Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study

Glioblastoma multiforme grade IV (GBM) is the most aggressive and common brain tumor in humans. In Sweden, with a population of 9 million, there are approximately 300 new GBM cases per year [1]. Histologically, GBM is characterized by the presence of necrotic areas in the brain tissue surrounded by anaplastic cells and hyperplastic blood vessels, and a disparate genetic signature, which illustrates the heterogeneity of this tumor [2, 3]. Current treatments such as surgical resection, radiation, and chemotherapy are relatively ineffective due to the aggressive nature of GBM. Despite increasing molecular knowledge of GBM tumors, few new therapeutic strategies have been offered these patients during the past decades and these patients still have a poor prognosis with a median survival of 12-15 months [4]. Recent studies have reported the presence of an active Human Cytomegalovirus (HCMV) infection in 90-100% of GBM tumors [5–7].

HCMV belongs to the herpesviridea family and maintains latency in pre-monocytic cells after a primary infection [8]. Viral reactivation can occur when latently infected monocytes undergo differentiation to macrophages or dendritic cells, which involves stimulation with inflammatory cytokines [9, 10]. Thus, it is possible that the inflammatory environment in the glioblastoma tumor triggers reactivation of latent HCMV.

Although there is compelling evidence that the HCMV protein US28 may be oncogenic, this virus has not been considered to be oncogenic. Rather, HCMV proteins confer a variety of biological functions that potentially affect tumor biology and contribute to cancer progression. HCMV proteins for example interact with p53, Rb and PTEN and influence cell-cycle regulation, induce chromosomal aberrations, cellular differentiation, migration, angiogenesis and confer immune evasion mechanisms [11–16]. Recent studies have demonstrated that HCMV-US28 induces cyclo-oxygenase-2 (COX2) expression, production of vascular endothelial growth factor (VEGF) and tumor formation in vivo, via activation of nuclear factor kappa B (NF-kB), STAT3 phosphorylation and accumulation of beta cathenin in the cell nucleus. Furthermore, we found that phosphorylated STAT-3 was correlated with poor survival of GBM patients [17–19]. Thus, this virus may either be an epiphenomenon of glioblastomas or may through its sophisticated strategies that affect tumor biology and provide immune evasion strategies contribute to cancer progression. We recently demonstrated that targeting HCMV infection in medulloblastoma tumors with an anti-viral drug and a COX-2 inhibitor prevented tumor growth by 72% in an animal model [20]. These observations suggest that HCMV may contribute to the tumorgenesis of medulloblatsoma tumors, and that the virus may be a novel target for cancer therapy. We hypothesized that if the virus affects tumor progression, a low grade HCMV infection in glioblastomas may be associated with longer patient survival. In this case-control study, we investigated if the grade of HCMV infection in GBM tumors was associated with survival over 18 months.

The etiology of GBM is unknown and the prognosis for GBM patients is still poor despite recent advances in medical treatment with temozolomide and radiotherapy [23, 24]. In our study, we demonstrate a strong association between HCMV infection grade and overall survival. In the cohort of GBM patients that survived > 18 months, 40% had no or low-grade HCMV-IEA infection as compared with 8% of patients with overall survival ≤ 18 months. Young age (< 50 years) and good neurologic function have been reported to be important prognostic factors in GBM patients. In our study, we observed a significant association between no or low-grade HCMV-IEA infection (p .0006) and RPA subclass III-VI (p < .0001) and survival > 18 months. Further regression analyses of factors influencing survival of patients with low-grade versus high grade-HCMV infection in GBM tissues included, age, extent of resection, gender and RPA subclass. We found an odds ratio of 6.6 for low-grade HCMV infection and 8.2 for RPA subclass III and IV (p .019 and p .002, respectively). However, no association was found between survival and radical surgery, age, gender or gamma knife treatment (Table 2).

