This study was approved by the Capital Health Research Ethics Board, Halifax, Nova Scotia, Canada, and by the Baylor College of Medicine Institutional Review Board, Houston. A total of 353 frozen specimens from various body sites were analyzed. Tissues from 196 malignant lymphomas, including 152 non-Hodgkin lymphoma (NHL) and 44 Hodgkin lymphoma (HL) samples, were retrieved from the Department of Anatomical Pathology, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada.

All cases were diagnosed during 1994 through 2001 and classified according to World Health Organization criteria by using morphologic and immunohistochemical evaluation (21). The NHL samples were classified as B-cell lymphoma (n = 133), NK/T-cell lymphoma (n = 18), or were unclassified (n = 1). The HL samples were classified as classical HL (n = 41) or nodular lymphocyte predominant HL (n = 3). Also included in our study were 157 nonlymphoma frozen tissue specimens, including 110 benign lymph node biopsy specimens from healthy patients or patients with inflammatory disease, 27 lymph nodes from patients with metastatic carcinoma or melanoma, 7 biopsy specimens of inflammatory tissues, and 13 other neoplastic (non-MCC) tissue samples.

Lymphoma specimens were obtained from 96 women and 98 men; patients’ ages ranged from 15 to 88 years (mean 54.5 years). Of the 110 benign lymph nodes, 53 were from women and 57 from men, ranging in age from 17 to 85 years (mean 46 years). Lymph nodes with metastatic tumors were obtained from 16 women and 10 men, ranging in age from 22 to 86 years (mean 60.6 years). Of the 7 inflammatory tissue samples, 3 were from women and 4 were from men 19–84 years of age (mean 52 years). Finally, the non-MCC tumors were obtained from 8 men and 5 women, ranging in age from 22 to 72 years (mean 50.5 years). Data were not available for 3 patients, 2 with lymphoma and 1 with metastatic cancer. The cancers of all lymphoma patients were staged according to the Ann Arbor system (22).

Formalin-fixed, paraffin-embedded biopsy specimens of skin cancers (4 MCCs and 4 melanomas), obtained from the archives of the Department of Pathology, Baylor College of Medicine, Houston, Texas, USA, served as controls for PCR. Specimens were collected during 1998–2008.

The expression of MCPyV T-ag was detected by immunohistochemical analysis by using a BenchMark XT IHC (Ventana Medical Systems, Inc., Tucson, AZ, USA) system. Tissue sections on microslides were deparaffinized with xylene, hydrated in serially diluted alcohol, and endogenous peroxidase activity was quenched. The sections were then treated with slightly basic Tris-based buffer for 30 min for antigen retrieval. Sections next were incubated with CM2B4, a monoclonal antibody against MCPyV large T-ag (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:250, followed by a standard polymer detection kit (diaminobenzidine) that served as the chromogen. The slides were counterstained with hematoxylin, dehydrated, and mounted for examination. A sample from a patient with MCC served as a positive control.

DNA was extracted from frozen tissues by lysis buffer/proteinase K treatment and phenol-chloroform extraction as previously described (20), omitting the deparaffinization step. For the paraffin-embedded specimens, sections were deparaffinized, and DNA was extracted by using our previously described protocol (20). During sample processing, stringent precautions were taken to avoid cross-contamination between samples. The microtome was cleaned carefully, and the blade was replaced for sectioning of each tissue. Other safety measures included working within a biosafety hood located in a dedicated room free from plasmids and viruses and using a dedicated set of pipettors and barrier filter tips. In addition, a negative extraction control that lacked tissue was processed in parallel.

Samples were first screened for the single-copy human RNase P gene by real-time quantitative PCR (qPCR), as described (20,23). Briefly, 5 μL of each DNA sample (undiluted and 1:10 diluted) was used in 50-μL qPCRs. This strategy detected potential PCR inhibitors in the DNA preparations, determined the human cell equivalents in each DNA sample, and normalized MCPyV viral loads to human cell numbers.

