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10
Евразийский Союз Ученых (ЕСУ) # 5 (50), 2018
31
STUDY OF MOLECULAR BIOLOGICAL MARKERS IN OVARIAN CANCER _AS FACTORS OF PREDICTION OF DISEASE_
Kamishov Sergey Viktorovich
MD, PhD, senior researcher, chemotherapeutist
Republican Specialized Scientific and Practical Medical Center of Oncology and Radiology of the Ministry
of Health of the Republic of Uzbekistan, Tashkent, Uzbekistan
ИЗУЧЕНИЕ МОЛЕКУЛЯРНО-БИОЛОГИЧЕСКИХ МАРКЕРОВ ПРИ РАКЕ ЯИЧНИКОВ В КАЧЕСТВЕ ФАКТОРОВ ПРОГНОЗА ЗАБОЛЕВАНИЯ
Камышов Сергей Викторович
кандидат медицинских наук, старший научный сотрудник отдела химиотерапии Республиканского специализированного научно-практического медицинского центра онкологии и радиологии Министерства Здравоохранения Республики Узбекистан, Ташкент
АННОТАЦИЯ
Целью данного исследования явилось изучение экспрессии молекулярно-биологических маркеров опухоли у больных яичников в качестве факторов прогноза сопроводительной иммунотерапии на фоне полихимиотерапии. Материалы и методы. Наибольший эффект в увеличении 5-летней выживаемости больных раком яичников оказывала схема сопроводительной иммунотерапии, включающая ЭИФТ с плазмаферезом: такое влияние данного метода проявлялось как при положительных, так и при отрицательных уровнях, рассмотренных онкомаркеров. Результаты. Анализ показал, что изучение корреляции уровня онкомаркеров и 5-летней выживаемости у больных, позволяет сделать вывод о том, что положительный уровень p53, VEGF и Ki-67 у больных раком яичников, наряду с высокой пролиферативной активностью опухоли, служит основанием для проведения сопроводительной иммунотерапии с ЭИФТ, что может существенно увеличить эффективность стандартных схем противоопухолевого лечения. Вывод: При положительном уровне данных онкомаркеров, наряду с высокой пролиферативной активностью опухоли, можно рекомендовать проведение сопроводительной иммунотерапии с ЭИФТ с плазмаферезом.
Ключевые слова: рак яичников, иммунитет, молекулярно-биологические маркеры, иммунотерапия, полихимиотерапия
ANNOTATION
The purpose of this study was to study the expression of molecular-biological tumor markers in ovarian patients as predictors of accompanying immunotherapy against polychemotherapy. Materials and methods. The greatest effect in increasing the 5-year survival of patients with ovarian cancer was provided by a scheme of accompanying immunotherapy, including EIPHT with plasmapheresis: this effect of the method was manifested both at positive and at negative levels of oncomarkers examined. Results. The analysis showed that the study of the correlation of the tumor markers level and 5-year survival in patients allows to conclude that the positive level of p53, VEGF and Ki-67 in patients with ovarian cancer, along with high proliferative activity of the tumor, is the basis for carrying out accompanying immunotherapy with EIPHT, which can significantly increase the effectiveness of standard antitumor treatment regimens. Conclusion: With a positive level of oncomarker data, along with high proliferative activity of the tumor, it is possible to recommend carrying out accompanying immunotherapy with EIPHT with plasmapheresis.
Key words: ovarian cancer, immunity, molecular-biological markers, immunotherapy, polychemotherapy
Topicality. At present time, various genes, proteins and other compounds have been identified, which are considered as additional prognostic factors in patients with malignant tumors, including those with on-cogynecological diseases. They are designated as immunological and molecular-biological markers. At the same time, much attention is paid to the study of molecular-biological markers that characterize apoptosis, cell proliferation and angiogenesis [1, P.1293; 2, P.4385; 7, P.707]. These include p53, B1-2, Bax, Raz1, Kl67, YEvP, He2 / neu. Discoveries in the field of cell biology and experimental oncology have determined the development of a new section of oncology. Many mechanisms for controlling cell division and apoptosis, maintaining genetic stability, ways of signal transmission from receptors to the nucleus, etc. have become known [2, P.1293; 3, P.1419; 7, P.707; 8, P.1;. However, in the specialized literature there are only single
publications devoted to the clinical significance of these markers in patients with ovarian cancer [1, P.1293; 2, P.1293; 9, P.1597]. Immunotherapy with time can be the most promising method of treating tumors, as it is physiologically adequate method, restoring the natural strength of the patient's body to combat the neoplastic process and infectious complications, often arising in the treatment [5, P.357; 7, P.707; 10, P.242]. It should be noted that, unlike the usual methods of immunotherapy, when immunomodulating drugs are taken in the form of tablets, or are administered intramuscularly or intravenously, the use of extracorporeal immunopharmacotherapy (EIPHT) methods makes it possible to selectively select directly from the blood cells of the immune system - leukocytes. This direction of immunotherapy has great prospects in oncological practice in connection with the possibility of re-
moving the consequences of cancer and chemoradia-tion intoxication, and also activate its own system of antitumor protection of the organism [5, P.357; 8, P.1; 9, P.1597]. However, in the literature there is very little information on the use of the EIPHT method in the treatment of cancer. Many methods of immunotherapy in the field of oncology are used empirically, we note that there are no clear criteria for indications and contraindications in the treatment of malignant tumors of various localizations. Determining the optimal doses of drugs, the sequence of various effects on the immune system, their duration, and the influence of immunotherapy methods on the immediate and long-term results of antitumor therapy, require the efforts of many researchers.
