Glioblastoma multiforme (GBM) is an aggressive and malignant tumor of glial origin with a grim prognosis (43
). It accounts for nearly half of all central nervous system (CNS) malignancies in adults. Equivalent to a grade IV diffuse astrocytoma, GBMs consist primarily of neoplastic astrocytes, the most abundant type of glia, but tumor specimens also include other nonneoplastic cell types, including neurons, oligodendrocytes, macrophages, and glial and neural stem cells (42
). The heterogeneous nature of these tumors may at least partially explain why they are refractory to current therapeutics and has led to the hypothesis that GBMs may originate from stem-like cells, perhaps in the subventricular zone of the CNS (9
). The etiology of GBM is unknown, although exposure to ionizing radiation, electrical, or magnetic fields has been proposed as a risk factor (22
). Recently, several reports have detected a potential association between GBMs and human cytomegalovirus (HCMV), a common betaherpesvirus.
Viruses are causative agents of human cancers (21
). At least 15% of all human tumors have a viral etiology. Human cancer viruses include Epstein-Barr virus (EBV), hepatitis B virus (HBV), human T-lymphotropic virus type 1 (HTLV-1), human papillomavirus (HPV), hepatitis C virus (HCV), Kaposi's sarcoma-associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCV). True viral infection (whether productive or latent) where the entire genome is maintained, as well as abortive infections where only select regions of the viral genome are present, can lead to cellular transformation and cancer development. Not surprisingly, human cancer viruses can produce within cells one or more of the molecular hallmarks of cancer (10
) that promote cellular plasticity (through genomic instability, inflammation, deregulation of cellular energetics, and induction of angiogenic and metastatic processes), proliferation (by sustaining proliferative signaling, evading growth suppressors, and enabling replicative immortality), and survival (avoidance of immune detection and inhibition of apoptosis). However, many other viruses not yet directly associated with human tumors may also initiate such molecular events, leading to speculation that more human cancers have a viral etiology or association than is currently appreciated. One such putative relationship between a virus and a cancer that is receiving increased examination is that of HCMV and GBM.
HCMV asymptomatically infects the majority of the human population, and virus-induced sequelae are generally observed only under conditions of insufficient host immune function (7
). Examples include birth defects in congenitally infected neonates, retinitis and blindness in AIDS patients, and graft rejection in transplant patients receiving immunosuppressive therapy. However, an emerging concept hypothesizes that chronic conditions, although perhaps not directly caused by HCMV, are likely to be exacerbated by infection with this virus (32
). Disease states linked to HCMV infection include immunosenescence (40
), certain cardiovascular diseases (36
), and cancer (34
). Either HCMV infection or the ectopic expression of individual viral proteins can produce all of the molecular hallmarks of cancer (13
). For example, viral infection or HCMV-encoded proteins promote genomic instability (8
), inflammation (2
), angiogenesis (4
), and cell migration (39
), modulate cellular energetics (41
), proliferation (11
), and life span (35
), and inactivate cellular immune functions (25
) and apoptotic pathways (3
). Therefore, HCMV represents an intriguing candidate human cancer virus.
The major experimental method identifying HCMV in GBM samples has been immunohistochemical (IHC) detection of the 72-kDa viral immediate-early 1 (IE1) protein. Individual studies have detected IE1 in the following number of GBM specimens: 27 out of 27 (5
), 42 out of 45 (20
), 21 out of 21 (29
), 10 out of 10 (35
), 20 out of 21 (31
), or 8 out of 49 (16
). The viral phosphoprotein of 65 kDa (pp65) has also been detected by flow cytometry in 5 out of 5 GBM samples (6
) and by IHC in 8 out of 8 (5
), 30 out of 33 (20
), or 25 out of 59 (16
) GBM samples. Furthermore, the protein product of the 28th gene in the unique short region of the genome (US28) was found in 20 out of 21 GBM samples (31
). In situ
hybridization (ISH) for either the HCMV IE locus (5
), total viral genomic DNA (5
), or an undisclosed region(s) of the viral genome (29
) has also been used to score for the presence of HCMV, obtaining positive results in every (29 out of 29) GBM sample examined. Finally, PCRs have detected the 55th gene in the unique long section of the genome (UL55 that encodes glycoprotein B [gB]) in 7 out of 9 (5
) or 21 out of 34 (20
) GBM specimens. Many interpret these results as solid evidence indicating that HCMV is present in GBMs.
However, other studies using similar approaches have failed to detect HCMV in GBM samples. PCR and IHC for IE1 or pp65 failed to detect the presence of HCMV in 22 GBM samples (24
). Likewise, an independent study (15
) that used IHC for pp65, ISH for IE1 or pp65, and PCR for gB failed to detect HCMV in 8 GBM samples. An additional investigation (28
) detected IE1 by IHC in only 9 out of 81 GBMs and by ISH in only 7 out of 81 GBMs, although those 7 were also positive by IHC. Furthermore, complicating issues have created uncertainty about the results of the above studies that detected a strong association of HCMV with GBM tumors. For example, the IHC images presented are very difficult for all but a trained pathologist to decode, and thus, it could be argued that the conclusions drawn from such experiments are more subjective than objective. Furthermore, the viral antigens are detected only when ultrasensitive IHC methods are employed. Perhaps most troubling, the normally nuclear IE1 and pp65 proteins detected with this technique are almost invariably cytoplasmic when detected in GBM tissue, calling into question the assay's specificity. Thus, any association of HCMV with GBM is considered with a healthy skepticism.
The previous studies discussed above often suffer from small sample sizes, low numbers of HCMV loci analyzed, subjective assays, and insufficient quantitative analysis. Thus, we sought to independently determine whether HCMV was statistically more likely to be present in GBM samples as opposed to other cancerous or noncancerous brain tissues by testing for the presence of multiple viral genomic loci by PCR in GBM specimens. Our results lead us to conclude that all regions of the HCMV genome are present in the vast majority of GBM samples but that only a small minority of cells in any individual sample harbors HCMV DNA.