MCPyV was detected by qPCR with primers and TaqMan probe (Applied Biosystems, Foster City, CA, USA) designed to detect sequences from the unique coding region of the small tumor antigen (t-ag) gene of MCPyV. The sequences of the oligonucleotides were as follows: LT3-fwd primer 5′-AGTGTTTTTGCTATCAGTGCTTTATTCT-3′, corresponding to nt 632–659 of MCPyV350 (GenBank accession no. EU375803); LT3-rev primer 5′-CCACCAGTCAAAACTTTCCCA-3′, corresponding to nt 702–682; and fluorogenic probe 5′-FAM-TGGTTTGGATTTCCTC-MGB-3′, corresponding to nt 661–676. The pCR.MCV350 plasmid described by Feng et al. (1) was used as a positive control. Amplifications were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) by using the following cycling parameters: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and of 1 min at 60°C. All PCRs were performed in duplicate. Reactions were considered positive if >10 viral genome copies/reaction were detected.

Conventional PCR was performed on a subset of DNA samples to validate the presence of MCPyV. The primer set used was the following: MC_F2 (5′-CTCATCCTCTGGATCCAGTAGC-3′) and MC_R2 (5′-CAGAAGAGATCCTCCCAGGTG-3′) specific for a conserved region between nt positions 1142 and 1267 of the large T-ag gene of MCPyV (GenBank accession no. EU375803) and gave a 126-bp fragment. The pCR.MCV350 positive control plasmid was added to the control PCR outside the clean room after tubes containing test DNAs and negative controls (without DNA template) were closed. PCRs were performed in a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems). Thermal cycling parameters were as follows: 94°C for 2 min, followed by 45 cycles at 94°C for 15 s, 60°C for 15 s, and 72°C for 15 s, with a final extension step at 72°C for 7 min. Amplified fragments were visualized by electrophoresis on a 3% agarose gel and stained with ethidium bromide. All PCR products were then purified by using the DNA Clean & Concentrator-5 system (Zymo Research, Orange, CA, USA) and sequenced by Lone Star Labs, Inc. (Houston, TX, USA).

The z score of difference for proportions was used to test for a difference between selected groups and an outcome. Fisher exact test was used to test the distribution of clinical stage of disease and 5-year survival category between MCPyV-status groups; a p value <0.05 was considered significant.

DNA was extracted from the specimens and screened for suitability for qPCR analysis by amplification of the cellular RNase P gene. Serial dilutions were tested to determine whether PCR inhibitors were present in the DNA samples and to select noninhibitory dilutions for MCPyV analysis. Total DNA yields ranged from 41 × 10⁴ to 403 × 10⁶ cell equivalents (median 51 × 10⁶) for the lymphoma samples, from 64 × 10³ to 216 × 10⁶ cell equivalents (median 29 × 10⁶) for the benign lymph node samples, from 13 × 10⁵ to 117 × 10⁶ cell equivalents (median 21 × 10⁶) for the lymph node samples with metastatic cancer, from 30 × 10⁵ to 78 × 10⁶ cell equivalents (median 39 × 10⁶) for the other inflammatory tissues, and from 16 × 10⁵ to 126 × 10⁶ cell equivalents (median 20 × 10⁶) for the other neoplastic tissues.

We tested 4 MCC and 4 melanoma samples (fixed and paraffin-embedded) for the presence of MCPyV by qPCR. MCPyV sequences were detected in 2 of 4 MCC samples; none were detected in the 4 melanoma samples (Table 1). MCCs contained an average of 0.29 (range 0.02–0.56) MCPyV genome copies per cell. MCPyV in 1 sample was confirmed by conventional PCR and sequence analysis. The sequence of the amplified fragment showed 98% similarity with the MCC350 reference sequence (GenBank accession no. EU375803) and 100% homology with MCC349 (GenBank accession no. FJ173813).

