Cytomegaloviruses and malignant brain tumors
Summary. The review analyzes in detail the data on cytomegalovirus (CMV) as the cause of the development of malignant brain tumors. The use of modern methods of immunohistochemistry and polymerase chain reaction makes it possible to detect both individual proteins and CMV genes in tumor tissue, while virus cannot be isolated from tumor tissue using classical virological methods. The paper discusses the theories of “hit-and-run” and “microinfections”, which explain the mechanism of action of CMV. The data on various molecular mechanisms of transformation of normal cells into tumor cells under the action of CMV are presented. The presence of CMV was shown not only in tumor cells, but also in neural stem cells, monocytes and macrophages. The possibility of using immunotherapy with T-lymphocytes and CMV-based dendritic cellular vaccines for the treatment of cancer patients is discussed in detail. Clinical data on their effectiveness are presented. Three possible mechanisms of the action of immunotherapeutic drugs containing CMV antigens are considered.
Submitted: June 25, 2019
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Abbreviations used: CMV — cytomegalovirus; IL — interleukin; NSC — neural stem cells; PCR — polymerase chain reaction.
Today, thanks to the use of new molecular genetic techniques and the improvement of the known immunohistochemical methods, a lot of data on the role of viral infection in oncogenesis and immunotherapy of malignant tumors have appeared. Taking part in a scientific discussion about viruses and cancer, More and Chang (2010)  argued that only 7 viruses are completely oncogenic and cause human tumors, which make up only 10–15% of all tumors known in the world. In principle, oncology, according to these authors, can be divided into infectious and non-infectious. Comparing the development of a viral infection with carcinogenesis, one can find many common characteristics, such as inflammation, innate immunity reactions, immunity suppression, etc. . The role of many other viruses that are detected in tumor tissue, including cytomegalovirus (CMV), in tumor induction or oncostimulation has not yet been definitively proven, despite a large number of publications .
It is known that antibodies to CMV in the blood are detected in 80–85% of the adult population, and the infection itself is asymptomatic in healthy individuals. This virus is detected in tumor tissue in many cancers — in breast, intestinal, prostate, salivary gland cancer as well as in glioblastomas and medulloblastomas [2–9].
The presence of viral antigens in tumor parenchyma can be determined by immunohistological methods, but it is rarely possible to isolate CMV using classical virological methods, which led supporters of the CMV virus oncogenesis theory to propose “hit-and-run” hypothesis, which explained the frequent absence of the active virus in tumors and in the blood . Somewhat later, the term “microinfection” of CMV in a tumor was also proposed to explain the conflicting data on the role of the virus in the induction of tumors [11–13]. It was stated that the reason for the contradictory data lies in the imperfect research methods that were used by various authors. However, even the use of modern immunohistochemistry, gene-molecular methods such as polymerase chain reaction (PCR) did not always allow detection of CMV infection in tumors [14, 15].
Proponents of the CMV theory of neuro-oncogenesis give much evidence in its favor and point out the important role of CMV in the pathogenesis of the neoplastic process. Thus, it has been shown that US-28, a CMV-coded chemokine receptor, can bind chemokines of glial cells, which leads to their activation and stimulation of proliferation, the synthesis of pro-angiogenic factors, in particular, vascular endothelial growth factor. Injection of glial cells to mice expressing US-28 protein causes the development of tumors  and activates signal transducer and activator of transcription 3 — STAT-3 . It was also shown that the conservative CMV proteins encoded by the 23/122 genes responsible for viral replication are able to induce the growth of glioblastomas [18, 19]. It has been shown that CMV infection induces phosphorylation of intracellular kinases, and IE-2 CMV protein binds to histone deacetylase-2, which enhances the transcriptional activity of cells . At the same time, microRNA associated with CMV infection could be involved in the induction of tumors [21, 22].
