Role of HCMV in neurological decline of glioma patients during radiochemotherapy - Detection of HCMV in myelomonocytic cells from the peripheral blood of glioma patients

Document Type
Doctoral Thesis
Granting Institution
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Medizinische Fakultät
Issue Date
Neiswirth, Monika

Background The human cytomegalovirus (HCMV), also called human herpesvirus 5, belongs to the Herpesviridae and can cause the so-called cytomegaly. As with all Herpesviridae, the HCM virus persists in the human organism for a lifetime. This leads to lifelong seropositivity, which can be detected by laboratory chemistry. With increasing age, up to 90 % of the population have had contact with HCMV and are thus carriers of a latent infection. In the event of reactivation, healthy individuals usually do not experience symptoms. However, in immunocompromised individuals such as fetuses, AIDS or transplant patients, severe courses may occur with the risk of HCMV retinitis, which can lead to blindness, or life-threatening HCMV pneumonia. In addition, HCMV can affect the immune response and is associated with higher expression of inflammatory markers. In cancer patients with latent HCMV infection, nucleic acids and proteins of the virus have been detected in tissues of brain metastases from primary breast and colon cancer and in tissues of glioblastoma. Glioblastoma is the most aggressive and most commonly diagnosed primary brain tumor in adults. Over the past decade, the median survival time has been approximately 10 to 15 months, even with therapy. The current standard therapy is to first remove the tumor surgically, if possible. This is followed by radiotherapy in combination with the chemotherapeutic agent temozolomide. During and after radiotherapy of patients with brain tumors, sudden onset of severe neurological deficits was observed, which also led to a worsening of overall survival. Interestingly, these patients simultaneously showed reactivation of HCMV in serological tests. Therefore, immediate treatment with antiviral drugs was given, which improved the condition and also the survival of these patients within a short period of time. These findings suggest a relationship between HCMV infection/reactivation and the resulting encephalopathy. In which compartment this reactivation takes place and what triggers the neurological symptoms is not known. On the one hand, the virus may reactivate in the glioma tissue itself; on the other hand, immune cells may also be involved. HCMV persists mainly in myelomonocytic cells and their differentiation into macrophages or dendritic cells, triggered by inflammatory mediators, can provoke viral reactivation. In addition, irradiation of the brain makes the blood-brain barrier more permeable, allowing immune cells to migrate in greater numbers and also thus further promoting reactivation. This occurs particularly in an immunosuppressive environment generated during glioma therapy. Furthermore, it is important to further investigate whether there are specific risk factors for the development of HCMV-associated encephalopathy. It has been shown that patients who had significantly lower blood basophil counts pretherapeutically, had a higher risk of developing HCMV-associated encephalopathy. In addition, seropositive patients had a higher proportion of CD8+ T cells and a lower proportion of CD4+ T cells, resulting in an inverted CD4:CD8 ratio and reduced resistance to infection. Whether the timing of HCMV infection and the total number of reactivations a person undergoes over a lifetime have an impact on these factors is unclear.

Material and Methods For the detection of even low levels of HCMV, a suitable digital droplet PCR (ddPCR) protocol was established. Various positive controls and primer/probe constellations were tested. Serum and peripheral blood mononuclear cells (PBMCs) from patients with intracranial malignancies were used for analysis. PBMCs were sorted into three major subclasses of monocytes: classical (CD14+ / CD16-), intermediate (CD14+ / CD16+), and nonclassical monocytes (CD14- / CD16+). In addition, CD14- / CD16- cells were defined as a fourth subgroup. The determination of viral load from whole blood was performed by the central laboratories of the University Hospital Erlangen. The ratio of CD4+ and CD8+ T cells in donors with different HCMV status was determined by flow cytometry.

Results A suitable protocol for ddPCR for the detection of HCMV could be established. Despite cell sorting into three major myelomonocytic subclasses in which HCMV mainly persists, detection was not successful in seropositive healthy volunteers. Detection was also unsuccessful in asymptomatic healthy volunteers who showed serological reactivation of the virus. In contrast, analysis of blood samples from glioblastoma patients detected single virus-infected cells. It was shown that HCMV can first be detected in whole blood during the course of therapy and only later in serum.

Conclusion and discussion Because HCMV is present at an extremely low copy number in the latent stage, the probability of detecting an HCMV-infected cell is low. Moreover, in healthy subjects, a rapid immune response may prevent the onset of symptoms. Since the symptoms of encephalopathy appear at the onset of radiotherapy, it can be assumed that reactivation in the cellular compartment, rather than spread of the virus and infection of other cell types, is primarily responsible for the neurological symptoms. However, no monocytic cell series has been identified that is primarily involved in HCMV reactivation. In the future, it will be necessary to further develop the detection method of HCMV in order to also identify latently infected cells in seropositive patients. In patients who developed HCMV-associated encephalopathy during brain irradiation, HCMV could be detected in monocytes. However, no cell population could be defined that seems to play a predominant role in this. Therefore, in further studies with a larger number of patients, additional cell types need to be analysed to further elucidate the genesis of HCMV-associated encephalopathy. In addition, seropositive patients should be clinically examined, and blood samples obtained at closer intervals during therapy to determine the time of highest risk for HCMV-associated encephalopathy and to identify possible promoting influences.

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