Based on the foregoing, the aim of the study was to study the expression of molecular-biological tumor markers in patients with ovarian cancer in the context of accompanying therapy as factors predicting the disease.
Material and methods of investigation. Molecular biological tumor markers were studied in patients with ovarian cancer (OC) T2-3N0-1M0 stages (II-III clinical stages) who were admitted to hospitalization in on-cogynecology and chemotherapy departments in 20092014. Immunohistochemical methods of investigation were carried out on histological preparations of the operational-biopsy material of the primary tumor of patients, obtained before the initiation of therapy. Samples of tumor tissue were fixed in neutral buffered formalin with conventional standard wiring and paraffin waxing. Histological preparations were stained by usual methods and immunohistochemical studies were performed. Paraffin sections were dewaxed and rehy-drated using a standard procedure. To visualize the immunohistochemical reaction used DAB + system [DakoCytomation, Denmark]. The staining results were evaluated using a light microscope "Leica" (Germany) under magnification x10; x20; x40. For the marker, the localization of staining in the cell (nucleus, cytoplasm, membrane) was evaluated. The number of positive cells was evaluated in zoos containing their maximum number. In patients with cervical cancer, monoclonal mouse antibodies to p53 (clone DO-7), Bc1-2 (clone 124), VEGF (clone VG1), Ki-67 (clone M1B-1) (DakoCytomation, Denmark) were used in immunohistochemical evaluation of p53 expression. Immunotherapy in the complex of standard polychemo-therapy was carried out in three groups of patients: group 1 - 28 patients without immunotherapy (control group); Group 2 - 32 patients with OC in combination with extracorporeal immunopharmacotherapy (EIPHT); Group 3 - 34 patients with OC in combination with EIPHT + plasmapheresis (EIPHT + PPh).
The results obtained and their discussion. In
ovarian cancer, the highest rates of 5-year survival were observed in patients who underwent EIPHT with plasmapheresis. Thus, in the control group of patients, the survival rate with negative p53 level was 62.8 ± 4.2%, whereas in the groups with EIPHT and EIPHT + PPh these values were 75.4 ± 3.8 (p> 0.05) and 79 , 4 ± 4.4%
(p> 0.05), respectively. With a positive value of this on-comarker, the 5-year survival was 44.9 ± 4.1% for the control group without immunotherapy and 62.8 ± 4.1 (p <0.05) and 64.8 ± 3.2% (p > 0.05) for groups with the use of immunotherapy, respectively. The p53 protein, being the product of the TP53 tumor suppressor gene, is expressed in all cells of the body. In the absence of damage to the genetic apparatus, the p53 protein is in an inactive state, and when DNA damage occurs, the DNA is activated. The result of activation of p53 is the stopping of the cell cycle and DNA replication; with a strong stress signal - the initiation of apoptosis. Violations of the mechanism of apoptosis development can occur when the key gene of this process p53 loses its function. Increasing the expression of mutated p53 in a tumor is accompanied by its greater aggressiveness, as the number of tumor cells undergoing apoptosis decreases. In OC, according to different researchers, mutant p53 is detected in more than half of patients already at the early stages of the disease. In some works devoted to ovarian cancer, it has been shown that the amplification of HER-2 / neu, occurring in 10-50%, indicates an unfavorable prognosis of the course of the disease. However, there are also conflicting data, so the practical significance of HER-2 / neu testing remains controversial today [3, P.1419;7, P.707; 9, P.1597]. Recently, it was found that some mucinous ovarian carcinomas have amplification of the HER2 / neu gene and overexpression of its protein [1, P.1293; 2, P.4385]. In recently published results of a genomic analysis of 50 samples of light-cell ovarian cancer, the presence of HER2 / neu amplification in 14% of cases was shown [8,16]. It turns out that at a rare frequency of HER2 / neu overexpression and amplification of its gene in the general ovarian carcinoma group in a subgroup of mucinous and clear cell carcinomas, this receptor is much more common. The 5-year survival rate in the control group of patients when the HER2 / neu marker was examined in the control group was 68.7 ± 2.6% with a negative level and 59.2 ± 4.4% with its positive value. Accordingly, in groups with immunotherapy, this indicator was 69.3 ± 5.2 (p <0.05) and 75.3 ± 3.7% (p <0.05) at a negative level and 68.9 ± 