We determined the presence of HPV DNA in samples of lesional and healthy perilesional skin from immunocompromised and immunocompetent patients. We studied 60 patients (30 renal transplant recipients and 30 immunocompetent patients), who were referred from the Nephrology department to the Dermatology department at the University Hospital Doctor Peset, Valencia, Spain, between January 2010 and January 2012 for evaluation of suspected of NMSC lesions. The study was approved by the hospital's Ethics Committee. Informed consent was obtained from all patients.

Patients were interviewed using a structured questionnaire to collect information on potential confounding variables: age, sex, skin type (Fitzpatrick scale), occupational sun exposure (considered high for patients who spend or had spent most of their working day outdoors), and current and past history of warts and actinic keratosis.

Patient characteristics are shown in Table 2.

Two samples were taken from each patient as follows: after surgical excision of the suspicious lesion a 3-mm punch was used to take a sample from the center of the lesion (tumor sample) and another from the uninjured portion of the same piece of excised tissue (healthy perilesional skin) (Fig. 1). The samples were stored at -80°C in individual cryotubes until processing. The remainder of the excised tissue was fixed in 10% formalin and embedded in paraffin for histopathological diagnosis. A total of 120 samples were obtained: 60 from transplant recipients and 60 from immunocompetent patients (ie, 30 tumor samples and 30 healthy perilesional skin samples from each group).

Figure 1.

1, Tumor lesion. 2, Healthy perilesional skin.

(0,08MB).

Each group of 30 tumor samples collected from immunocompromised and immunocompetent patients consisted of 7 SCC, 10 SCC in situ, and 13 basal cell carcinomas (BCC).

Skin samples were defined as being from exposed areas when they were taken from the face, the neck or nape of the neck, the chest, the upper back, the dorsum of the forearm, and the shins.

DNA extraction and HPV typing was performed as previously described.14 DNA was extracted from samples using a Maxwell 16 FFPE Tissue LEV DNA purification kit. Different HPV types were identified by nested polymerase chain reaction (PCR) followed by sequencing. DNA amplification was achieved using 100 ng of DNA in a final volume of 25μL (for the 2 rounds of amplification) containing 3.5mM MgCl2, 10mM Tris-HCL, 50mM KCl, 0.1% Triton X-100, 0.2mM of each deoxynucleotide triphosphate, 0.75μM of each primer, 5% dimethyl sulfoxide (DMSO), and 1.25 U Taq DNA polymerase (Promega). A volume of 2.5μL was transferred from the first to the second amplification. The first round of amplification consisted of 45 cycles of denaturation (1.5min at 94°C), annealing (1.5min at 50°C), and extension (1.5min at 72°C). The second round of amplification consisted of 30 cycles of denaturing (1.5min at 94°C), annealing (1.5min at 50°C), and extension (1.5min at 72°C). The primers used in the first round of amplification were FAP64 and FAP59, which have been previously described,15 with positions corresponding to nucleotides 5981-6001 (sense primer, FAP59) and 6458-6436 (antisense primer, FAP64). The primers used in the second round of amplification were FAP6085F and FAP6319R,16 with positions corresponding to nucleotides 6085-6104 (sense primer, FAP6085F) and 6319-6296 (antisense primer, FAP6319R). The position of these primers corresponds to the HPV-8 genome. The amplified 235-base pair product was detected on 3% agarose gel and was purified on a Purelink column (Invitrogen). The sequencing reaction was performed in a final volume of 10μL using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's instructions. The amplification profile used was as follows: 25 cycles of 10seconds at 94°C, 10minutes at 50°C, and 2minutes at 60°C. On completion of the sequencing reaction, the product was purified in alcohol (100% and 70% ethanol). The final product was resuspended in deionized formamide, sequenced using an ABI PRISM ® 310 Genetic Analyzer (Applied Biosystems), and the sequence searched against the NCBI database using the BLASTn algorithm.

