ANTICANCER RESEARCH 38 : 4111-4114 (2018) doi:10.21873/anticanres.12701 DNA from Human Polyomaviruses, MWPyV, HPyV6, HPyV7, HPyV9 and HPyV12 in Cutaneous T-cell Lymphomas MASSIMILIANO BERGALLO 1, VALENTINA DAPRÀ 1, PAOLO FAVA 2, RENATA PONTI 2, CRISTINA CALVI 1, PAOLA MONTANARI 1, MAURO NOVELLI 2, PIETRO QUAGLINO 2, ILARIA GALLIANO 1 and MARIA TERESA FIERRO 2 1Department of Pediatrics, Infectious Diseases Unit, Regina Margherita Children’s Hospital, University of Turin, Turin, Italy; 2Department of Medical Sciences, Dermatology Section, University of Turin, Turin, Italy Abstract. Background/Aim: The etiopathogenesis of characterized by longstanding, scaly patch lesions mycosis fungoides and Sézary syndrome remains obscure. preferentially involving the buttocks and body areas Different viruses have been proposed to have a role in the infrequently exposed to sunlight. Disease progress is slow etiopathogenesis of cutaneous T-cell lymphomas (CTCL). In over years, from patches to plaques and eventually tumors or the present study, the presence of five recently discovered erythroderma. Lymph node and visceral involvement, as well human polyomaviruses 6 (HPyV6), human polyomaviruses 7 as large cell transformation, usually occur in the late stages of (HPyV7), human polyomaviruses 9 (HPyV9), human the disease (1). Sézary syndrome (SS) is an erythrodermic polyomaviruses 12 (HPyV12), and Malawi polyomavirus cutaneous T-cell lymphoma with leukemic involvement, with (MWPyV), have been analyzed in 55 CTCL in order to an aggressive clinical behavior and poor prognosis (2, 3). confirm the skin tropism and the possible pathological The etiopathogenesis of MF and SS remains obscure. association of these new polyomaviruses. Materials and Persistent antigen stimulation could lead to a continuous Methods: Human polyomaviruses DNA were amplified from proliferation of T-cells and chronic inflammation and, skin lesions were recovered from a total of 55 patients (32 ultimately, to the development of a malignant T-cell clone males and 23 females, average age 63±15 years) affected by (4). Another hypothesis suggests that specific viral agents CTCL. Results: When assayed for the presence of 5 different may serve as triggering factors (5). Different viruses have HPyVs, (HPyV6, HPyV7, HPyV9, MWPyV, and HPyV12) been suggested to have a role in the etiopathogenesis of HPyV9, HPyV10 and HPyV12 DNA sequences were not CTCL, mainly the human T-cell leukemia virus and the found in any skin specimens. HPyV6 and 7 DNA was Epstein-Barr virus (6, 7). Contradictory results have arisen detected in 1/55 (1.8%) of skin specimens. Conclusion: The from studies investigating the role of the Epstein-Barr virus low-level presence of HPyV6 and HPyV7 DNA, and lack of in CTCL (8). Our group demonstrated in previous reports detection of polyomaviruses HPyV9, MWPyV and HPyV12 that the Epstein-Barr virus, parvovirus variants (B19, in our series do not support a significant role of these HPyVs LaL1/K71, V9), human herpesvirus 7 (HHV-7), and human subtypes in the etiopathogenesis of skin cancers. polyomaviruses (HPyVs) HPyV6, HPyV7 and TSPyV were not involved in CTCL pathogenesis (9-11). Cutaneous T-cell lymphoma (CTCL) is a group of More specifically, the first HPyVs, polyomavirus BK malignancies derived from skin-homing T cells. Mycosis (BKPyV) and polyomavirus JC (JCPyV), were discovered in fungoides (MF) and Sézary syndrome (SS) are the most 1971, but it was not until 2007, that two more HPyVs, common CTCL variants. Mycosis fungoides (MF) is polyomavirus KI (KIPyV) and polyomavirus (WUPyV), were discovered in nasal aspirates (12, 13) followed by Merkel Cell polyomavirus (MCPyV), isolated from Merkel Cell Carcinomas (MCC) in 2008 (14). Since then, seven new HPyVs have been Correspondence to: Massimiliano Bergallo, Department of Public characterized from samples derived from the skin (Human Health and Pediatric Sciences, University of Turin, Medical School, polyomavirus 6 (HPyV6), human polyomavirus 7 (HPyV7) and 10136 Turin, Italy. Tel: +39 0113135414, Fax: +39 0113135416, e-mail: [email protected] Trichodysplasia Spinulosa polyomavirus (TSPyV)) (15-17), blood (human polyomavirus 9 (HPyV9)) (18, 19), feces Key Words: Human polyomavirus, cutaneous T-cell lymphomas, (Malawi polyomavirus (MWPyV) and STL polyomavirus mycosis fungoides, Sézary syndrome. (STLPyV)) (20, 21), and from the gastrointestinal tract (human 4111 ANTICANCER RESEARCH 38 : 4111-4114 (2018) Table I. Primer and probe sequences used for the amplification of human polyomaviruses (HPyVs). Target Orientation Sequences Location HPyV6 Sense 5’-AAGTGGGAAGTGCTGGATATATAAGAG-3’ KX379631.1 1624-1650 Antisense 5’-TCCACAGGCCCAAAAGTACAT-3’ 1719-1699 Probe FAM-CCCCTGCTGGTGTAGAAGGTTCCCA-TAMRA 1673-1697 HPyV7 Sense 