A role for HCMV in malignant disease initiation has long been proposed since the findings of the oncogenic potential of the HCMV viral particles or gene particles in in vitro cultured cells.9,22–25
Furthermore, the establishment of lifelong latency of HCMV within progenitor cells in the bone marrow, brain, and possibly other tissues seems consistent with a predisposing risk factor for viral transformation of immortalized and pluripotent cell types.26–30
However, a simpler and more plausible explanation for virus detection in cancer patients is a secondary reactivation of virus after cancer-related, and possibly treatment-related, immunosuppression. GBMs are known to exert a variety of local and systemic immunosuppressive effects in patients, all of which could contribute to the establishment of an environment permissive of HCMV reactivation.31
Among other immunosuppressive factors, we have found that newly diagnosed patients with GBM are often profoundly lymphopenic, with particular deficits in their CD4+ T-cell compartment that result in impaired cell-mediated immunity.32,33
Given the known role of the cellular immune system in maintaining viral latency, cell-mediated immunologic defects in patients with GBM and other cancers may cause viral reactivation and propagation. In addition to being susceptible to tumor-related immunosuppression, patients with GBM are often placed on corticosteroids at the time of diagnosis, which has a known capacity to elicit further immunosuppression that could lead to HCMV reactivation.34
Thus, our findings and those reported by Cobbs et al.5
could simply be attributed to a subclinical reactivation of virus secondary to tumor-related or treatment-related immunosuppression. However, viral reactivation secondary to treatment-related immunosuppression is an unlikely mechanism because newly diagnosed patients with GBM at our institution have usually undergone less than 2 weeks of corticosteroid therapy prior to resection, and the detection of virus in the blood and throughout the tumor specimens at the time of initial resection seems indicative of viral reactivation and a more prolonged course of viral replication.35
Studies in patients undergoing bone marrow transplantation and organ transplantation who are placed on much more rigorous immunosuppressive regimens demonstrate a time course of HCMV reactivation, typically on the order of several weeks to months posttherapy.36,37
A few lines of evidence, however, support a more closely linked association of HCMV with GBM. The frequency of detection of virus in tumor samples in our studies is higher than the expected frequency of latently infected individuals in the population (50%–70%), so one would expect that if HCMV were simply secondarily reactivated by immunosuppression in these patients that a frequency more closely linked to the general population would be found. The seropositive status of patients whose tumors were examined from our tumor bank is unknown, however, and much larger epidemiologic studies beyond the scope of this report would be needed to provide any meaningful investigation of this type of analysis.
As reported by Cobbs et al.,5
we have also detected HCMV proteins and nucleic acids in the tumors—but not surrounding normal brain—of patients with MGs. Preferential viral replication within astrocytomas may be explained by the relative permissiveness of astrocytes and neural progenitors to HCMV infection compared with other brain-cell types.38–40
Of interest, astrocytoma cell lines have been used for years to propagate HCMV in vitro because they are one of the few permissive cell lines that allow for culture of the virus.41,42
Another plausible explanation for preferential viral tropism in brain tumors is recent identification of the epidermal growth factor receptor (EGFR) as a cellular binding and incorporation site for the entry of HCMV into cells.43
GBMs almost uniformly demonstrate amplified EGFR expression, while normal brain is largely negative.44–46
We noted that IHC was more sensitive in our hands in the detection of CMV than PCR (). We attribute this difference to the fact that normal brain and necrotic tissue, which may be included in the gross tumor specimens provided during resection, are devoid of CMV; thus, sampling error may result in missing CMV-infected viable tumor when directly extracting DNA from small quantities of tissues for PCR. This sampling error is avoided during IHC evaluation since viable tumor tissue is selected by a trained neuropathologist prior to immunohistochemical evaluation. We have also observed that IE1 is generally more ubiquitously expressed in GBM tissue than pp65, and in a minority of tumor samples, focal reactivity could be observed (). While evaluation of the demographics and prognosis of the few cases where a focal pattern was observed did not reveal any distinguishing characteristics, a more extensive evaluation of the levels of CMV in GBM tissue—based on quantitative PCR analysis, immunohistochemical evaluation of large areas of tumor, or intracellular FACS analysis of CMV proteins in dissociated tumor tissue—may reveal whether the levels of CMV or staining patterns have any prognostic or predictive value. Such analysis is the focus of future research.
