Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR, Immunohistochemistry, and In Situ Hybridization

Abstract

Purpose: Reports of cytomegalovirus (CMV) detection in high-grade gliomas (HGG)/glioblastoma have been conflicting. We undertook a comprehensive approach to determine the presence or absence of CMV in tissue, plasma, and serum of HGG patients.Experimental Design: In a retrospective arm, 25 fresh frozen tissues from glioblastoma patients were tested for CMV by real-time PCR. Tissue microarrays from 70 HGG patients were tested by IHC and 20 formalin-fixed paraffin-embedded (FFPE) glioblastoma tissues by IHC and chromogenic in situ hybridization (CISH), targeting CMV-encoded IE1/2 and pp65. In a prospective arm, 18 patients with newly diagnosed HGG provided tissue and blood samples.Results: All retrospectively collected tissues were negative for CMV by all methods. In the prospective cohort, 18 patients with newly diagnosed HGG provided blood samples at the time of diagnosis and during follow-up. Of 38 plasma specimens, CMV DNA was detected in 3 of 18 samples at baseline and 1 of 20 follow-up samples. Serum CMV IgG was positive in 8 of 15 (53%) of patients. Among the FFPE samples tested in the prospective arm, all were negative for CMV by IHC, CISH, and PCR.Conclusions: Utilizing 6 highly sensitive assays with three orthogonal technologies on multiple specimens and specimen types, no evidence for CMV in glioblastoma tissues was found. Our findings call for multicenter blinded analyses of samples collected from different geographical areas with agreed upon study designs and determination of causality or lack thereof of CMV in HGG/glioblastoma for future guidance on the necessary antiviral and/or CMV-based therapies. Clin Cancer Res; 23(12); 3150-7. ©2016 AACR.

Figure 1. Analytical validation of CISH and IHC in human fibroblast cell lines

Human foreskin fibroblasts were uninfected or infected with a human CMV Towne for 72 h. Cells were then fixed in neutral buffered formalin overnight and processed into paraffin blocks. CISH assay depicts targeting IE1 DNA in uninfected (A) and CMV-infected (B), and pp65 DNA in uninfected (C) and CMV-infected cells (D). Representative figures for IHC staining of IE1- and pp65 proteins in uninfected (E,G) and CMV-infected (F,H) cells are shown (all images are original magnification of ×400).

Figure 2. CMV detection by CISH and IHC from CMV-positive human tissues

CISH assays targeting IE1 DNA (A), pp65 DNA (B); IHC staining for IE1 (C), pp65 (D) proteins performed on a colonic biopsy with known CMV infection (all images are ×100).

Figure 3. Lack of CMV detection in human high grade gliomas

IE1 (A) and pp65 (B) CISH assays performed on FFPE samples of a representative high grade glioma were negative for their corresponding DNA segments. By immunohistochemistry the same sample was negative for IE1 (C) and pp65 (D) proteins as well (all images are ×200).