HCMV infection was detected exclusively in tumor cells and endothelial cells in the tumor part, but not in the non-tumor part of the tissue, which suggest that HCMV infection is restricted to the tumor cells. HCMV proteins may affect many central mechanisms in tumor biology and confer immune evasion mechanisms. For instance HCMV-IE72 and IE86 proteins interact with p53 and Rb that result in enhanced cellular proliferation [12, 14]. In this study, we found that p53 mutation was associated with HCMV-LA expression (p .026 in patients surviving > 18 months), which implies a potential viral effect on p53. Interestingly, HCMV has been shown in vitro to cause mutations, in particular in p53 in cells that are transformed by IE72, IE86 and adenovirus E1A proteins [16]. These and other HCMV proteins also affect several additional pathways in the cellular machinery linked to tumor biology, such as cell cycle control, enhanced proliferation and migration of the cells, stimulation of telomerase activity, induced expression of COX-2 and 5-lipoxygenase and production of prostaglandin E2, leukotriene B4, and accumulated beta cathenin with potential key functions in HCMV induced oncogenesis or cancer progression [12, 15–17, 25–29]. In collaboration with Smit's group, we recently described an additional potential oncomodulatory role of HCMV US28, which is a viral G protein-coupled receptor encoded by HCMV (18). HCMV-US28 expression in the cells stimulated activation of STAT-3 and secretion of IL-6 and VGEF that led to enhanced proliferation and angiogenesis of HCMV infected cells. Interestingly, HCMV-US28 was found to be expressed in GBM tissue sections and GBM patients that had high grade phosphorylated STAT-3 (pSTAT-3) in their tumors had shorter time to tumor progression and overall survival (18). Interestingly, US28 expressing 3 T3-cells injected into nude mice formed tumors [29]. Smit's and Lira's groups established a transgenic mouse with US28 expressed in the intestine [29]. These animals developed adenomas and adenocarcinomas, further providing evidence that HCMV US28 may be oncogenic [29]. Furthermore, Soroceanu et al have recently demonstrated expression of US28 in 60% of GBM specimens and suggested that the invasive tumorigenic and angiogenic properties of US28 mediated by US28-CCL5 paracrine signaling may contribute to glioma progression [30].

A recent study by Dziurzynski et al showed that HCMV infection in glioblastoma stem cells (gCSCs) results in induction of viral IL-10 that activates HCMV-IE1 in monocytes and affects polarization of macrophages toward a M2 phenotype of macrophages. The authors claim that immunosuppressive M2 macrophages in GBM patients may contribute to gliomagenesis via induction of VEGF and enhanced angiogenesis, and increase immunosuppression by production of TGF-beta [31]. Both HCMV IL-10 and HCMV-US28 stimulate activation of STAT-3 (pSTAT-3) and thereby link them to tumorigenesis [32]. pSTAT-3 down regulates the anti-tumor response, suppresses macrophage activation, reduces NK cell and neutrophil cytotoxicity and impairs dendritic cell function [33].

In this study, we demonstrate that low-grade HCMV infection is strongly associated with long-term survival in glioblastoma patients, and hence the level of HCMV infection may provide a useful prognostic marker for survival of glioblastoma patients. This observation also implies that HCMV may be important in glioblastoma progression, and that the virus may provide a target for cancer therapy. In support of this hypothesis, we have recently demonstrated that the growth of HCMV infected human meduloblastoma xenografts can be inhibited by therapies targeting HCMV. Treatment of animals carrying HCMV positive human medulloblastoma xenografts with the anti-viral drug valganciclovir and the COX-2 inhibitor Celecoxib (that also targets HCMV replication) resulted in a 72% inhibition of tumor growth without using chemotherapy [20].

In summary, our results suggest that low grade HCMV infection in glioblastoma tumors is strongly associated with longer survival of GBM patients. This observation further supports the hypothesis that this virus may play a pathogenetic role in GBM tumors rather than representing an epiphenomenon. Targeting HCMV with anti-viral drugs may affect the viral load in the tumor and thereby prevent progressive disease. In support of this statement, we have recently completed the first clinical phase I/II trial on anti-viral therapy in glioblastoma patients, which indicate improved overall survival in patients receiving long-term treatment of valganciclovir.