Detection of human neurotropic JC virus DNA sequence and expression of the viral oncogenic protein in pediatric medulloblastomas

Abstract

Medulloblastoma represents greater than 25% of childhood intracranial neoplasms and is considered a highly malignant tumor. This tumor, which arises predominantly in the cerebellar vermis, preferentially affects children between the ages of 5 and 15. Although the etiology of medulloblastomas in humans remains unknown, results from several experiments have indicated that the human neurotropic JC virus (JCV) is able to induce cerebellar neoplasms in rodents that exhibit a phenotype similar to that of human medulloblastomas. JCV is a polyomavirus that is widespread in the human population, with infection occurring most frequently in early childhood. In this study, we have examined the possible association of JCV with human medulloblastomas. By using PCR techniques we demonstrate that 11 of 23 samples of tumor tissue contain DNA sequences corresponding to three different regions of the JCV genome. More importantly, we demonstrate the presence of DNA sequences encoding the N- and C-terminal regions of the JCV oncogenic protein, T antigen, in 11 of 23 samples and the production of T antigen in the nuclei of 4 samples of tumor tissue. These observations provide evidence for a possible association of JCV with human medulloblastomas.

Figure 1

Histological classification of medulloblastoma samples. Tumor specimens were classified as classic with a high degree of cellularity but no secondary architectural features (A), neuroblastic indicated by the presence of indian filing (see arrows in B) and rosettes (B Inset), or desmoplastic exhibiting pale islands (C). (C) Arrow and arrowhead indicate dark, poorly differentiated cells in the mantle zone surrounding a pale island and the pale island itself, respectively. (A Inset) The presence of apoptotic bodies. (C Inset) The reticulin impregnation characteristic of the compact, mantle-like zone of desmoplastic medulloblastoma. In all variants, a high nuclear-to-cytoplasmic ratio was observed [hematoxylin and eosin, original magnification, ×200; ×100 (C and C Inset); ×1,000 (all other Insets)].

Figure 2

Structural organization of the JCV genome and the position of the oligonucleotide primers and probes used for PCR amplification and Southern blot analysis. The numbers within the inner circle represent the map positions, with 0.0 being the EcoRI site (25). Thick arrows depict coding regions for the viral early protein T antigen (solid) and late proteins (shaded). The position of the viral control region between the initiation sites for early and late proteins is shown. The locations of the PCR primers are shown by thin arrows outside of the circle. The sizes of the amplified DNAs and the DNA probes used for Southern blot analysis for the detection of PCR products are depicted.

Figure 3

Detection of JCV DNA sequences in human medulloblastomas. DNA from nine clinical samples was analyzed for the presence of JCV sequences by PCR using primers derived from the N terminus of T antigen (A), the C terminus of T antigen (B), and the late region of the viral genome (C). PCR products were analyzed by Southern blot analysis by using specific DNA probes as shown in Fig. 2. The nucleotide composition of the amplified DNA for each region is illustrated. Nucleotides depicting the position of PCR primers are shown in bold, and underlined nucleotides show the region detected by probes used for Southern blotting. Lane 10 represents a negative control (A, B, and C).

Figure 4

Immunohistological analysis of human of medulloblastomas. Immunostaining of tumor samples with anti-T antigen antibody (A, B, and C). Arrows indicate T antigen-positive cells. Arrowhead in A and A Inset show the presence of mitotic figures. Immunostaining of tumor samples with neuronal markers including synaptophysin (D), class III β-tubulin (E), and glial fibrillary acidic protein (GFAP, F) is shown [hematoxylin counterstain, original magnification: ×400 (A, B, E, and F); ×200 (C and D); ×1,000 (Insets)].