated in NMSC, such as TP53, PTCH1, and telomerase (Madan et al., 2010). The functional activity of oncoproteins encoded for by HPV types in genera α and γ have not yet been characterized.

The clinic-based study design may introduce selection bias if referral patterns differ between cases and controls. However, in the current study, the cases and a portion of the controls were selected from University of South Florida (USF) clinics that serve the same underlying community. BCC cases were significantly older than controls and tended to be male, but all analyses adjusted for these factors. In addition, the age-adjusted analysis restricted to a narrower age range revealed findings similar to those obtained from the overall study population, indicating that the observed associations are not due to confounding by age. Men were significantly more likely than women to be seropositive for genus-γ HPV types. However, the only statistically significant interaction observed was between sex and genus-mu seropositivity in relationship with BCC. Similar to the current study, a single study from the United States (Karagas et al., 2010) reported a nonstatistically significant higher seroprevalence for any HPV type in genus-β among men compared with women. However, the study did not discuss potential interaction effects between sex and genus-β seropositivity in relation to BCC (Karagas et al., 2010).

Several strengths of the current case–control study should also be noted. This is the first study to examine associations between BCC and seroreactivity for cutaneous HPV types outside of genus-α and -β utilizing the same multiplex serologic methods used in recent previously published studies (Casabonne et al., 2007; Karagas et al., 2010), allowing for direct comparisons across studies with regard to genus-α and -β seroreactivity. A major strength of our study was the unique opportunity to investigate the specificity of serologic response and the presence of DNA for the same HPV type in the BCC tumor tissues. Only one study in the published literature has presented similar results among a Swedish/Austrian population; however, the concordance between seropositivity and DNA status was presented for NMSC overall and not by skin cancer type (Andersson et al., 2008). Furthermore, the type-specific multiplex genotyping assay used for genera β and γ HPV DNA detection afforded many strengths including high sensitivity, allowing for the detection of low amounts of viral genomes and high specificity for the identification of specific HPV types in both single and multiple infections (Gheit et al., 2007). Another strength of the current study includes the full-body skin screening exams undergone by all control subjects to identify previously undetected cases of skin cancer.

In conclusion, the current study provided evidence suggesting a potential association between cutaneous HPV infection and BCC. However, cutaneous HPVs are ubiquitous in healthy, immunocompetent populations, and the mechanisms by which HPV may have a role in NMSC carcinogenesis are not yet well understood. To rule out the possibility of reverse causality, additional studies are necessary to address the functional role of, and the natural history of, cutaneous HPV infections as they relate to NMSC development.

MATERIALS AND METHODS

Study design and population

To investigate the association between cutaneous HPV infection and BCC, a clinic-based case–control study was conducted in Tampa, FL. The study design and population have been previously described in detail (Rollison et al., 2012). Briefly, histologically confirmed BCC cases were recruited from the USF Dermatology clinic (n = 236). Control subjects were recruited from the affiliated USF Family Medicine and Moffitt Lifetime Cancer Screening and Prevention clinics and could not have a history of any type of cancer, including skin cancer (n = 281). To exclude prevalent cases of undetected skin cancer, all potential control participants underwent a full-body skin cancer screening at the time of study enrollment. If a patient had a suspicious lesion detected during the skin screening that was later determined to be benign on the basis of pathology review, the patient was included as a control (n = 77). If a patient’s screen-detected lesion was histological-confirmed BCC, then that patient was included as a case (n = 8). All eligible study participants were aged 18–80 years.

Participants completed a self-administered questionnaire, including information on demographic (e.g., age, sex, race, education, and US residency), lifestyle (e.g., history of smoking and alcohol consumption), and skin cancer (e.g., eye, hair, and untanned skin color, occupational sunlight exposure, history of blistering sunburn, cutaneous sensitivity, and tanning ability to sunlight exposure) risk factors. Analyses were restricted to those individuals who reported being White, with the exception of two non-White BCC cases and two non-White controls retained to match to the non-White cases. Blood samples were obtained from 226 (92.2%) BCC cases and 340 (95.0%) controls. The final sample size for the analysis of cutaneous HPV seroreactivity was 224 BCC cases and 300 controls. For BCC patients undergoing surgical excision, a 3-mm punch of the residual BCC tumor was obtained and flash-frozen in liquid nitrogen (n = 242). Analyses were restricted to tumor specimens that tested positive for β-globin (98.3%), corresponding to 238 BCC tumors from 230 individual patients. Information on both HPV seroreactivity and DNA status of the tumor was available for 195 BCC cases. All study procedures adhere to the Declaration of Helsinki Principles. All study participants provided written informed consent, and the institutional review board at USF approved all study procedures.

HPV antibody measurement

Sera were tested for antibodies to the major capsid protein L1 of cutaneous HPV type(s) within genera α (2, 3, 7, 10, 27, 57, and 77), β (5, 8, 9, 15, 17, 20, 23, 24, 36, 38, 49, 75, 76, 92, 96, and 107), γ (4, 48, 50, 65, 88, 95, 101, and 103), mu (1), and nu (41). Sera were also tested for antibodies to the VP1 capsid protein of two human polyomaviruses, JC virus and KI virus, to test the specificity of associations observed between cutaneous HPV and BCC. The antibody detection method used was based on glutathione-S-transferase capture ELISA (Sehr et al., 2001, 2002) in combination with fluorescent bead technology (Luminex) (Waterboer et al., 2005, 2006), as described previously. Individual cutoff values to define HPV type–specific seropositivity were applied as described previously to allow for the direct comparison of cutaneous HPV seroprevalence across studies that used the same assay (Casabonne et al., 2007; Michael et al., 2008).

