Features of immune status in patients with metastatic and glial brain tumors at the preparatory stage of radiotherapy

© Грязов А. А., Лісяний М. І., Грязов А. Б., 2020. РЕЗЮМЕ Актуальність. Дослідження останніх десятиліть показали, що імунні клітини є важливими учасниками онкопроцесу та запалення, пов’язаного з раком. Зусилля були зосереджені на розумінні того, як імунні клітини впливають на результат розвитку пухлини на різних стадіях захворювання: рання неопластична трансформація, клінічно виявлені пухлини, метастатичне поширення та на етапах хірургічного та променевого лікування. Мета роботи – оцінити стан імунної системи у хворих із пухлинами головного мозку перед початком променевої терапії та радіохірургії та порівняти особливості імунітету при метастатичних і гліальних пухлинах головного мозку. Матеріали та методи. У дослідженні представлені результати імунограм 61 пацієнта. З них 18 пацієнтів із первинними гліальними пухлинами та 23 пацієнти зі вторинними метастатичними пухлинами в головний мозок. Як контрольна група представлені результати 20 умовно здорових пацієнтів, які не мали онкозахворювань. Вік пацієнтів склав 24–75 років. Усі пацієнти мають гістологічне підтвердження діагнозу пухлини. Оперативне втручання проводилось за 1,0– 3,0 роки до обстеження. Оцінка стану імунної системи у хворих на пухлини мозку проводилась з урахуванням клітинної, гуморальної та фагоцитарної ланки імунітету. Для оцінки клітинного імунітету визначено відносну та абсолютну кількість основних субпопуляцій лімфоцитів, таких як CD3+ – загальні Т-лімфоцити, CD4+ – Т-лімфоцити-хелпери, CD8+ – цитотоксичні лімфоцити, CD16+ – натуральні кілерні лімфоцити, CD19+-В-лімфоцити. Визначення гуморальних показників включало оцінку кількісних показників IgG, IgM та IgA. Кількісна оцінка фагоцитарної ланки імунітету включала фагоцитарну активність нейтрофілів (а саме: НСТ-тест, індукуюча (зимозан) та спонтанна активність мієлопероксидази нейтрофілів). Особливості імунного статусу у хворих з метастатичними та гліальними пухлинами головного мозку на підготовчому етапі променевого лікування Грязов А. А., ORCID: 0000-0002-2210-1430, e-mail: precisemaningame@gmail.com Лісяний М. І., ORCID: 0000-0002-0498-62047, e-mail: nimun.neuro@gmail.com Грязов А. Б., ORCID: 0000-0003-1785-6705, e-mail: grandoc61@gmail.com Державна установа «Інститут нейрохірургії ім. А. П. Ромоданова Національної академії медичних наук України», Київ, Україна

Purpose -assessing the status of the immune system in patients with brain tumors before radiation therapy and radiosurgery and comparing the features of immunity in metastatic and glial brain tumors. Materials and methods. The study presents the immunogram findings of 61 patients. Out of those: 18 patients with primary glial tumors and 23 patients with secondary metastatic tumors to the brain. The outcomes of 20 conditionally healthy non-cancer patients are presented as a control group. The age of patients is 24-75. All patients were histologically diagnosed with the tumor. Surgery was performed 1.0-3.0 years before the examination. Assessment of the immune system in patients with brain tumors was performed taking into account the cellular, humoral and phagocytic component of innate immunity. When assessing cellular immunity, the relative and absolute count of major lymphocyte subpopulations, such as CD3+general T-lymphocytes, CD4+ -T-lymphocytes-helpers, CD8+ -cytotoxic lymphocytes, CD16+ -natural killer lymphocytes, CD19+-B-lymphocytes, were calculated. Determining the humoral parameters included an assessment of quantitative values of IgG, IgM and IgA. Quantitative assessment of the phagocytic component of innate immunity included phagocytic activity of neutrophils (i. e. NBT test (Nitroblue Tetrazolium test), inducing (Zymosanum) and spontaneous neutrophil myeloperoxidase activity). Results. When comparing the immune parameters of the number of T-and B-subpopulations of lymphocytes in patients with primary malignant brain tumors and secondary metastatic tumors, no statistically significant difference has been detected between these params. Glioblastomas show higher levels of СD4+-and CD8+-lymphocytes in comparison with other tumour groups as well as higher levels of IgG and IgA than in other tumors, while IgM concentration is almost at the same level in three groups of patients. There is a tendency for reducing IgG and IgM level in the blood of patients with metastatic tumors. Both groups of cancer patients under study show inhibition of myeloperoxidase activity of neutrophils in the setting of maintaining the function of NBT cell activity. Conclusions. According to the findings obtained via studying immunological indicators of brain tumors, both metastatic and primary malignant glial ones, there are partial changes in various immune system components such as cellular, humoral and phagocytic activity. However, no statistically significant difference was detected between immune status indicators, that substantiates the need for further study of this issue. At the stage of preparation for radiation therapy, no significant changes in the immune system of the patients with brain tumors, that would make such treatment impossible and be considered as one of contraindications, are observed. Зв'язок роботи з науковими програмами, планами і темами Робота виконана в рамках планової науково-дослідної роботи Державної установи «Інститут нейрохірургії ім. А. П. Ромоданова Національної академії медичних наук України «Розробка методу комплексного лікування метастатичних пухлин головного мозку з використанням передопераційного опромінення».

