Merkel cell polyomavirus (MCV) T-antigen seroreactivity, MCV DNA in eyebrow hairs, and squamous cell carcinoma

Study population

The study population has been described in detail previously 11]. Briefly, a clinic-based case–control study was conducted at Moffitt Cancer Center,
Tampa, Florida in 2007–2009. Cases included newly diagnosed, histologically confirmed
cases of SCC, a majority of which were immunocompetent 11], and were identified through the University of South Florida (USF) Dermatology Clinic.
Controls included patients without history of cancer and had a negative skin cancer
screening exam at the USF Family Medicine Clinic or Moffitt’s cancer screening clinic.
Of the 173 SCC cases and 300 controls included in the MCV capsid antibody analysis
11], MCV T-Ag serology results were available for 171 SCC cases and 300 controls. Eyebrow
hair samples were available for 169 SCC cases and 292 controls. All study participants
completed a comprehensive questionnaire on demographics, lifestyle and skin cancer
risk factors.

Ethics, consent and permissions

All participants provided written informed consent, and the study protocol was approved
by the Institutional Review Board at USF.

MCV serology

Serum antibodies (IgG) to small T-Ag, full-length large T-Ag, large T-Ag exon 1 and
large T-Ag exon 2 of MCV (isolate 344) were measured using a fluorescent bead-based
multiplex assay, as described previously 15], 11], 16]. Briefly, seroreactivity to MCV T-Ag was expressed as median fluorescence intensity
(MFI). The cut-offs for seropositivity were determined using an independent reference
sample of 42 MCC patients (200 MFI for small T-Ag, 200 MFI for large T-Ag exon 1,400
MFI for large T-Ag exon 2 and 400 MFI for full-length large T-Ag).

MCV DNA measurement in SCC tumor tissues

As described previously, MCV DNA from eyebrow hairs and fresh frozen SCC tumor tissue
was detected using a highly sensitive and specific assay which combines multiplex
polymerase chain reaction (PCR) and bead-based Luminex technology 11], 17]. MCV viral load (absolute copy number per sample) in SCC tumor tissue was determined
using a multiplex quantitative real-time PCR targeting the N-terminus and C-terminus
of the MCV T-antigen sequence 18]. The ratio of N-terminus to C-terminus copy numbers of MCV DNA was determined to
examine the presence of C-terminus deletions. Two samples of formalin fixed paraffin
embedded MCC tumor tissues were analyzed as positive controls. Data on both MCV T-Ag
serology and MCV tumor DNA were available from 144 SCC cases, while data on MCV DNA
in eyebrow hair and MCV DNA in SCC tumor were available from 141 SCC cases.

Statistical analysis

Associations between MCV T-Ag seropositivity, MCV DNA in eyebrow hairs and SCC were
estimated by odds ratios (OR) and 95 % confidence intervals (CI) calculated using
logistic regression with adjustment for age and gender. Odds ratios were calculated
separately for MCV DNA-positive SCC cases and MCV DNA-negative SCC cases using multinomial
logistic regression, also adjusting for age and gender. Analyses were conducted using
SAS software, version 9.3 (SAS institute Inc., Cary, North Carolina) and R software,
version 2.15.1 19].