Finally, CMV plays a role in the development of intratumoral immunosuppression, whether this suppression creates conditions for the development and maintenance of CMV in a tumor, or vice versa, CMV infection induces immunosuppression and thereby promotes tumor growth and suppression of antitumor immunity . CMV-infected blood monocytes begin to synthesize the so-called CMV-dependent interleukin (IL)-10, which binds to the corresponding receptor on the cell and activates the STAT-3 transcription factor [24, 25], which is a key molecule in carcinogenesis and in immune suppression of the tumor [26, 27].
As known, the STAT-3 factor is important for the activation and migration of neural stem cells (NSC) [28, 29]. In addition, CMV-induced IL-10 inhibits the synthesis of pro-inflammatory cytokines  and the proliferation of monocyte precursors . This factor contributes to the conversion of proinflammatory monocytes M-1 into immunosuppressive M-2 monocytes, which suppress the functions of immune cells [32, 33]. It is believed that the presence of M-2 monocytes in the tumor is an unfavorable prognostic sign [33, 34].
According to the latest theories of oncogenesis, it is believed that glioblastomas develop from tumor stem cells [34, 35], which can occur as a result of disruption of normal differentiation of NSC into astrocytes under the influence of mutagenic factors or viruses . Thus, it is known that brain NSCs express a platelet-derived growth factor receptor binding one of the CMV gB proteins, which leads to virus penetration into stem cells and its reproduction in the cell followed by activation of the phosphoinositidine-3 kinase (PI3K) signaling pathway involved in proliferation [36–38].
There are two stages of the interaction of CMV and tumor [3, 39]. At the first stage, CMV binds to the NCS via the platelet growth factor receptor and activates the STAT-3 factor, as stated above, which ensures the migration of these cells and the synthesis of IL-10. IL-10 acts on monocytes turning them into M-2 cells. The second stage of interaction of CMV and tumor is associated with M-2 cells, which accumulate in the tumor focus and indirectly stimulate tumor growth, affecting angiogenesis, the immune response, migration and invasion of tumor cells . It is assumed that the interaction of CMV and host cells can lead to tumor induction or modulation of tumor growth, which can be realized, as noted above, by various mechanisms leading to inhibition or stimulation of one or another intracellular process [3, 39].
Thus, it is possible that the tumor microenvironment, namely, macrophage-monocytic cells and NSC may be the reservoir of the virus in the tumor, although there is an assumption that all or most of the tumor cells contain CMV proteins .
The theory of CMV involvement in the development of malignant brain tumors is not recognized by all researchers. In this regard, the study of 40 glial tumors, 31 meningiomas and 6 neurinomas for the presence of CMV or its antigens in tumors using PCR methods and immunohistochemistry is indicative . In parallel, the authors investigated 76 blood samples to check for the presence of the virus in the blood and the possibility of its transition from blood to a tumor. PCR method could not be able to detect virus DNA both in tumors and in the blood of these patients. Immunohistochemistry also did not reveal the presence of CMV antigens. When studying the level of antibodies to CMV, as well as to Epstein — Barr virus, herpes zoster virus it was not possible to obtain significant differences in the level of antibodies in the general population. These results do not support the hypothesis suggesting an association of herpes viruses and primary brain tumors .
Nevertheless, other studies confirm the presence of CMV proteins and genes in glial tumors, which are detected by immunohistochemical methods or PCR [38–40]. CMV can be present not only in cancer cells, but as noted above, in monocytes, normal and cancer stem cells, the number of tumor cells affected by CMV is very variable and depends largely on both the research method and the type of gene or proteins that were used [38, 40].
The detection of CMV and its antigens in glioblastoma cells served as the basis for the development of new approaches to virotherapy and immunotherapy of CMV-containing tumors. The very first studies were directed to the use of antiviral drugs alone or in combination with other inhibitors of proliferation or inflammation and immunotherapy [41–43]. However, despite the positive results in pilot studies in patients with glioblastomas, it was not possible to obtain a visible positive effect from specific antiviral therapy. A retrospective analysis of 50 patients with glioblastoma multiforme who received valganciclovir for 6 months together with standard treatment showed that the overall survival of patients was 25.0 months compared with 13.5 months in the control group [44, 45].