Connection with scientific programs, plans and topics
Included into the research project plan State Organization «Romodanov Neurosurgery Institute of the National Academy of Medical Sciences of Ukraine» that is «Development of a method of comprehensive treatment of metastatic brain tumors with preirradiation».

INTRODUCTION
Cancer is still considered as one of the principal causes of death worldwide, and, in view of aging population, its annual loss of 8.2 million is only expected to increase [1]. Primary and metastatic tumors are complex ecosystems consisting of neoplastic cells, extracellular matrix (ECM) and «additional» non-tumor cells, which include resident mesenchymal cells, endothelial cells and infiltrated inflammatory immune cells. The cross dialogue between cancer cells and additional cells furthers tumor development and forms it. During tumor formation, the tissue structure changes to a highly specialized microenvironment, characterized by damaged ECM and chronic inflammation [2].
It has been made clear that RT can help tumors become «visible» to the immune system [7][8][9][10][11][12]. After RT, there is a tendency for increasing the pool of peptides antigen presentation, which is reflected by MHC-I molecules [4]. Via this mechanism, adaptive immune responses can facilitate the elimination of cells and tumor metastases that do not express MHC-II. CD4+-T-cells can help to kill tumor cells through several mechanisms. In spite of the central role of CD4+-T-cells in antitumor adaptive immunity, the tumor antigens such as TAA can be presented to dendritic cells by CD8+-T-cells and molecules in MHC-I class; this process can be handled without involvement of the previous CD4+-T-cells. The data listed above indicate the importance of assessing the state of CD-4 and CD-8 cellular component of the immune system at different stages of tumor treatment, especially before RT. At the same time, there are lots of issues regarding the status of the immune system, the count of CD3+-, CD4+-, CD8+-and other immune cells in the blood in tumors of different origin and location, including brain tumors, which should be researched further in order to provide successful treatment [8,13,14].
Purpose -assessing the status of the immune system in patients with brain tumors before radiation therapy and radiosurgery and comparing the features of immunity in metastatic and glial brain tumors.

MATERIALS AND METHODS OF RESEARCH
The study presents the immunogram findings of 61 patients with brain tumor, out of these: 18 patients with primary glial tumors (glioblastomas -9, anaplastic astrocytomas -2, anaplastic oligoastrocytomas -2, anaplastic oligodendrogliomas -2, diffuse astrocytomas -3) and 23 patients with secondary metastatic tumors to the brain with the primary focus being localized in the breast -9 patients, in the lungs -5 patients, in the skin (melanoma) -3 patients, in the intestine -2 patients, in the uterus -1 patient, in the pleura (mesothelioma) -1 patient, anonymous metastasis -1 patient. The outcomes of 20 conditionally healthy non-cancer patients are presented as a control group. The age of patients is 24-75. Surgery was performed 1.0-3.0 years before the examination. All patients were histologically diagnosed with the tumor in accordance with the current histological classifications of tumors. Informed written consent for diagnostic tests was provided to the patients; stored in clinical records. діагностичних досліджень пацієнтами дана і зберігається в історії хвороби. Оцінка стану імунної системи у хворих на пухлини мозку проводилась з урахуванням клітинної, гуморальної та фагоцитарної ланки імунітету.
When assessing cellular immunity, the relative and absolute count of major lymphocyte subpopulations, such as CD3+ -general T-lymphocytes, CD4+-Tlymphocytes-helpers, CD8+ -cytotoxic lymphocytes, CD16+ -natural killer lymphocytes, CD19+-B-lymphocytes, were calculated. The concentration of these subpopulations was evaluated by means of the applicable monoclonal antibodies produced by Becton Dickinsom USA, according to the guidelines and study protocols adapted to assessing these cells in peripheral blood (Pinegin).
The level of serum immunoglobulins of IgM, IgA class was counted using standard sets of monospecific sera produced by «Microgen» of the Ministry of Health of the Russian Federation in agar plates, according to the guidelines and instructions of the manufacturer. Phagocytic activity of leukocytes and neutrophils was assessed by means of NBT test via colorimetric method of Hordiienko S. N. in the modification of Lisianyi M. I. Myeloperoxidase activity of neutrophils was performed by colorimetric method on the activity of intracellular peroxidase in comparison with horseradish peroxidase according to the method, that was adaptive to enzyme immunoassay analyzer-reader (Freemel).
Statistical processing of the obtained outcomes was carried out in accordance with Statistica-8 SW along with counting the mean peripheral (M) square deviation and Student's test. The significance level p < 0.05 was chosen as a criterion for differences in indicators.

