Association between Human Polyomaviruses and Keratinocyte Carcinomas: A Prospective Cohort Study

Merkel cell polyomavirus (MCPyV) was originally discovered in Merkel cell carcinomas (MCC) and is a causative agent in some MCC (1). We previously reported case–control associations between MCPyV seropositivity and MCPyV DNA-positive cutaneous squamous cell carcinoma (cuSCC; ref. 2) as well as between MCPyV DNA in eyebrow hairs (EBH) and MCPyV DNA-positive cuSCC (3). However, the temporal relationship between MCV and cuSCC could not be assessed using the case–control design. Therefore, we conducted a prospective cohort study of human polyomaviruses (HPyV), including MCPyV, and keratinocyte carcinomas, including cuSCC and basal cell carcinoma (BCC). We incorporated markers of skin damage resulting from chronic UV radiation (UVR) exposure to examine associations between HPyV infection and UVR exposure in keratinocyte carcinoma development.

The Viruses in Skin Cancer (VIRUSCAN) Study has been described previously (4). Briefly, 1,179 patients who underwent skin cancer screenings, age 60+, were enrolled from the University of South Florida (USF, Tampa, FL) Dermatology Clinic between 2014 and 2017 (4). Patients with a history of both cuSCC and BCC were ineligible, but they could have had one or the other in the past. Study follow-up visits coincided with routine skin cancer screening exams at USF (Tampa, FL) and occurred every 6–12 months through September 2018 (4). The current analysis includes 1,008 participants seen at least once during the 4-year follow-up period. Participants provided written informed consent in accordance with the Belmont Report. Study methods were approved by the USF Institutional Review Board.

Participants completed a baseline questionnaire on skin cancer risk factors (4), and recent UVR exposure was estimated quantitatively using a spectrophotometer at baseline (5). A blood sample, plucked EBH and forearm skin swabs (SSW) were collected at baseline for measurement of HPyV infection, as described previously (4). Recuts of pathologically confirmed keratinocyte carcinoma tumors were obtained from the USF Pathology department (4). The study dermatopathologist (J.L. Messina) reviewed keratinocyte carcinoma tumors and graded the degree of solar elastosis in the adjacent normal tissue, a measure of cumulative UVR exposure (4).

Serum was analyzed using multiplexed serologic assays (6) for assessment of antibodies to the VP1 capsid protein corresponding to four HPyV types typically found in the skin [HPyV6, HPyV7, MCPyV, and trichodysplasia spinulosa-associated polyomavirus (TSV)]. DNA was extracted from the EBH, SSW, and tumor samples, and multiplex assays (7) were used for the measurement viral DNA, corresponding to five HPyV types (HPyV6, HPyV7, HPyV9, MCPyV, and TSV).

Associations between markers of baseline HPyV infection and incident keratinocyte carcinoma (separately for cuSCC and BCC) were modeled using multivariate Cox regression, including age- and sex-adjusted models (“min-HR”; Table 1) and fully adjusted models including confounders identified via backward elimination (“full-HR”; Table 1). HPyV prevalence in keratinocyte carcinoma tumors and the percentage of baseline HPyV infections that were concordant with subsequent keratinocyte carcinoma tumors positive for the same HPyV type were calculated. Associations between HPyV tumor viral DNA and levels of solar elastosis were examined.

VIRUSCAN Study participants were 48.6% male, 96.8% White, 95.3% non-Hispanic with a median age at baseline of 68 years (interquartile range = 9). No associations were observed between individual markers of baseline HPyV infection (serum antibodies, EBH, and SSW) and incidence of cuSCC or BCC (Table 1). Participants with MCPyV in both their EBH and SSW were more likely to develop cuSCC compared with those who did not have MCPyV in either marker [HR = 1.66, 95% confidence interval (CI) = 1.00–2.77]; however, statistical significance was lost in the fully adjusted model; this pattern was not observed with risk of BCC (HR = 1.23, 95% CI = 0.72–2.12). Evidence of MCPyV infection was observed across all three markers (serology, EBH, and SSW) for 27.2% of study participants, however this “triple positivity” was not significantly associated with subsequent cuSCC (HR = 1.15, 95% CI = 0.55–2.38) or BCC risk (HR = 1.54, 95% CI = 0.72–3.30).

