Detection of human cytomegalovirus in different histological types of gliomas

Abstract

The association between human cytomegalovirus (HCMV) infection and glioblastoma has been a source of debate in recent years because of conflicting laboratory reports concerning the presence of the virus in glioma tissue. HCMV is a ubiquitous herpesvirus that exhibits tropism for glial cells and has been shown to transform cells in vitro. Using sensitive immunohistochemical and in situ hybridization methods in 50 glioma samples, we detected HCMV antigen and DNA in 21/21 cases of glioblastoma, 9/12 cases of anaplastic gliomas and 14/17 cases of low-grade gliomas. Reactivity against the HCMV IE1 antigen (72 kDa) exhibited histology-specific patterns with more nuclear staining for anaplastic and low-grade gliomas, while GBMs showed nuclear and cytoplasmic staining that likely occurs with latent infection. Using IHC, the number of HCMV-positive cells in GBMs was 79% compared to 48% in lower grade tumors. Non-tumor areas of the tissue contained only four and 1% of HCMV-positive cells for GBMs and lower grade tumors, respectively. Hybridization to HCMV DNA in infected cells corresponded to patterns of immunoreactivity. Our findings support previous reports of the presence of HCMV infection in glioma tissues and advocate optimization of laboratory methods for the detection of active HCMV infections. This will allow for detection of low-level latent infections that may play an important role in the initiation and/or promotion of malignant gliomas.

Figure 1. Immunohistochemical and In Situ Hybridization (ISH) staining for HCMV

(A) Immunohistochemical stain of a GBM section without an antibody (negative control). (B) Immunohistochemical stain of a GBM section with an antibody against actin (positive control). (C) Immunohistochemical stain of a GBM section with an antibody against the HCMV IE1-72 protein. (D) ISH stain of a GBM section with a probe against black beetle virus DNA (negative control). (E) ISH stain of a GBM section with an Alu DNA probe (positive control). (F) ISH stain of a GBM section with an HCMV DNA probe. Panels A, B, and C represent immunohistochemical staining of serial sections from the same GBM specimen, and panels D, E, and F represent ISH staining of serial sections from the same GBM specimen. We acquired images of stained GBM sections using a microscope with a Nikon Plan Fluor 20×/0.50 (Panels A-F) (Nikon, Melville, NY, USA).

Figure 2. Immunohistochemical staining for HCMV by Tumor Histology

(A) Immunohistochemical stain of a GBM section with an antibody against the HCMV IE1-72 protein. (B) Immunohistochemical stain of an anaplastic glioma section with an antibody against the HCMV IE1-72 protein. (C) Immunohistochemical stain of a low-grade glioma section with an antibody against the HCMV IE1-72 protein. (D) Immunohistochemical stain of a lung tissue section for HCMV IE1-72 representing an active infection. Of note in the GBM specimens is that the presence of HCMV infection is highly cytoplasmic within the area of the tumor but not areas of nontumor brain or necrotic tissue within the same specimen. We captured images of stained glioma sections using a microscope with a Nikon Plan Fluor 20×/0.50 (panel A) or Nikon Plan Fluor 40×/0.75 lens (Panels B-D) (Nikon, Melville, NY, USA).