RESULTS AND DISCUSSION
Studying the immune status in patients with brain tumors of primary and metastatic origin has made it possible to detect the changes in various components of the immune system. Thus, when comparing the absolute number of individual subpopulations of lymphocytes, the imbalance in composition of CD3+-, CD4+-, CD8+-Tlymphocytes and CD19+-CD3+B-lymphocytes, in comparison with a group of non-cancer patients, in particular, was revealed. An increase in the indicated level of T-lymphocyte subpopulations along with decreased level of CD19+-CD3+B-lymphocytes in peripheral blood were detected. When comparing the immune parameters in patients with primary malignant brain tumors, that is glioblastomas and anaplastic astrocytomas, with secondary metastatic tumors, there were minor differences in the number of T-and B-subpopulations of lymphocytes, namely, it was found that glioblastomas showed the highest CD4+-and CD-8+-levels of lymphocytes compared to other tumor groups, although no statistically significant difference between these indicators was found (ref Table. 1, p < 0.05).
При визначенні показників гуморального імунітету, а саме -рівнів різних типів імуноглобулінів у периферичній крові, установлена залежність від характеру пухлинного процесу. При гліобластомах виявляється більш високий рівень IgG та IgA, ніж при інших пухлинах, тоді як концентрація IgМ практично знаходиться на одному рівні в усіх трьох групах хворих. Спостерігається тенденція до зниження рівня of tumor location, such as brain or breast. Indicators of CD16+-content of natural killer cells are somewhat different. Thus, in metastatic tumors as well as in anaplastic astrocytomas, the level of CD16+-cells in the blood was decreased, while in glioblastomas the number of CD16+lymphocytes was a 1.4-1.5-fold increased compared to anaplastic astrocytomas. The same changes in the immune system params are found not only in assessing the absolute number of certain subpopulations of lymphocytes, but also in the relative (percentage) level of lymphocytes also confirming the changes depending on the nature of the tumor process, i.e. primary or metastatic tumors (ref Table. 2, p < 0.05). At the same time, when comparing the percentage and absolute content of certain subpopulations of lymphocytes, it should be noted that evaluating the absolute content of immune cells in the blood more clearly illustrates the immune system status in comparison with its percentage level, however, to get the whole picture of the peculiarities of disorders occurring in the immune system, both absolute and percentage level of specific subpopulations of lymphocytes in the peripheral blood should be counted.
Проаналізувавши отримані дані імунного статусу у хворих із пухлинами мозку на етапі підготовки до радіотерапевтичного лікування, яке буде направлене на пухлинне вогнище, можна узагальнити їх таким чином. У хворих перед початком Having analyzed the obtained data on the immune status of patients with brain tumors at the stage of preparing for radiotherapy treatment, which will be directed to the tumor nidus, we can summarize them as follows. Thus, patients retain the function of T-cell immunity before treatment. However, a minor decrease in B-lymphocytes level should be noted. There are also changes in the phagocytic activity of neutrophils: on the one hand, inhibition of myeloperoxidase activity in the setting of preserving their NBT activity, which is responsible for phagocytic activity.
2. При визначенні активності неспецифічної фагоцитарної ланки імунної системи було of the patients with brain tumors, that would make such treatment impossible and be considered as one of contraindications, are observed. Moreover, there is a certain activation of the immune system, which is confirmed by increased levels of almost all subpopulations of T-lymphocytes in the peripheral blood. Changes in the humoral and phagocytic component in these patients are insignificant and also they are not the factor limiting this stage of treatment. The reasons, mechanisms of development and maintenance of such imbalance in the composition and activity of immune cells in the body are known [15,16] and they are associated with tumor factors in the first instance, and then with the nature of previous treatment already provided for these patients, especially in case of metastatic tumors.
Another feature of the obtained outcomes is that no statically significant difference was revealed between primary malignant brain tumors and metastatic brain tumors, where the primary focus was more frequently located in the breast. Immune indicators of patients with metastatic tumors were similar to those in patients with anaplastic astrocytomas, which belong to anaplasia degree III. The most malignant glial tumors, that is glioblastomas (stage IV of anaplasia), had somewhat increased level of T-lymphocyte subpopulations, which is difficult to explain, since these tumors are known to have the most significant imbalance in immune cell composition and inhibition of specific antitumor immunity [17].
Probably, further study of this issue and clarifying the features of treatment will make it possible to answer the following: why in glioblastomas, tumors of degree IV of anaplasia, immune levels are higher than in less malignant anaplastic astrocytomas and metastases.
In patients with metastatic and primary brain tumors, certain changes in the cellular, humoral and phagocytic parts of the immune system are recorded suggesting the differently directed effects of the tumor process on the immune system and these changes are specific depending on the histostructure of glial tumors.

CONCLUSIONS
1. The most significant changes in the cellular immunity level are observed in glial tumors of anaplasia degree IV, glioblastomas, which are characterized by an increased number of helper (CD4+) and cytotoxic (CD8+) cells in comparison with other patients with metastatic and astrocytic tumors and the control group data.
2. Changes in the humoral immune system component were characterized by higher levels of IgG and IgA in glioblastomas and a tendency for decreased levels of