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. 2012 Dec;122(12):4645-53.
doi: 10.1172/JCI64116. Epub 2012 Nov 1.

Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus

Scott J Rodig  1 , Jingwei Cheng, Jacek Wardzala, Andrew DoRosario, Jessica J Scanlon, Alvaro C Laga, Alejandro Martinez-Fernandez, Justine A Barletta, Andrew M Bellizzi, Subhashini Sadasivam, Dustin T Holloway, Dylan J Cooper, Thomas S Kupper, Linda C Wang, James A DeCaprio
Affiliations

Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus

Scott J Rodig et al. J Clin Invest. 2012 Dec.

Abstract

A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen.

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Figures

Figure 1
Figure 1. Detection of MCPyV in MCC cell lines.
(A) Diagram of the MCPyV early region. Numbers correspond to nucleotide position in the MCPyV genome (HM355825). Rectangles show domain structure of large (LT) and small T (ST) antigen with approximate position of epitopes for CM2B4 and Ab3 antibodies and LXCXE (RB-binding) motif. Ab3 was generated against recombinant LT 1–260 corresponding to nucleotides 196–429 and 861–1,406. MCPyV isolated from MCC tumor contains truncations of LT that delete the helicase domain. PCR primer sets with nucleotide boundaries are indicated. (B) Western blot with Ab3 or CM2B4 of cell lysates prepared from MCC cell lines showing a predominant signal for truncated large T antigen between 36 and 50 kDa. U-2OS-LT contains a full-length large T antigen cDNA. Actin blot indicates protein loading. (C) MCPyV genome copy number in cell lines with indicated qPCR primer sets. All values were normalized to MKL-2. Error bars represent mean ± SEM.
Figure 2
Figure 2. Detection of MCPyV in MCC.
(A) Number of cases with immunohistochemical staining intensity for Ab3 and CM2B4. 3+, strong; 2+, moderate; 1+, weak, 0, negative. Ab3: n = 58; CM2B4: n = 57. (B) Histogram showing number of cases with highest MCPyV copy number value for any qPCR primer set. Copy number of viral genomes per tumor cell indicates less than or equal to the value indicated. n = 60.
Figure 3
Figure 3. Comparison of Ab3 and CM2B4 immunohistochemistry staining of MCC.
(A) UPI 26 and UPI 18: 3+ staining with CM2B4 and Ab3; UPI 30: primary and metastatic (Met) lymph node–negative staining with CM2B4 and 2+ with Ab3; UPI 27: 1+ staining with CM2B4 and Ab3. (B) UPI 37: no staining with CM2B4, 1+ with Ab3 at 0.6 μg/ml, and 2+ with Ab3 at 2.4 μg/ml. (C) UPI 15 and UPI 42: negative staining with CM2B4 and Ab3. Original magnification, ×400; scale bar: 50 μm.
Figure 4
Figure 4. Negative staining with Ab3 of normal lymph node and tonsil and GI neuroendocrine and small cell lung carcinomas.
(A) Lymph node and tonsil stained with Ab3. (B) GI neuroendocrine tumors stained with Ab3. Results are shown for cases C (left) and E (right). (C) SCLC specimen Q and (D) N stained with CM2B4 or Ab3. (E) MCC (UPI 36) stained with CM2B4 and Ab3. Original magnification, ×200; scale bar: 100 μm.
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