A total of 196 frozen malignant lymphoma samples were tested for MCPyV sequences. The classification of those samples is summarized in Table 2. Viral DNA was detected in 13 (6.6%) of the lymphomas (Tables 1, 2). As determined by qPCR, the viral copy numbers were relatively low. An average of 4.6 copies/10⁴ cells (range 0.16–27 copies/10⁴ cells, median 0.94 copies/10⁴ cells) was detected in the MCPyV-positive lymphomas. Among the lymphomas, the overall frequency of MCPyV between NHL and HL cases was similar (6.6% and 6.8%; p = 1.0) (Table 2).

MCPyV was identified most frequently in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (20.8%), a tumor type known to occur in MCC patients (Table 2). Patients with CLL/SLL had a high prevalence of MCPyV compared to all other B-cell lymphomas (20.8% vs. 2.8%; p = 0.01). The virus loads in CLL/SLL were similar to those found in patients with other types of lymphoma. Characteristics of MCPyV-positive and MCPyV-negative CLL/SLL patients are shown in Table 3. None of the 24 CLL/SLL patients had MCC.

A total of 110 frozen benign lymph node samples, 27 lymph nodes with metastatic tumors, and 20 other inflammatory or neoplastic tissues were tested in parallel (Table 4). Eleven of 110 (10%) benign lymph nodes were MCPyV positive (Tables 1, 4). Viral loads in MCPyV-positive lymph nodes averaged 2.3 copies/10⁴ cells (range 0.44–6.0 copies/10⁴ cells, median 2.1 copies/10⁴ cells). Of the 11 benign lymphoid specimens positive for MCPyV DNA, 8 were among 61 (13.1%) reactive hyperplasia samples and 3 were from 29 (10.3%) normal lymph nodes. All other samples tested were negative for MCPyV (Table 4).

Sequence analysis confirmed the qPCR results. Conventional PCR with MCPyV-specific primers was performed on 9 viral DNA-positive samples (4 lymphoma, 5 reactive hyperplasia). DNA amplification products of the expected size (126 bp) from the large T-ag gene were obtained, and sequence analysis confirmed the identity of MCPyV. Three of 4 MCPyV-positive lymphoma samples shared 100% sequence homology with strain MCC344 (GenBank accession no. FJ173807), whereas sequences of the remaining sample were identical to those of MCC349 (GenBank accession no. FJ173813). Among the 5 MCPyV-positive reactive hyperplasia samples, 4 had 100% homology to strain MCC344 and 1 to strain MCC339 (GenBank accession no. EU375804).

A series of 17 lymphoid specimens (7 of which were positive for MCPyV DNA by PCR), consisting of 11 lymphomas and 6 reactive hyperplasia samples, were tested for expression of MCPyV T-ag. Immunohistochemical staining with the CM2B4 monoclonal antibody was carried out without knowledge of the PCR results. One sample, classified as an angioimmunoblastic T-cell lymphoma, expressed detectable T-ag in scattered lymphocytes (Figure). This sample was positive for MCPyV DNA by PCR. The remaining samples tested were negative for T-ag expression by immunohistochemical test.

Five-year clinical follow-up information was available for 114 of 196 patients who had malignant lymphoma. Of those patients not included in follow-up analysis, 14 had died from other causes, 47 had received a diagnosis of lymphoma within the past 5 years, and 21 were lost to follow-up. Among the 114 patients with a 5-year follow-up, 49 had died of the disease and 65 were alive (24 with lymphoma and 41 without). Analyses showed no difference in the distributions among survival categories relative to MCPyV status (Table 5). Of the MCPyV-positive patients for whom follow-up data were available, 3 (42.9%) of 7 were alive and in remission compared with 38 (35.5%) of 107 MCPyV-negative patients. There were 3 (42.9%) deaths among the MCPyV-positive group and 46 (43.0%) deaths among the MCPyV-negative group (p = 0.88).