Merkel cell polyomavirus and Merkel cell Carcinoma, France.
A new polyomavirus, provisionally named Merkel cell polyomavirus (MCPyV), has been recently identified in tumor tissue from patients with MCC. Furthermore, clonal integration of viral DNA within the tumor genome was observed in most of the cases (7). To assess the implication of MCPyV in MCC, we tested tumor biopsy samples collected from 9 patients with MCC. Patient median age was 65 years, and 2 patients were immunocompromised (patient 1 had a lymphoma, which was treated with rituximab; patient 7 had psoriatic rheumatism, which was treated with corticosteroids and methotrexate). As controls, biopsy samples from 15 patients with diverse proliferative or inflammatory skin or mucosa lesions were tested (Table).
DNA was extracted from fresh tissue samples by using the QIAamp DNA Mini Kit (QIAGEN, Courtaboeuf, France) according to the manufacturer's instructions. Paraffin was removed from previously formaldehyde-fixed, paraffin-embedded biopsy samples with xylene, and the samples were rehydrated with decreasing concentrations of ethanol. The extracts were tested for MCPyV DNA by PCR using 3 sets of primers initially described by Feng et al. (7) to target the predicted T-antigen (LT 1 and LT3 primer pairs) and the viral capsid (VP1 primer pair) coding regions. Extracted DNA (5[micro]L) was added to 45 [micro]L of the reaction mixture, which contained 5 [micro]L 10 x PCR buffer, 10 [micro]L 5x Q-solution (QIAGEN), 2.5 mmol/L Mg[Cl.sub.2], 200 [micro]mol/L each dNTP, 2.5 units Taq DNA polymerase (QIAGEN), and 15 pmol of each primer. Touchdown PCR conditions were as follows: 95[degrees]C for 5 min followed by 35 cycles of denaturation at 95[degrees]C for 30 s; annealing at 61[degrees]C (10 cycles), 59[degrees]C (10 cycles), and 57[degrees]C (15 cycles) for 30 s; extension at 72[degrees]C for 1 min; and a final extension step at 72[degrees]C for 10 min. Amplification products were subjected to electrophoresis in a 2% agarose, 1 x Tris-borate-EDTA gel stained with ethidium bromide and examined under UV light. The sizes of the fragments amplified with the LT1, LT3, and VP1 primers pairs were 439, 308, and 351 bp, respectively. A negative control was included in each experiment; positive samples were confirmed by analyzing a second stored sample aliquot, and the amplified fragments were sequenced by using the same primers used for the amplification. The sequences were submitted to GenBank under accession numbers AM992895-AM992906. Total DNA level in sample extracts was measured by using the LightCycler control DNA kit targeting the [beta]-globin gene (Roche Diagnostics, Meylan, France).
MCPyV sequences were detected in 8 of the 9 patient samples and in none of the control samples (Table). Results for all 8 patients were positive with the LT3 primer pair, whereas they were positive for only 5 with the VP1 primer pair and only 1 with the LT1 primer pair (Table). Because the LT1, VP1, and LT3 primer pairs generate the longer, intermediate, and shorter DNA fragments, respectively, the difference in sensitivity could result from the deleterious effect of formaldehyde fixation on DNA; this effect would increase with the length of the fragment to be amplified. The negative result obtained for patient 8 might suggest that some MCC patients are not infected with MCPyV. This explanation is in accordance with the findings of Feng et al., who reported 80% prevalence of MCPyV in patients with MCC (7). Nevertheless, the single negative result observed in our study might alternatively be explained by insufficient tissue or by DNA degradation through the fixation and embedding process. Indeed, the level of [beta]-globin gene DNA was much lower in the sample from this patient (441 pg/[micro]L) than in samples from the other patients (median 13,500 pg/[micro], interquartile range 8,902-19,750 pg/[micro]L).