4.5 (p> 0.05) and 78.4 ± 2.7% (p> 0.05) - with its positive value. The epidermal growth factor receptor EGFR is a transmembrane glycoprotein localized on chromosome 7p12. EGFR functions via dimerization, activating tyrosine kinase, by participating in the regulation of normal and neoplastic cell proliferation. The EGFR receptor family consists of 4 members: EGFR / ErB1 / HER1, ErbB2 / Neu / HER2, ErB3 / HER3 and ErB4 / HER4. In normal physiological conditions, activation of HER receptors is controlled by temporary low expression of their lig-ands. When cells are transformed, overexpression of these proteins and an increase in the number of receptors on the cell surface are observed. EGFR is present in many normal tissues and expressed expression is observed in solid tumors. According to literature data, expression of EGFR is observed in approximately 40% of malignant tumors of the gastrointestinal tract, lung, ovary, uterus [6, P.8878]. When examining the EGFR marker, it was shown that, with its negative value in the group without immunotherapy, the 5-year survival was
68.4 ± 4.8%, and at a positive - 58.4 ± 6.0%. In the EIPHT group, the corresponding indices were 76.4 ± 4.9 (p> 0.05) and 66.7 ± 4.6% (p> 0.05), and in the group with EIPHT + PPh - 76.8 ± 4.4 (p <0.05) and 69.0 ± 4.8% (p> 0.05).
One of the most studied indicators of tumor growth aggressiveness is cell proliferation, which can be assessed using the mitotic index and the Ki-67 index. The Ki-67 antigen is expressed practically in all phases of the mitotic cycle, and accordingly reflects the proliferative tumor pool. The proliferative index of Ki-67 is considered as an independent prognostic indicator of recurrence, general and disease-free survival, a predictive factor for determining sensitivity to chemotherapy (CT) and radiation therapy (RT). The Ki-67 index allows to assess the degree of malignancy of the tumor and to predict the course of the disease together with other factors. It has been shown that a high level of the Ki-67 index is associated with an unfavorable forecast. In particular, with a high level of Ki-67, there is a deterioration in the indices of disease-free and overall survival in patients with breast, ovarian, colon, bladder, soft tissue sarcomas, etc. In malignant ovarian tumors, in most cases, a high level of Ki- 67. The Ki-67 tumor marker was not detected in tumors of 6 patients who had a 5-year survival rate of 68.4 ± 6.9% in the control group. At its positive level in the group without immunotherapy, this indicator was 41.8 ± 3.8%. In the groups with EIPH and EIPHT + PPh at negative Ki-67, survival rates were 78.6 ± 4.5 (p> 0.05) and 79.8 ± 5.9% (p> 0.05), respectively, and at positive - 58,9 ± 4,3 (p <0,05) and 62,4 ± 3,5% (p <0,05). At a low level of the PA index, which was calculated from Ki-67, the 5-year survival rate in the group without immunotherapy was 67.3 ± 5.7, with a high level of 52.4 ± 2.4%. In groups with immunotherapy, 5-year survival at a low PA index was 78.2 ± 4.8 (p> 0.05) and 78.8 ± 5.0% (p> 0.05), with a high index - 66.2 ± 2.8 (p <0.05) and 69.9 ± 4.7% (p> 0.05). Molecular biological markers p53, VEGF and Ki-67 were present in most patients with OC (83.3, 86.7 and 80.0%, respectively). In this case, the markers HER-2 / neu and EGFR were detected in 20.0 and 30.0% of the patients, respectively. It was shown that the positive level of p53, VEGF and Ki-67 markers had a noticeable negative effect on the 5-year survival of patients with OC, in the case of HER-2 / neu and EGFR, this effect was not so pronounced, indicating a smaller prognostic significance in this disease. The greatest effect in increasing the 5-year survival of patients with cervical cancer, as well as OC, was provided by a scheme of accompanying immunotherapy, including EIPHT with plasmapheresis: this effect of this method was manifested with both positive and negative levels of expression. Analysis of the correlation between the
level of cancer markers and 5-year survival of patients allows us to conclude that the positive level of p53, VEGF and Ki-67 in patients with OC, along with high proliferative activity of the tumor, may serve as a basis for carrying out this category of accompanying immu-notherapy with EIPHT , which can significantly increase the effectiveness of standard antitumor treatment regimens. It should be noted that, with a positive level of oncomarkers, along with high proliferative activity of the tumor, it is possible to recommend carrying out accompanying immunotherapy with EIPHT with plasmapheresis.
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