Nevertheless, on the basis of ideas about the different antigenicity of CMV-infected and non-infected tumor cells, new immunotherapy technologies were developed. It has been established that cytotoxic T-lymphocytes specific for CMV are able to lyse ex vivo CMV-infected glioblastoma cells , which served as the basis for using T-lymphocytes from blood of patients with CMV gliomas for autologous immunotherapy . While T-lymphocytes of patients with gliomas were not sufficiently active, due to immunosuppression, their additional activation with IL-2 and CMV peptides led to an increase in cytotoxicity against tumor cells containing CMV antigens [41, 42]. These works drew the attention of researchers to two positions, namely that the patient’s T-cells can be activated ex vivo with CMV antigens and cytokines and that the presence of CMV antigens in a tumor cell makes it susceptible to the action of lymphocytes and therefore such tumor cells can be target cells for cytotoxic lymphocytes [38, 41]. Unfortunately, the of such T-cell adoptive therapy in the clinic did not give a significant positive result [41, 42], whereas the use of dendritic cells enriched with CMV antigens allowed to induce a sufficiently strong immune response, which led to the accumulation of cytotoxic lymphocytes in the tumor and lymph nodes . The survival rate of patients who received vaccine based on dendritic cells associated with CMV increased to 36.5 months whereas in the control without vaccination was 18.5 months [43, 44].
The use of a dendritic cell vaccine enriched with СMV peptides in combination with T-cell therapy or temozolomide chemotherapy caused both CD-8 superactivation and prolonged survival of patients without tumor progression up to 25.3 months whereas in the control group without vaccination, the survival rate was 16.5 months [45–47]. It has been shown that the use of CMV dendritic cell vaccine against the background of chemotherapy with temozolomide leads to a stable specific immune response [48–50]. Several other variants of CMV dendritic and T-cell vaccines are being tested .
To explain the mechanism of action of different immune vaccines that are associated with CMV proteins and antigens, several theories have been proposed, among which the most popular are the direct, indirect, and cross-antigen-specific theory . The essence of the direct theory is the destruction of CMV-positive cells by immune cells, including tumor stem cells, which contain much CMV. Immune cytotoxic and apoptotic destruction of glioblastoma cells containing CMV leads to a decrease in tumor size and lengthening the patient’s life. The hypothesis of indirect immunotherapeutic action indicates that in addition to the direct effect on tumor cells in the tumor focus, the activity of macrophage and T-killers increases, as well as the synthesis of cytokines, in particular, interferons, which leads to the destruction of tumor cells that do not contain CMV [51, 52]. The cross-reaction hypothesis is that after the death of tumor cells, many tumor antigens are released which cause a second wave of activation of antitumor reactions that kill tumor cells regardless of their CMV content .
Summarizing the above results about the immunotherapeutic efficacy of CMV containing immune vaccines and technologies, it must be said that it is still early to draw definite conclusions. The results indicate only the existence of a large number of approaches to improve the specified method of immunotherapy of tumors. At a minimum, the effectiveness of immunotherapy of tumor cells directed against CMV can be realized by different mechanisms, both through specific anti-CMV immune responses and by cross-immune responses, which can lead to the destruction of both positive and negative CMV tumor cells.
At the same time, the proposal about the possibility of using antiviral CMV immunotherapy in the combined treatment of glioblastomas is not recognized by all researchers, and, according to Ranganathan et al. , such a recommendation is premature, and a few publications on this issue should be considered as preliminary and not convincing yet.
It is also known that CMV can persist in other human malignant tumors, not only in medulloblastomas and glioblastomas. This is probably the universal property of this virus to accompany any malignant processes in the body. In other words, we can talk only about the association of CMV with the malignancies, and tumors containing CMV are recommended to be called “CMV-associated glioblastomas or medulloblastomas” . The practical use of this association for the development of new methods for the treatment of malignant brain tumors is still under intensive study, and this gives us hope for obtaining new immunotherapeutic drugs.
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