Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma

Abstract

Human cytomegalovirus (HCMV) has been described to be associated with several human malignancies, though the frequency of detection remains controversial. It is unclear whether HCMV plays an active role in malignant tumor progression or becomes reactivated under pathologic conditions that result in chronic inflammation or immunosuppression. In this study, we report on the investigation of detecting HCMV in the tumors and peripheral blood of patients with newly diagnosed glioblastoma multiforme (GBM). Using immunohistochemistry, in situ hybridization, and polymerase chain reaction amplification of viral DNA, the detection of HCMV was investigated in tumor and blood specimens from patients with GBM as well as in the peripheral blood of normal volunteers and patients undergoing craniotomy for diagnoses other than GBM. We found that a high percentage (>90%) of GBM tumors, not surrounding normal brain, are associated with HCMV nucleic acids and proteins. Furthermore, a significant proportion of patients (80%) with newly diagnosed GBM have detectable HCMV DNA in their peripheral blood, while sero-positive normal donors and other surgical patients did not exhibit detectable virus, suggesting either a systemic reactivation of HCMV within patients with GBM or shedding of viral DNA from infected tumor cells into the periphery. These results confirm the association of HCMV with malignant gliomas and demonstrate that subclinical HCMV viremia (presence of viral DNA in blood without clinical symptoms of infection) is a previously unrecognized disease spectrum in patients with GBM.

Fig. 1

(A) Immunohistochemical detection of human cytomegalovirus (HCMV) proteins: (a) negative control (no primary antibody; objective lens ×10); (b) antismooth-muscle actin (mouse IgG2a mAb; ×10); (c) glioblastoma multiforme (GBM) specimen 1 stained with anti-HCMV IE1 (mouse IgG2a mAb, ×10); (d) higher magnification of anti-IE1 staining shows positive tumor cells and endothelial cells but negative lymphocytes and vascular intima (×20); (e) GBM specimen 2 stained with anti-HCMV IE1 showing staining of perivascular tumor cells but lack of detection in necrotic areas (×10); (f) perivascular tumor cells stained with anti-IE1 mAb (×20); (g and h) GBM specimen 3 stained with anti-HCMV pp65 mAb showing nuclear and perinuclear staining of tumor cells scattered throughout the GBM specimen (×10 and ×20, respectively); (i and j) CMV-infected lung stained with antismooth-muscle actin mAb (×10); (k) CMV-infected lung stained with anti-HCMV IE1 (×20); (l) CMV-infected lung stained with anti-HCMV pp65 mAb (×20). (B) HCMV detection in matched GBM and normal brain. Representative histochemical sections from two GBM specimens containing areas of normal brain and tumor were stained for detection using isotype control antibodies (patient 1, left column), or anti-HCMV pp65 (patient 1 tumor, middle column; patient 2, right column). Focal areas of reactivity against the HCMV pp65 antibody was observed throughout the tumor-involved areas, but normal brain was devoid of immunoreactivity to the HCMV-specific antibodies (IE1 staining showed identical findings with more ubiquitous detection of IE1 in the tumor, not shown). All photographs taken at ×40 objective magnification.

Fig. 2

In situ hybridization. Detection of human cytomegalovirus (HCMV) nucleic acids using HCMV IE1 and whole genomic probe. First row: CMV-infected lung hybridized with (a) a nonsense DNA probe (negative control) and (b) poly-AAA probe (positive control); (c) HCMV IE1 probe demonstrates hybridization to large cytomegalic cells throughout the lung specimen. Second row: (d) GBM specimen hybridized with nonsense DNA probe and (e) poly-AAA antisense probe; (f) detection of HCMV IE1 in a GBM specimen shows hybridization of tumor cells throughout the specimen. Third row: (g) GBM specimen stained with nonsense whole genomic probe (negative control); (h) alu DNA probe (positive control) and (i) pan HCMV genomic DNA probe, demonstrating HCMV detection within tumor cells but not vasculature within the specimen. Fourth row: Competition experiment showing ability to block detection of IE1 hybridization using a 50-fold excess of unlabeled specific competitor DNA probe but not using an excess of the nonsense probe; (j) lack of detection with nonsense DNA probe; (k) HCMV IE1 antisense probe with 50-fold excess nonsense competitor probe; (l) HCMV IE1 antisense probe with 50-molar excess of IE1 competitor probe. Results show localization of HCMV DNA within GBM tumor cells and capacity to specifically compete hybridization with HCMV-specific competitor probe.

Fig. 3

Detection of human cytomegalovirus (HCMV) in primary glioblastoma multiforme (GBM) cultures. Short-term GBM cultures from two patients were stained with a variety of monoclonal antibodies to detect HCMV proteins or astrocyte markers (GFAP). Cells were fixed, permeabilized, and stained with equimolar concentrations of isotype control antibodies (mouse IgG), anti-GFAP antibody, anti-HIV p17, anti-HCMV glycoprotein B (gB), anti-CD45, and anti-HCMV pp28. Photos demonstrate strong nuclear reactivity of astrocytic tumor cells to anti-HCMV gB and pp28 with scant cytoplasmic staining in two primary GBM cultures. Isotype controls and HIV p17 staining were negative in all samples examined. CD45 immunoreactivity was observed in scattered hematopoietic cells in one culture (patient 1 GBM, bottom left panel), but did not stain astrocytic tumor cells.