Fluorescence in situ hybridization confirms the presence of Merkel cell polyomavirus in chronic lymphocytic leukemia cells

To the editor:

We would like to complement our recently reported data on the detection of the Merkel cell polyomavirus (MCPyV) in chronic lymphocytic leukemia (CLL) cells.¹  We have shown that MCPyV could be identified by PCR in highly purified CLL cells of 19 of 70 (27.1%) CLL patients. In 6 cases, a 246 bp deletion in the helicase region of the large T-antigen (LTAg) of MCPyV was found. Truncating mutations or deletions in the LTAg of MCPyV lead to a loss of the helicase activity and thus prevent viral replication.²  In addition, in 2 of these CLL cases deleted LTAg was detected concomitant to wild-type MCPyV.

Here we confirm the presence of the MCPyV genome in CLL cells by using fluorescence in situ hybridization (FISH). MCPyV-FISH was performed according to a recently published FISH protocol³  to detect human papilloma virus (HPV) in formalin-fixed and paraffin-embedded (FFPE) tissue. The MCPyV DNA probe we used was isolated from a Merkel cell carcinoma (MCC) and contained a 4 bp deletion creating a stop codon before the helicase domain. FISH was applied to all 6 FFPE bone marrow trephines (EDTA decalcified) in which we previously detected the 246 bp LTAg MCPyV deletion. Three MCPyV DNA-positive MCCs, as detected by PCR,⁴  were used as positive controls showing punctate nuclear signals in MCC indicative for integrated MCPyV in the tumor cells (Figure 1A). Negative controls included omission of the MCPyV probe and several MCPyV-negative tissues, that is, a mantle cell lymphoma and 2 colon carcinomas. None of the negative controls revealed a FISH signal (Figure 1C). Of the 6 CLL cases harboring the 246 bp deletion in the MCPyV-LTAg, 4 revealed punctate signals in the nucleus (Figure 1E,G), comparable to those observed in MCC. The observation that nuclei sometimes show FISH signals differing in intensity (granular staining pattern) might suggest that in addition to MCPyV-DNA integration, transcribed viral RNA is also present.^3,5  FISH analysis on the remaining 2 MCPyV-positive CLLs were inconclusive, most likely because of tissue overfixation and/or poor DNA quality.

In analogy to the punctate nuclear signals observed in anogenital and oropharyngeal lesions by HPV-FISH^3,5  and in MCC by MCPyV-FISH, we have interpreted the present findings as integration of MCPyV in CLL cells. This finding additionally strongly supports a possible involvement of MCPyV in a significant subset of CLL.