As observed with human papilloma-viruses, a gene disruption caused by viral DNA integration into the host genome might be an alternative hypothesis to explain the lack of amplification of an MCPy V genome region (8). Sequences of the amplified PCR product were 99%-100% identical to those reported by Feng et al. (7), which indicates that this virus is genetically stable.
In summary, we detected MCPy V DNA sequences in 8 of 9 tumor samples from patients with MCC but in none of 15 control samples. Our results confirm the likely association of MCPyV with MCC. The epidemiologic characteristics as well as the carcinogenic role played by this newly discovered virus need to be more thoroughly investigated.
Vincent Foulongne, Nicolas Kluger, Olivier Dereure, Natalie Brieu, Bernard Guillot, and Michel Segondy
Author affiliation: University of Montpellier I, Montpellier, France
DOI: 10.3201/aid 1409.080651
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Address for correspondence: Vincent Foulongne, Laboratory of Virology, Hopital St-Eloi, 34295 Montpellier CEDEX 5, France; email: email@example.com
Table. Merkel cell polyomavirus from 9 patients with Merkel cell carcinoma and 15 control patients, France * Patient no. Age, y Sex Diagnosis 1 57 M MCC, primary 2 61 F MCC, primary 3 57 F MCC, primary 4 82 F MCC, primary 5 78 M MCC, metastatic 6 80 F MCC, primary 7 65 M MCC, primary 8 81 M MCC, primary 9 60 M MCC, primary 10 61 M Hyperkeratosis 11 49 M Seborrheic keratosis, penis 12 60 M Nonspecific lesion, esophagus 13 40 M Nasal papilloma 14 58 M Anal condylomas 15 44 M Epidermodysplasia verruciformis 16 19 M Cutaneous warts 17 57 F Cutaneous warts 18 64 F Cutaneous nodule 19 6 F Pharyngeal papillomatosis 20 27 M Vocal cord polyp 21 58 M Lichen 22 63 M Skin inflammation 23 72 M Skin inflammation 24 57 M Skin inflammation Patient no. Sample 1 Fresh biopsy, buttock 2 FFPE biopsy, buttock 3 FFPE biopsy, lower eyelid 4 FFPE biopsy, cheek 5 Fresh biopsy, jugular lymph node 6 Fresh biopsy, upper eyelid 7 FFPE biopsy, temple 8 FFPE biopsy, forearm 9 FFPE biopsy, forearm 10 Fresh biopsy, foot 11 Fresh biopsy, penis shaft 12 Fresh biopsy, lesion 13 Fresh biopsy, papilloma 14 Fresh biopsy, condyloma 15 Fresh biopsy, skin 16 Fresh biopsy, wart 17 Fresh biopsy, wart 18 Fresh biopsy, skin 19 Fresh biopsy, pharynx 20 Fresh biopsy, polyp 21 Fresh biopsy, skin 22 Fresh biopsy, skin 23 Fresh biopsy, skin 24 Fresh biopsy, skin PCR primer pair Patient no. LT1 LT3 VP1 1 + + + 2 -- + -- 3 -- + + 4 -- + -- 5 -- + + 6 -- + + 7 -- + -- 8 -- -- -- 9 -- + + 10 -- -- -- 11 -- -- -- 12 -- -- -- 13 -- -- -- 14 -- -- -- 15 -- -- -- 16 -- -- -- 17 -- -- -- 18 -- -- -- 19 -- -- -- 20 -- -- -- 21 -- -- -- 22 -- -- -- 23 -- -- -- 24 -- -- -- * MCC, Merkel cell carcinoma; +, positive Merkel cell polyomavirus PCR amplification; FFPE, formaldehyde-fixed paraffin-embedded; --, negative Merkel cell polyomavirus PCR amplification.
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|Author:||Foulongne, Vincent; Kluger, Nicolas; Dereure, Olivier; Brieu, Natalie; Guillot, Bernard; Segondy, Mi|
|Publication:||Emerging Infectious Diseases|
|Date:||Sep 1, 2008|
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