Regardless of whether HCMV is an early or late event associated with gliomagenesis, the presence of the virus within tumor cells holds significance for several reasons: (1) HCMV is known to down-regulate the immunogenecity of infected cells through inhibition of antigen presentation, down-regulation of surface MHC expression, elaboration of TGF-β from infected cells (particularly astrocytes), and secretion of a viral interleukin 10 homologue (vIL-10).47–49
All of these factors may contribute to the immunologic evasion of infiltrative tumor cells and make MGs more difficult for the immune system to eradicate. (2) HCMV could modulate other properties that could contribute to a more malignant phenotype in tumor cells, including increasing angiogenesis, invasiveness, and cell proliferation, as well as decreasing susceptibility of infected tumor cells to cell death through blockade of apoptopic pathways.17
(3) The presence of viral antigens specifically in tumor cells lends the potential for targeting HCMV as a tumor-associated antigen in gliomas, lending the vast array of reagents and extensive experience in immunotherapeutic targeting of HCMV as tools to leverage against malignant brain tumors.50
Three other groups recently investigating the presence of HCMV in gliomas have failed to confirm the findings published by Cobbs et al.5
and reported by us in this manuscript.12–14
While the reasons for these discrepancies are unclear, one possibility is differences in the sensitivities of the assays employed by the different investigators’ laboratories. We have found, for instance, that detection of HCMV by IHC in brain tumors requires optimal antigen retrieval as well as blockade of nonspecific binding of isotype controls; IHC protocols using less optimized processes revealed negative detection in tumors. Nonoptimized staining protocols, however, were sufficient for detection of HCMV in cases of HCMV pneumonia used as positive controls in this study. While quantification of viral load was not examined in this study, these results, and the fact that GBM patients do not exhibit clinical signs of HCMV infection, suggest that very low levels of virus may propagate within these patients and require more sensitive detection methods than are necessary for detection in cases of symptomatic viral infection. Also, extensive comparison of primer sets and PCR detection methods revealed variability among the sensitivities of various primer sets and PCR conditions using known concentrations of viral standards. Our efforts at optimizing the recovery of low levels of viral DNA and PCR amplification of HCMV DNA have demonstrated that detection of HCMV levels present in patients with MG is not a trivial issue; therefore, controls having limiting quantities of virus should be used to ensure retention and detection of small numbers of viral copies per sample. Ultrasensitive detection techniques run the risk of detecting latent viral genomes persistent in a very small fraction of cells present in normal hosts. However, our inability to detect HCMV viral DNA in the whole blood of normal hosts or patients with nonmalignant brain tumors indicates that our detection methods are likely not sensitive enough to pick up latent virus and demonstrates a specific association of HCMV with GBM tumors.
We found that direct isolation of DNA from tumor samples and blood was consistently more reliable in detection than using DNA purification prior to PCR amplification, where detection of low-copy viral DNA required considerably larger sample size in order to purify sufficient viral DNA (Mitchell et al., unpublished data). Because we have confirmed the amplification of HCMV by DNA sequencing in more than 21 GBM samples, we are confident that our detection is due neither to artifact nor contamination by laboratory viral DNA. Consistent negative results obtained in samples from normal patients further support this conclusion. Finally, it is possible, based on the demographic profile of patients examined within various laboratories reporting on HCMV detection, that wide variation in HCMV association may exist, although we do not think this is likely to explain the inability of some laboratories to detect HCMV in association with GBM.
These findings warrant further study to determine whether the presence of viral DNA in the blood or tumors of patients, or quantification of viral load among HCMV-positive patients with GBM, holds any prognostic or predictive significance. Studies are underway in our laboratory to determine the clinical significance of HCMV detection in patients with GBM, along with efforts aimed at targeting these tumors through use of HCMV-targeted immunotherapy.