DNA extraction

DNA extraction from fresh-frozen BCC tumor tissues was conducted with the QIAGEN BioRobot EZ1 with the EZ1 DNA Tissue Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). Briefly, frozen tissues were incubated in proteinase K and a buffer G2 (QIAGEN) at 56 °C until the tissue was completely lysed. To monitor the possible occurrence of cross-contamination between the different specimens during DNA extraction, tubes containing only buffer were also included.

HPV DNA typing

HPV typing was performed by a type-specific multiplex genotyping assay (Schmitt et al., 2006; Gheit et al., 2007; Ruer et al., 2009; Schmitt et al., 2010). For specificity evaluation, cloned HPV genomes and human specimens were used. PCR products were obtained even when only 10 copies of the viral genome for each HPV type were used as a template, demonstrating high sensitivity for type-specific detection. The assay detects HPV DNA of 25 genus-β types (5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96), 16 genus-γ types (4, 65, 95, 60, 48, 50, 88, 101, 103, 108, 109, 112, 116, 119, 121, and 123), 5 genus-α types (2, 3, 10, 27, and 57), and the single type in genus-mu (1). Two primers for the amplification of β-globin were added to provide a positive control for the quality of the template DNA (Saiki et al., 1988).

Statistical analysis

Logistic regression (LR) was used to estimate crude ORs and 95% CIs between BCC and demographic, lifestyle, and phenotypic characteristics. Seroprevalence for individual HPV types was estimated as the proportion of patients who tested positive for antibodies to a single HPV type. Subsequently, genus-specific HPV seroprevalence was estimated as the proportion of patients who tested positive for antibodies to at least one HPV type in a given genus. Using LR, OR and 95% CI were estimated for the associations between BCC and genus-specific and type-specific cutaneous HPV seropositivity. LR was also used to assess the association between BCC and the number of HPV types in a given genus for which an individual tested seropositive. To test for trends in risk for BCC associated with seropositivity to an increasing number of cutaneous HPV types within a given genus, an ordinal variable was included in the model. Bonferroni correction of P-values was used to account for multiple comparisons, reducing the statistical significance level for genus-specific HPV seropositivity to P<0.01 and type-specific HPV seropositivity to P<0.002 (Iannacone et al., 2012).

BCC cases that provided tumor tissues were classified as either positive or negative for the presence of HPV DNA for type(s) in genera α, β, and γ. The eight BCC cases that provided two tumor tissues were categorized as genus-specific HPV DNA positive if at least one of the two specimens tested positive for any HPV type belonging to genera α, β, or γ. To calculate OR and 95% CI for the association between seropositivity to any genus-α, β, or γ HPV type and BCC, LR was used, stratifying by the presence and absence of DNA in the tumor tissue. The analysis was restricted to HPV types included in both antibody and PCR assays for genus-α (2, 3, 10, 27, 57), genus-β (5, 8, 20, 24, 36, 9, 15, 17, 23, 38, 75, 76, 92, 96), and genus-γ (4, 48, 50, 65, 88, 95, 101, 103).

Genus-β type-specific concordance was calculated among the BCC cases as the proportion that tested seropositive for a given HPV type and had DNA in their tumor tissue corresponding to the same HPV type. Using LR, additional stratified analyses were then conducted to compare genus-β type-specific HPV seropositivity between controls and BCC cases that had DNA in their tumors corresponding to the same HPV type for which antibodies were detected. LR was also used to assess the associations between genus-β type-specific seropositivity and BCC among cases with HPV DNA–negative tumor tissues. Similar analyses were conducted for genus-α and genus-γ HPV types and BCC.

Skin cancer risk factors were investigated as potential confounders of the associations between genus-specific HPV seroreactivity and BCC. With the exception of age and sex, none of the factors investigated altered the ORs and corresponding 95% CIs for serological associations by more than 10%. To examine residual confounding resulting from the case–control differences in age and sex, analyses were conducted stratified by sex and restricted to a narrower age range of 40–69 years. The SAS statistical software package (version 9.2; SAS Institute, Cary, NC) was used to conduct all study analyses.

ACKNOWLEDGMENTS

This work was supported by a James and Esther King New Investigator grant awarded to DER (06NIR-08) by the Florida Department of Health. The authors thank the supporting staff at USF and LCS for their assistance with patient recruitment, especially Kristen Jonathan, Jill Weber, and Carolyn Gerow. This research was also supported in part by the Miles for Moffitt Foundation Funds.

Abbreviations

BCC

basal cell carcinoma

CI

confidence interval

HPV

human papillomavirus

LR

logistic regression

NMSC

non-melanoma skin cancer

OR

odds ratio

SCC

squamous cell carcinoma

TS-MPG

type-specific multiplex genotyping

USF

University of South Florida

Footnotes

The work was conducted in Tampa, Florida, USA.

CONFLICT OF INTEREST

The authors state no conflict of interest.

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