Prevalence of HPyV DNA was 16.7% and 11.9% in incident cuSCC and BCC tumors, respectively, with MCPyV being the most frequently detected HPyV type in cuSCC (14.5%) and BCC (9.7%). The prevalence of the other HPyV types ranged from 0% to 1.8% in keratinocyte carcinoma tumors. Interestingly, among HPyV DNA-positive tumors, 79%–92% harbored the same HPyV type measured in baseline SSW or serum antibodies while only 33%–50% of HPyV DNA-positive keratinocyte carcinoma tumors harbored the same HPyV type measured in baseline EBH, with MCPyV infections following the same pattern (82%–94% in SSW or serum antibodies vs. 41%–55% in EBH). The percentages of baseline HPyV infections that were present in subsequently developed cuSCC were 3.7%, 2.4%, and 1.0% as measured in baseline EBH, SSW, and serum antibodies, respectively (Fig. 1). Lower proportions of baseline HPyV infections were concordant with BCC tumors (Fig. 1). Presence of HPyV DNA in keratinocyte carcinoma tumor tissues was not associated with levels of solar elastosis in the adjacent normal tissue (HPyV DNA was detected in 16.5% vs. 12.7% keratinocyte carcinoma tumors with lower versus higher solar elastosis levels, P = 0.37).

In this prospective cohort study, baseline HPyV infections, including MCPyV, were highly prevalent across multiple biomarkers of infection. However, HPyV infections did not predict subsequent development of keratinocyte carcinoma, including cuSCC or BCC. A very small percentage of baseline HPyV infections were concordant with a subsequent HPyV virus–positive keratinocyte carcinoma tumor, and HPyV-positive tumors were not associated with cumulative sun damage. Taken together, our results do not support a major role for HPyV infection in keratinocyte carcinoma development among immunocompetent individuals.

Our null findings are inconsistent with the positive associations observed in previous case–control studies (2, 3), but consistent with one prospective serologic study (8). If HPyV is not directly involved in the etiology of the cancer but more readily replicates in the microenvironment of the tumor, then this could explain the positive associations observed in the case–control studies but not in the prospective studies. The VIRUSCAN Study is the largest prospective study to measure HPyV infection at both baseline and in the subsequently developed keratinocyte carcinoma tumors, although statistical power was limited for some analyses, and the exact timing of the initiation of baseline HPyV infections was unknown. Future VIRUSCAN analysis will leverage EBH and SSW samples collected longitudinally to explore the risk of keratinocyte carcinoma associated with incident and persistent HPyV infection.

R.P. Amorrortu reports grants from NIH during the conduct of the study. M.J. Schell reports grants from NCI during the conduct of the study. N.A. Fenske reports grants from NIH during the conduct of the study. V.K. Sondak reports personal fees from Merck, BMS, Eisai, Novartis, Regeneron, and Replimune, and grants from Neogene Therapeutics outside the submitted work. A.R. Giuliano reports grants and other support from Merck & Co, Inc outside the submitted work. D.E. Rollison reports grants from NIH during the conduct of the study, as well as other support from NanoString Technologies, Inc. outside the submitted work; in addition, D.E. Rollison has a patent #63/041,676 pending. No disclosures were reported by the other authors.

R.P. Amorrortu: Validation, visualization, writing–original draft, project administration. Y. Zhao: Data curation, formal analysis, validation, visualization, methodology, writing–review and editing. J.L. Messina: Conceptualization, formal analysis, investigation, methodology, writing–review and editing. M.J. Schell: Conceptualization, methodology, writing–review and editing. N.A. Fenske: Resources, writing–review and editing. B.S. Cherpelis: Resources, writing–review and editing. V.K. Sondak: Conceptualization, writing–review and editing. A.R. Giuliano: Conceptualization, methodology, writing–review and editing. M. Pawlita: Conceptualization, investigation, methodology, writing–review and editing. S. McKay-Chopin: Investigation, writing–review and editing. T. Gheit: Conceptualization, resources, investigation, methodology, writing–review and editing. T. Waterboer: Conceptualization, resources, investigation, methodology, writing–review and editing. M. Tommasino: Conceptualization, resources, investigation, methodology, writing–review and editing. D.E. Rollison: Conceptualization, resources, supervision, funding acquisition, methodology, writing–original draft, project administration.

This work was supported by the NCI at the NIH (R01-CA177586) awarded to D.E. Rollison. This work was also supported in part by the Tissue Core and the Participant Research, Interventions, and Measurement (PRISM) Core at the H. Lee Moffitt Cancer Center and Research Institute, a comprehensive cancer center designated by the NCI and funded in part by Moffitt's Cancer Center Support Grant (P30-CA076292). The funders were not involved in the study design, data collection/analysis, or manuscript preparation. Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.

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