Primary central nervous system lymphoma expressing the human neurotropic polyomavirus, JC virus, genome

Abstract

B lymphocytes are known as a potential site for latency and reactivation of the human neurotropic polyomavirus, JC virus (JCV). In light of recent studies on the oncogenicity of JCV and the transforming ability of the JCV early protein, T antigen, we investigated the association of JCV with B-cell lymphomas of the central nervous system. Examination of 27 well-characterized clinical specimens by gene amplification and immunohistochemistry revealed the presence of DNA sequences corresponding to the JCV early genome and the late Agnoprotein in 22 samples and the JCV late genome encoding the viral capsid proteins in 8 samples. Expression of T antigen and that of Agnoprotein by immunohistochemistry were each detected in six specimens. No evidence of the production of viral capsid proteins was observed, ruling out productive infection of JCV in the tumor cells. The results from laser capture microdissection verified the presence of JCV T-antigen sequences in tumor cells with positive immunoreactivity to antibodies against the viral proteins T antigen and Agnoprotein. Due to previous reports demonstrating an association of the Epstein-Barr virus (EBV) with transformation of B lymphocytes, EBV DNA sequences and the EBV transforming protein, latent membrane protein 1 (LMP1), were analyzed in parallel. EBV LMP1 DNA sequences were detected in 16 of 23 samples, and LMP1 expression was detected in 16 samples, 5 of which exhibited positive immunoreactivity to JCV proteins. Double labeling demonstrated coexpression of JCV T antigen and EBV LMP1 in the same cells. The detection of the JCV genome in large numbers of B-cell lymphomas and its coexistence with EBV suggest a potential role for JCV in the pathogenesis of primary CNS lymphoma.

FIG. 1.

PCR amplification and Southern blot hybridization to detect JCV DNA fragments from tissue sections of primary CNS lymphomas. (A) Schematic representation of the JCV genome showing the early and late coding regions separated by the noncoding regulatory region. The location and nucleotide position of the primers used for PCR amplification are noted by using the nomenclature for the Mad-1 reference strain of JCV (GenBank no. J02226). (B) PCR products were analyzed by Southern blotting with three different probes specific for each of the polyomaviruses—JCV, BKV, and SV40—for which representative control data are shown. (C) PCR-Southern blot hybridization of DNA extracted from B-cell lymphoma samples show the presence of JCV T-antigen but not SV40 or BKV T antigens (left). Samples were also analyzed for the presence of JCV VP1, JCV Agnoprotein, and EBV LMP1 (right).

FIG. 2.

LCM and PCR amplification of JCV DNA from immunohistochemically positive tumor cells. Samples immunostained for the presence of JCV T-antigen and Agnoprotein are shown prior to LCM (A and D, respectively) and after LCM (B and E, respectively). The captured T-antigen- and Agnoprotein-positive cells (C and F, respectively) were analyzed by PCR-Southern blotting for JCV T antigen. (G) JCV T antigen could be detected by PCR-Southern blot analysis in both T-antigen-positive and Agnoprotein-positive cells.

FIG. 3.

Histological characterization and immunohistochemical detection of JCV and EBV proteins in primary CNS lymphoma samples. (A) A low-power magnification view of the tumor shows abundant neoplastic lymphocytes arranged in a concentric perivascular pattern that diffusely infiltrates the brain parenchyma. (B and C [inset]) Immunohistochemical expression of the Pan-B cell marker, CD20, demonstrates robust cytoplasmic reactivity in the neoplastic cells. At higher magnification, the cells appear round with irregular pleomorphic nuclei and mitotic figures are observed (C). Immunohistochemistry to detect the JCV gene products, T antigen and Agnoprotein, demonstrates intense reactivity for T antigen in the nuclei of neoplastic lymphocytes and Agnoprotein in the cytoplasm of the tumoral cells with a perinuclear pattern (T antigen [D and G]; Agnoprotein [E and H]). (F and I) Samples stained with antibody for the EBV LMP1 show cytoplasmic staining within neoplastic lymphocytes in the cells trapped in the Virchow-Robin space, as well as in cells infiltrating the brain parenchyma. Positive controls for T-antigen and Agnoprotein staining, respectively, included sections of JCV T-antigen-induced murine tumor (J) and a case of PML (K). (L, left and right sides, respectively) Negative controls included sections of normal brain adjacent to CNS lymphoma and tumor sections in which primary antibodies were omitted during the immunohistochemistry. Magnifications: A and B, ×100; D to F, L, ×400; C, G to K, ×1,000.

FIG. 4.

Double-labeling immunofluorescence for JCV T antigen and EBV LMP1 in primary CNS lymphoma cells. Samples of primary CNS lymphoma were evaluated by double immunolabeling for the presence of JCV T antigen (A) and EBV LMP1 (B). (C) Superimposition of panels A and B in which JCV T-antigen-positive cells are green and LMP1-positive cells are red. Representative tumor cells expressing T antigen alone are indicated with an arrow; arrowheads indicate cells expressing LMP1 only, and cells positive for both T antigen and LMP1 are indicated with an asterisk. Magnification, ×1,000.