DNA from BK Virus and JC Virus and from KI, WU, and MC Polyomaviruses as Well as from Simian Virus 40 Is Not Detected in Non-UV-Light-Associated Primary Malignant Melanomas of Mucous Membranes

Patients' tumor material.Thirty-eight mucosal melanoma samples with confirmed PCR-amplifiable DNA tested in two previous studies (7, 15) were selected from samples from 64 patients who were diagnosed from 1985 to 2003 and reported to the (compulsory) Swedish Cancer Registry. The present study was conducted with the permission of the Swedish Data Inspection Authority and the Human Ethical Committee at the Karolinska Institutet and was in compliance with their rules. All histological slides were reviewed, and only primary melanomas, not metastases, were used for further analysis.

Preparation of samples.One 4-μm-thick and two 20-μm-thick consecutive sections were cut from each paraffin block of formalin-fixed tissue. The 4-μm sections were stained with hematoxylin and eosin to guide the dissection of tumor tissue to be used for DNA analysis. Tumor tissues were dissected with a scalpel under light microscopy in each case in order to avoid potential contamination with virus from surrounding tissues and from heavily inflammatory cell infiltrates. This procedure resulted in approximately 90% pure tumor samples for further analysis. Similarly, microtome knives and scalpels were washed in 70% ethanol between each preparation, and each section was stretched on a drop of distilled water placed on each slide, instead of using a regular water bath, in order to avoid contamination. In seven cases, the whole biopsy specimen contained tumor tissue and further dissection was not necessary.

DNA extraction.DNA was extracted using the High Pure RNA extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol, but without DNase treatment. Briefly, the paraffin was removed by treatment with xylene and ethanol. The tissue pellet was disrupted by overnight incubation at 55°C in tissue lysis buffer with the addition of sodium dodecyl sulfate and proteinase K. On day 2, the DNA was bound to a membrane in collection tubes, washed, and eluted by elution buffer. The proteinase K treatment was repeated once (incubation for 1 h at 55°C) for the removal of trace proteins. The DNA was bound to the membrane in a fresh collection tube, washed, and eluted, and finally the DNA amount and purity were measured by NanoDrop technology (NanoDrop Technology Inc., Wilmington, DE). To avoid and check for virus carryover between the melanoma samples, tubes with a slice from an empty paraffin block were placed between the samples and treated in the same way as the melanoma samples throughout the experimental procedures.

Verification of amplifiable DNA.To avoid false-negative results due to DNA unsuitable for PCR analysis, a control PCR with β-microglobulin primers was run in 100 to 120 ng DNA for all samples in a previous study (7).

Nested PCR for the detection of BKV, JCV, and SV40.To detect the presence of BKV, JCV, or SV40, a nested PCR was performed as described previously (5, 12). In addition, each reaction tube contained 0.4 μg/μl purified bovine serum albumin (BSA) to avoid possible inhibition of the PCR by a high concentration of melanin in the DNA extracts. A urine sample positive for BKV, used as a positive control, was kindly provided by Anna-Lena Hammarin (SMI, Karolinska Institutet, Stockholm, Sweden).

PCR assay for the detection of KIPyV and WUPyV.To simultaneously detect the presence of KIPyV and WUPyV, a standard PCR was performed with a single primer pair detecting part of the VP1 region of both viruses (Table 1). The reverse primer KIPyV2404.R had a length of 27 bases to compensate for one mismatch with the WUPyV sequence at base 20. The 50-μl reaction mixture consisted of 50 mM NaCl, 10 mM Tris-HCl (pH 8.3), 3 mM MgCl₂, 200 μM of each deoxynucleoside triphosphate, 1 U of Taq DNA polymerase, 0.3 μM of each primer, and 500 to 1,000 ng of amplifiable DNA, corresponding to 1 × 10⁵ to 2 × 10⁵ cells per reaction. In addition, each reaction mixture contained 0.4 μg/μl purified BSA to prevent possible inhibition of the PCR by a high concentration of melanin in the DNA extracts. An initial 1-min denaturation at 94°C was followed by 40 cycles of 30 s at 94°C, 30 s at 53°C, and 45 s at 72°C, which, in turn, were followed by a final step of 5 min at 72°C. To produce a positive control, KIPyV bp 1395 to 2159 was cloned into pBSKS.

PCR assay for the detection of MCPyV.To detect the presence of MCPyV, a standard PCR was performed with a primer pair detecting part of the large T (LT) region of MCPyV (Table 1). The 50-μl reaction mixture consisted of 50 mM NaCl, 10 mM Tris-HCl (pH 8.3), 3 mM MgCl₂, 200 μM of each deoxynucleoside triphosphate, 1 U of Taq DNA polymerase, 0.3 μM of each primer, and 500 to 1,000 ng of amplifiable DNA, corresponding to 1 × 10⁵ to 2 × 10⁵ cells per reaction. In addition, each reaction tube contained 0.4 μg/μl purified BSA to prevent possible inhibition of the PCR by a high concentration of melanin in the DNA extracts. An initial 1-min denaturation at 94°C was followed by 40 cycles of 30 s at 94°C, 30 s at 53°C, and 45 s at 72°C, with a final step for 5 min at 72°C. Plasmid pUC57MC1, containing MCPyV bp 1681 to 1870 (constructed upon request by GenScript Corporation), was used as a positive control.

Evaluation of the sensitivities of the KIPyV and WUPyV PCR assay and the MCPyV PCR assay.To define the detection levels of the two newly designed PCR assays for KIPyV, WUPyV, and MCPyV, the assays were performed with increasing concentrations of the respective plasmids, from 5 copies to 1 billion copies. In order to investigate if there was any inhibiting effect of genomic DNA on PCR sensitivity, the detection level was also evaluated with 100 ng, 500 ng, and 1,000 ng of genomic DNA.