Categorical variables are presented as frequencies and percentages and continuous variables as the mean and standard deviation. Differences in the distribution of the variables between groups were evaluated using the χ2 test or the Fisher exact test for categorical variables and analysis of variance for continuous data. The odds ratio (OR) and 95% CI were calculated. The OR, adjusted for occupational sun exposure, skin type, and tumor location, was also estimated. Multiple logistic regression analysis was used to identify variables independently associated with the presence of HPV in skin samples. Analyses were performed using the SPSS statistical analysis package (version 19). Statistical significance was set at P<.05.

HPV DNA was detected more frequently in immunocompromised than immunocompetent patients. HPV was detected in 44 of the 60 samples (73.3%) from transplant recipients as compared with 32 of the 60 samples (53.3%) from immunocompetent patients. The difference between groups was significant (adjusted OR, 3.4; 95% CI, 1.2-9.6). Comparison of the different HPV types revealed that the most prevalent form was the β-PV genus, although the relative prevalence of HPV types did not differ significantly between immunocompromised and immunocompetent patients (Table 3).

HPV DNA was detected more frequently in healthy perilesional skin than in lesional skin in both immunocompromised and immunocompetent patients. Comparison between lesional and healthy perilesional skin samples of the prevalence of different HPV genera revealed that the β-PV genus, and in particular β-PV species 1, predominated in both groups and no differences were observed between groups.

Table 4 shows the rates of detection of HPV DNA and the adjusted OR value for skin tumor and perilesional skin samples from immunocompromised and immunocompetent patients. We adjusted the OR according to phototype, sun exposure, and sample location.

HPV types belonging to the β-PV genus were the most common and were found in 34/60 (56.5%) of the samples from immunocompromised patients and in 26/60 (43.3%) of the samples from immunocompetent patients. Within the β-PV genus species 1 was the most prevalent, and HPV-20 the most common type; no significant differences were detected between groups. The α-PV genus was detected in only 1 sample (in an immunocompetent patient). The γ-PV genus was strongly associated with immunocompromised patients, although this effect was not statistically significant. In 6 instances PCR for HPV was weakly positive, but sequencing could not be performed due to the limited amount of viral DNA available. Four samples of HPV that were not classified in Genbank were detected (Table 4).

Mixed infections (involving multiple HPV types) were detected in 29 of the 120 samples tested, and were more common in immunocompromised (17/60) than immunocompetent (12/60) patients.

The age profile and male to female ratio were similar in the immunocompromised and immunocompetent groups. No significant differences in sun exposure, tumor location, phototype, or history of warts or actinic keratosis were observed between groups. The location of the tumor in an exposed area was the only variable that was significantly associated with an increased risk of HPV in immunocompromised patients. In the immunocompetent group a high level of occupational exposure, tumor location in an exposed area, and a low phototype were significantly more common in HPV-positive patients. In immunocompromised patients the risk of HPV positivity was higher in patients with actinic keratoses and warts, although this difference was not statistically significant (Table 5).

We collected samples from 60 skin lesions: 30 from immunocompromised patients (7 SCC, 10 SCC in situ, and 13 BCC) and 30 from immunocompetent patients (7 SCC, 10 SCC in situ, and 13 BCC). The presence of HPV was more common in SCC in situ in both immunocompromised and immunocompetent patients; HPV DNA was detected in 8/10 (80%) SCC in situ samples from immunocompromised patients and in 6/10 (60%) SCC in situ samples from immunocompetent patients. HPV prevalence was lowest in BCC samples from both immunocompromised and immunocompetent patients, although this effect was not statistically significant (Table 6).

The authors declare that no experiments were performed on humans or animals during the course of this study.

The authors declare that they have followed the protocols of their place of work pertaining to the publication of patient data and that all patients included in the study were appropriately informed and provided written informed consent to participate in the study.

The authors declare that no private patient data appear in this article.