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Евразийский Союз Ученых (ЕСУ) #5 (50), 2018
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THE CONTRIBUTION TO THE STUDY OF MAIN MOLECULAR _BIOLOGICAL MARKERS IN CERVICAL CANCER_
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
ВКЛАД В ИЗУЧЕНИЕ ОСНОВНЫХ МОЛЕКУЛЯРНО-БИОЛОГИЧЕСКИХ МАРКЕРОВ
ПРИ РАКЕ ШЕЙКИ МАТКИ
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 исследования явилось изучение экспрессии молекулярно-биологических маркеров опухоли у больных раком шейки матки, и их влияние на течение сопроводительной терапии. Материал и методы: Наибольший эффект в увеличении 5-летней выживаемости больных РШМ оказывала схема сопроводительной иммунотерапии, включающая ЭИФТ с плазмаферезом: такое влияние данного метода проявлялось как при положительных, так и при отрицательных уровнях, рассмотренных онкомаркеров. Результаты и обсуждение. Проведённый анализ корреляции уровня онкомаркеров и 5-летней выживаемости больных, позволяет сделать вывод о том, что положительный уровень p53, VEGF и Ki-67 у больных РШМ, наряду с высокой пролиферативной активностью опухоли, может служить основанием для проведения данной категории пациенток сопроводительной иммунотерапии с ЭИФТ, что может существенно увеличить эффективность стандартных схем противоопухолевого лечения. Вывод: При положительном уровне данных окомаркеров, наряду с высокой пролиферативной активностью опухоли, можно рекомендовать проведение сопроводительной иммунотерапии с ЭИФТ с плазмаферезом.
Ключевые слова: рак шейки матки, адаптивный клеточный иммунитет, молекулярно-биологические маркеры, иммунотерапия
ANNOTATION
The aim of the study was to study the expression of molecular-biological tumor markers in patients with cervical cancer, and their effect on the course of accompanying therapy. Material and methods: The greatest effect in increasing the 5 -year survival of patients with cervical cancer was provided by a scheme of accompanying immunotherapy, including EIPHT with plasmapheresis: this effect of the method manifested itself in both positive and negative levels of oncomarkers. Results and discussion. The analysis of the correlation of the tumor markers level and 5-year survival of patients allows us to conclude that the positive level of p53, VEGF and Ki-67 in patients with cervical cancer, along with high proliferative activity of the tumor, may serve as a basis for conducting this category of patients of accompanying immunotherapy with EIPHT, which can significantly increase the effectiveness of standard antitumor treatment regimens. Conclusion: In case of a positive level of these markers, along with high proliferative activity of the tumor, it is possible to recommend carrying out accompanying immu-notherapy with EIPHT with plasmapheresis.
Key words: cervical cancer, adaptive cellular immunity, molecular-biological markers, immunotherapy.
Topicality. 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.3999; 3, P.357; 8, P.33; 10, P.395]. At present, various genes, proteins and other compounds have been
identified, which are considered as additional prognostic factors in patients with malignant tumors, including those with oncogynecological diseases. They are designated as immunological and molecular-biological markers. Much attention is paid to the study of molecular-biological markers that characterize apoptosis, cell proliferation and angiogenesis [1, P.4385; 4, P.1; 5, P.1597; 6, P. 242; 7, 765]. These include p53, B1-2,
Bax, Raz1, Kl67, YEvP, Her2 / neu. The study of the degree of amplification of these genes and the expression of the corresponding receptors and proteins has become one of the priority sections of oncology and for the general population of patients with malignant neoplasms of the ovaries. At the same time, in the specialized literature there are only single publications devoted to the clinical significance of these markers in patients with ovarian cancer [1, P.4385; 2, P.3999; 3, P.357; 9, P.1005]. Using modern methods of immuno-therapy in the treatment of gynecological cancer diseases with the usage of a new opportunities of the molecular-biological markers can give the information about the prognosis of the flow of desease and to promote the appointment of rational and verified treatment which determines the relevance of this work [4, P.1; 6, P.242] . 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 that often arise during treatment. The acquisition of new unique immunomod-ulating drugs has created a qualitatively new basis for correcting immunity disorders, it has become possible to influence more selectively the individual components and links of this system [1, P.4385; 5, P. 1597; 10, P.395]. A promising trend in the treatment of malignant neoplasms at the present stage of development of im-munotherapy is a combination of methods of activation of specific and nonspecific immunity [8, P.33; 10, P.395]. In contrast to the usual methods of immunother-apy, when immunomodulating drugs are taken in the form of tablets, or are administered intramuscularly or intravenously, the use of extracorporeal immunophar-macotherapy (EIPHT) methods makes it possible to selectively select from the blood directly the cells of the immune system - the leukocytes. This direction of im-munotherapy has great prospects in oncological practice in connection with the possibility of removing the consequences of cancer and chemoradiation intoxication, and also activate its own system of antitumor protection of the organism [3, P.357; 9, P.1005]. 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 still used empirically, 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 cervical cancer and ovarian cancer, and their effect on the course of accompanying therapy.
Material and methods of investigation. Molecular biological tumor markers were studied in patients with cervical cancer (CC) T2-3N0-1M0 stages (II-III clinical stages) who were hospitalized in oncogynecology
and chemotherapy departments in 2008-2012. Im-munohistochemical 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. His-tological preparations were stained by usual methods and immunohistochemical studies were performed. Paraffin sections were dewaxed and rehydrated using a standard procedure. To visualize the immunohisto-chemical reaction used DAB + system [DakoCytoma-tion, 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) (DakoCyto-mation, Denmark) were used in immunohistochemical evaluation of p53 expression. Immunotherapy in the complex of standard polychemotherapy was carried out on three groups of patients: the 1st group - 26 patients with cervical cancer without immunotherapy (control group); the 2nd group - 26 patients with cervical cancer in combination with extracorporeal immunopharma-cotherapy (EIPHT); the 3rd group - 28 patients with cervical cancer in combination with EIPHT + plasmapheresis (EIPHT + PPh).