5’-TGCTGCAGTGCAAGAAGTTACA-3’ KX771235.1 2241-2262 Antisense 5’-CCAAGGCCTCCCTCAACA -3’ 2315-2298 Probe FAM-CAAATGCAGCCTGCTACTATCCCTCCAA-TAMRA 2269-2296 HPyV9 Sense 5’-CTAGGGAACAATTTGAATATCAGGAA-3’ HQ696595.1 1227-1252 Antisense 5’-ATAGTGTCCAGATCTAGGCTCTGAAC-3’ 1306-1281 Probe FAM-AAGTTAGGCTGAGGCGGGAGATAGGG-TAMRA 1254-1279 MWPyV Sense 5’-CATTGATGGACAGCCAATGG-3’ JX262162.1 2387-2406 Antisense 5’-TCCTGGAAGAGGTTCTGTTCCTT-3’ 2468-2446 Probe FAM-TGGGACTGATAATCAAGTACAGGATGTAACTGTGT-TAMRA 2408-2442 HPyV12 Sense 5’-GTGGGAAGCTGTCAGTGTGA-3’ JX308829.1 1731-1750 Antisense 5’-CCACCTACTGCAAACATGTG-3’ 1868-1849 Probe FAM-ACTACAGGATGGCCTACCCCATTGTCAGTC-TAMRA 1835-1806 polyomavirus 12 (HPyV12)) (22). Like most HPyVs, they are consent, and the study was approved by the Ethics Committee of also present in a large part of the general population, and their the ‘A.O.U. Città della Salute e della Scienza di Torino’. Cryostatic seroprevalence ranges from 20% to >90%, with HPyV9 being sections OCT-embedded (Tissue-TeK O.C.T. Compound, Sakura Eu, The Netherlands) from skin lesions were recovered from a total of the less common among them (23-25). Moreover, some HPyVs, 55 patients (32 males and 23 females, average age 63±15 years) i.e. , BKPyV, JCPyV, MCPyV and TSPyV, have clearly been affected by CTCL. All patients had been referred to the Section of associated with specific diseases and cancer (26-27). In Dermatology, at the Department of Medical Sciences of the immunocompromised individuals, reactivation of BKPyV is University of Turin. Diagnosis was histologically confirmed; 43/55 associated with haemorrhagic cystisis, BKV nephropathy, and patients had MF, 8/55 had SS; and 4/55 had primary cutaneous T- ureteral stenosis, while JCPyV is associated with progressive cell lymphoma (CTCL) non-MF/SS. multifocal leukoencephalopathy and TSPyV associated with DNA extraction. OCT-sections were incubated overnight at room Trichodysplasia spinulosa, a rare skin disease (27). As noted temperature with 500 μl of lysis buffer (100 mM NaCl, 10 mM Tris above, MCPyV is a frequent cause of the skin malignancy HCl pH8, 1 mM EDTA pH8, 1% SDS, 2% Triton X-100). The tubes Merkel cell carcinoma (MCC), mainly in immunosuppressed or were incubated at +100˚C for 10 min. Equal volumes of phenol- older individuals (14). However, despite that only MCPyV has chloroform were added to the sample and the tubes were centrifuged been associated with cancer, most other HPyVs express proteins at 12,000 rpm for 10 min. Upper aqueous phase was transferred into that may potentially contribute to cancer development; all, fresh tubes, followed by adding 1 volume of ice cold ethanol, and incubated at –20˚C for 1h and centrifuged at 12,000 rpm for 10 min except HPyV12, have putative binding sites for the at +4˚C. The DNA pellets were separated from supernatants and retinoblastoma protein and many also for p53 (16,17, 19, 22). washed twice with 70% ethanol; then, re-centrifuged at 12,000 rpm Herein, the presence of five recently discovered HPyVs, for 10 min and left to air-dry at room temperature. The pellets were HPyV6, HPyV7, HPyV9, HPyV12 and MWPyV, was dissolved in 20 μl of distilled water. The DNA samples were stored analyzed in 55 CTCL in order to confirm the possible skin at −20˚C until assayed. tropism and the possible pathological association of these Real-Time PCR. new polyomaviruses with the development of CTCL. Primers and probes sets for HPyV detection are shown in Table I. Sets of primers and a probe targeting Small tumor antigen (STa), a part of early expressed genome of Materials and Methods polyomaviruses, were designed using Primer Express Software Version 3.0 (Thermo Fisher, Paisley UK). The primers/probes Patients and samples. The present study was performed in concentration was compliance with the principles of good clinical practice and 900 nM/250 nM for each target. Real-Time PCR assays were according to the principles of the Declaration of Helsinki. All the performed using the GoTaQ qPCR MasterMix (Promega, Milano patients were included after providing their written informed IT), on the 7500 Real-Time PCR System (Life Technologies Ltd) 4112 Bergallo et al : Human Polyomaviruses in CTCL HPyV6, HPyV7, HPyV9, and MWPyV are all in general common in the population and sequence data suggests they all are potentially oncogenic, by binding to Rb and p53. Thus, the possibility that one or more of these viruses are causative for a subset of tumors is a plausible hypothesis (29). The tropism of these viruses is not well understood. HPyV6 and HPyV7 are, similarly to MCPyV, common on skin (30). HPyV9 is mainly, although rarely, found in the blood, but has also been isolated from skin (17) and in a serum sample from a kidney transplant patient (18). MWPyV, isolated