Significant Association of Multiple Human Cytomegalovirus Genomic
Loci with Glioblastoma Multiforme Samples
Padhma Ranganathan,a,bPaul A. Clark,eJohn S. Kuo,c,d,e,fM. Shahriar Salamat,gand Robert F. Kalejtaa,b,c,d
Institute for Molecular Virology,aMcArdle Laboratory for Cancer Research,bCarbone Comprehensive Cancer Center,cStem Cell and Regenerative Medicine Center,dand
Departments of Neurological Surgery,eHuman Oncology,fand Pathology and Laboratory Medicine,gUniversity of Wisconsin—Madison, Madison, Wisconsin, USA
counts for nearly half of all central nervous system (CNS) malig-
nancies in adults. Equivalent to a grade IV diffuse astrocytoma,
GBMs consist primarily of neoplastic astrocytes, the most abun-
plastic cell types, including neurons, oligodendrocytes, macro-
phages, 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
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
human T-lymphotropic virus type 1 (HTLV-1), human papillo-
mavirus (HPV), hepatitis C virus (HCV), Kaposi’s sarcoma-
associated herpesvirus (KSHV), and Merkel cell polyomavirus
the entire genome is maintained, as well as abortive infections
to cellular transformation and cancer development. Not surpris-
of the molecular hallmarks of cancer (10) that promote cellular
plasticity (through genomic instability, inflammation, deregula-
tion of cellular energetics, and induction of angiogenic and met-
astatic processes), proliferation (by sustaining proliferative
signaling, evading growth suppressors, and enabling replicative
lioblastoma multiforme (GBM) is an aggressive and malig-
nant tumor of glial origin with a grim prognosis (43). It ac-
immortality), and survival (avoidance of immune detection and
inhibition of apoptosis). However, many other viruses not yet
lecular events, leading to speculation that more human cancers
have a viral etiology or association than is currently appreciated.
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. How-
ever, an emerging concept hypothesizes that chronic conditions,
although perhaps not directly caused by HCMV, are likely to be
to HCMV infection include immunosenescence (40), certain car-
diovascular diseases (36), and cancer (34). Either HCMV infec-
duce all of the molecular hallmarks of cancer (13). For example,
viral infection or HCMV-encoded proteins promote genomic in-
stability (8, 30), inflammation (2), angiogenesis (4), and cell mi-
and life span (35), and inactivate cellular immune functions (25)
Received 22 August 2011 Accepted 3 November 2011
Published ahead of print 16 November 2011
Address correspondence to Robert F. Kalejta, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 854–864
and apoptotic pathways (3). Therefore, HCMV represents an in-
triguing 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
20 out of 21 (31), or 8 out of 49 (16). The viral phosphoprotein of
uct 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, 20), 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
unique long section of the genome (UL55 that encodes glycopro-
tein 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.
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
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,
cating 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 experi-
ments 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
or noncancerous brain tissues by testing for the presence of mul-
tiple 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.
MATERIALS AND METHODS
Sample collection and DNA extraction. This retrospective study was
conducted in agreement with the terms of a University of Wisconsin—
Madison (UW-Madison) institutional review board (IRB) protocol (M-
2009-1420). Tumor samples were obtained with prior patient consent.
The samples were deidentified and are untraceable to individual patients.
Only the date of collection of the sample and diagnosis information were
available. The samples were destroyed during the course of the analysis.
Sample preparation was performed in clean laboratories never before
used for HCMV research in order to prevent spurious contamination.
Paraffin-embedded archived astrocytoma grade 4 (GBM), meningioma,
schwannoma, oligodendroglioma, or nonneoplastic epileptic brain sam-
slides were examined. Sections containing the highest percentage of tu-
mor cells and the least amount of necrosis were chosen. The 75 GBM
samples analyzed here represent essentially the entire collection of usable
samples collected from 1994 through 2009 at UW-Madison. To purify
DNA from paraffin-embedded tissues, 10-?m sections were extracted
TABLE 1 PCR primer sequences used in this studya
Gene Forward primerReverse primer
aNucleotide sequences for primer pairs used to amplify segments of the indicated HCMV gene are shown. The expected size of the amplified product is listed in base pairs. Also,
the sequences of the primers (named “IE1”) employed in the comparative PCR (Fig. 1) and copy number (Fig. 10) studies are indicated.
HCMV Genome in Glioblastoma (GBM)
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