The results obtained and their discussion. The analysis showed that the highest rates of 5-year survival were observed in the group of patients with cervical cancer who had EIPHT with plasmapheresis (PPh). Thus, in the control group of patients, the survival rate with negative p53 level was 62.4 ± 4.8%, whereas in the groups with EIPHT and EIPHT + PPh these parameters were 70.1 ± 4.2 (p> 0.05) and 70 , 2 ± 4.2% (p> 0.05), respectively. With a positive value of this on-comarker, the 5-year survival was 44.2 ± 3.1% for the control group without immunotherapy and 56.2 ± 2.2 (p <0.05) and 56.5 ± 2.4% (p <0.05) for groups using immunotherapy methods, respectively. The p53 gene suppressor encodes a nuclear protein that modulates gene expression, responsible for DNA repair, cell division and apoptosis. To date, there is no consensus in the literature about the dynamics of p53 expression in the progression of cervical cancer. According to several authors, it can both increase and decrease with this disease [6, P.242; 9, P.1005].
Protein Bcl-2 has an important role in the regulation of apoptosis. It is shown that the high degree of expression of the tumor cell Bid (protein from the Bcl family, which plays an important role in the regulation of apoptosis and integrating signals for mitochondria) correlates with the unfavorable cervical prognosis. Bcl-2 can completely delay apoptosis caused by p53 and other stimulants, including cytostatic drugs, but does not stop apoptosis caused by cytotoxic T lymphocytes. In various studies, a sharp and significant increase in the expression of Bcl-2 in localized forms of cervical cancer was shown in comparison with the initial stages
and a subsequent decrease in expression in the locally advanced process. The 5-year survival in the control group of cervical cancer patients when examining the Bcl-2 marker in the control group was 54.2 ± 2.4% at a negative level and 40.9 ± 2.5% at its positive value. Accordingly, in groups with immunotherapy, this indicator was 69.4 ± 3.2% (p <0.05) and 67.6 ± 4.2% (p> 0.05) at a negative level and 52.7 ± 4, 1% (p> 0.05) and 64.2 ± 4.8% (p> 0.05) - with its positive value.
The main activator of angiogenesis is the vascular endothelial growth factor (VEGF), responsible for the proliferation and migration of endothelial cells, and is directly related to tumor invasion and metastasis. The data supporting the participation of VEGF and EGFR in the construction of the vascular bed, the growth and progression of malignant neoplasms have been accumulated. Moreover, the interaction of these ligands with transmembrane tyrosine kinase receptors is considered as the most important autocrine pathway of tumor promotion [14]. With a negative VEGF level in the control group, the 5-year survival rate of the patients was 68.4 ± 4.1% and with a positive -56.0 ± 3.60%. In the groups of patients who used accompanying immu-notherapy, the corresponding values for a negative VEGF were 77.6 ± 4.15 (p> 0.05) and 76.2 ± 5.5% (p> 0.05), with positive the level of this oncomarker was 61.8 ± 4.56 (p <0.05) and 68.8 ± 4.18% (p <0.05). The Ki-67 antigen is a nuclear protein, the expression of which is noted in the active phase of the cell cycle, including mitosis. According to literature data, the expression of Ki-67 increases with cervical lesions. The proliferative index of Ki-67 is considered as an independent prognostic indicator of the occurrence of relapse, general and disease-free survival, a predictive factor for determining sensitivity to chemotherapy (ChT) and radiation therapy. 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 worsening of the disease-free and overall survival rates of patients with breast, ovarian, colon, bladder, soft tissue sarcomas, etc. The Ki-67 tumor marker was not detected in tumors of 7 patients with cervical cancer who had a 5-year survival rate of 65.2 ± 5.8% in the control group. At its positive level in the group without immunother-apy, this indicator was 44.2 ± 6.1%. In the groups with EIPH and EIPHT + PPh at negative Ki-67, the survival rates were 76.4 ± 7.9 (p> 0.05) and 77.8 ± 6.7% (p> 0.05), respectively, and at positive - 57,8 ± 5,9 (p <0,05) and 63,3 ± 7,4% (p <0,05). At a low level of the PA index, which was calculated from Ki-67, the 5-year survival rate in patients with cervical cancer in the group without immunotherapy was 62.6 ± 5.8, with a high level of 44.8 ± 5.1%. In groups with immunotherapy, 5-year survival at a low PA index was 73.7 ± 8.2 (p> 0.05) and 78.1 ± 7.7% (p> 0.05), with a high index
- 54.5 ± 5.4 (p> 0.05) and 62.2 ± 6.4% (p <0.05). When studying the expression of molecular-biological tumor markers in patients with ovarian cancer, it was shown that a negative level of p53 was noted in 5 (16.6%) patients, positive in 25 (83.3%). Corresponding indices in the study of HER-2 / neu were found in 24 (80.0%) and 6 (20.0%) patients, in the study of EGFR - in 21 (70.0%) and 9 (30.0%), VEGF - in 4 (13.3%) and 26 (86.7%) and in studying Ki-67 - in 6 (20.0%) and 24 (80.0%) patients. Thus, in most patients with OC, the level of oncomarkers studied was positive, with the exception of HER-2 / neu and EGFR, which were negative in 80.0 and 70.0% of patients, respectively.
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