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Section 1. Clinical Medicine
Koshelev Stanislav Vitalevich, Oncologist-surgeon, Oncology department № 1, City Clinical Oncology Dispensary Minsk, E-mail: slavakoshelev@yahoo.ca Titov Leonid Petrovich,
the Head of the Laboratory of Clinical and Experimental Microbiology, RSPC for Epidemiology and Microbiology, Minsk, Member of NAN Belarus, MD, Professor, E-mail: leonidtitov@tut.by Goncharov Andrej Evgenevich, MD, the Head of the Laboratory of Immunology and Cell Biotechnology RSPC for Epidemiology and Microbiology, Minsk, Shapoval Evgenija Victorovna, MD, the Head of the Laboratory of Reconstructive Surgery, Belorussian Cancer Center of N. N. Alexandrov, Dubrovsky Aleksandr Cheslavovich, MD, the Head of the Department of Pathomorphology Belorussian Cancer Center of N. N. Alexandrov,
DC-IMMUNOTHERAPY IN COMPLEX TREATMENT OF EARLY HIGH AGGRESSIV BREAST CANCER
Abstract: A method of anti-relapse autoimmunotherapy with antigen-specific dendritic cells was developed for patients with a primary highly aggressive Breast Cancer (BC). It is established that the proliferation of antigen-specific CD3 + T-cell lymphocytes with intracellular cytokine production evaluates the immunological efficacy of the treatment. The 5-year recurrence-free survival of patients with early aggressive cancer in clinical trials of the Method was approaching the overall 5-year survival of patients with early BC by Cancer - Registry, that indicates the effectiveness of the method in the complex treatment.
Keywords: dendritic cells, early biologically agressive Breast Cancer, specific immune response, duration of a non-recurring period.
Breast cancer (BC) is the most common malig- detection and systemic treatment of BC is explained nant neoplasm in women in industrialized countries. by the steady increase in the number of cases in all The development of numerous projects for the early countries, an average of 1, 2% per year [1].
The wide screening of BC and subsequent systemic therapy helped to identify early forms of malignant process, increase the duration and improve the quality of life of patients. According to the Be-larusian Cancer Registry-2017 [2] 73,4% of newly diagnosed cases of BC belong to the I-II clinical stage of the disease, with a 5-year general survival rate of 88,2%. But the optimization of treatment of such patients is a widely discussed problem, as numerous international studies have established that 6-30% of such patients after the completion of primary treatment for a radical program, clinically undiagnosed micrometastases promote the progression of the malignant process [3].
The different disease-free survival rates of patients after systemic treatment of the same stage BC attracted the attention of clinicians to the study of the molecular biological characteristics of malignant cells, which contributed to the new approaches to diagnosis and treatment of tumors [4].
A long-term prospective study conducted in the Department of Clinical Mammology Belorussian Cancer Center of N. N. Alexandrov (1999-2008) revealed a close relationship between disease-free survival (n = 164) after primary comprehensive therapy for early breast cancer T1-2N0-1 G2 and presence of biological aggressiveness of tumors (Plog-rank < 0,0001 - 0,0006). The biological aggressiveness of tumors was determined by the
imbalance of the proliferative-apoptotic potential of malignant cells, characterized by high expression of growth factors against the background of a pronounced blockade of apoptosis. Highly aggressive tumors were recorded in 10% of patients with stage I and in 35% of patients with stage II breast cancer, which implies the introduction of additional adjuvant therapy in this category of patients [5]. The performed study allowed to determine the most vulnerable time intervals of primary disease progression after complex treatment of patients with breast cancer (BC) of early clinical stages. It was established that even at the average degree of histological malignancy of the removed tumors (stratification by criterion G2) more than 40% of the first cases of disease progression after primary radical treatment (over 10 years of observation) were determined during the 2-3 years of the postoperative period [6].
New treatment standards introduced since 2004 have reduced the percentage of early disease returns. But the trend of disease returns towards the third year after systemic treatment is traced in the analysis of the recurrence-free survival in the population group of patients (in the country) treated for BC T1-2N0-1 (with tumors of varying degrees of malignancy G) - 75.4 ± 1.5% for the third year of the postoperative period [7].
Figure 1. The first cases of tumor progressi after primary radical treatment of breast cancer T12N01G2
The modern stage of increasing the effectiveness of complex antitumor therapy is largely associated with discoveries in the field of molecular biology and oncoimmunology, the development of innovative technologies in the diagnosis of malignant process and the treatment of cancer patients. Identification of antigenic differences in malignantly transformed and normal cells is used for the recognition and directed destruction of tumor cells.
The real ways of inducing an immune effect on a particular tumor are determined, one of which is Dendritic-Cellular Immunotherapy (DC-IT), aimed directly at the tumor cells antigens. This treatment involves the creation in the body of a significant population of specific cytotoxic lymphocytes (CTL) against antigens of malignant cells. But the specific activation of CTL occurs only when antigens are presented to lymphocytes on the surface of antigen-presenting cells (APCs) in association with HLA molecules, costimulatory and adhesive molecular structures. This event is the determining factor in the directed activation of T cells and the formation of an effective immune response, including the antitumor effect. Dendritic-Cells are the most strong CTL activators [8; 9].
New biotechnological methods represent the possibility of obtaining human DC in vitro in sufficiently large amounts from the monocyte-mac-rophage fraction of the blood to conduct directed antigen-specific autoimmunotherapy. The primers of DC can be any antigens highly expressed in tumor cells against which vaccination is performed. The key point of directed immunotherapy is the identity of antigens - DC primers - with antigenic determinants of malignant cells.
The principle of antitumor immunotherapy with DC-autovaccines is the cultivation of DC in vitro from autologous monocyte precursors, their priming (under laboratory conditions) with antigens identical to the patient's tumor antigens, and subsequent reinjection to the patient in the mode of vaccine therapy.
The purpose of this study was to determine the efficacy and safety of using autologous Dendritic Cells primed with tumor-associated antigens for anti-relapse immunotherapy of Early High Aggressive Breast Cancer.
Materials and methods: The study includes 2 section:
1) a series of in vitro experiments with blood samples of aggressive breast cancer patients to study the effect of mature unprimed and primed DC on the proliferation of autologous cytotoxic T lymphocytes and the production of intracellular cytokines IL-2, INF-y and TNF (determined by monoclonal antibodies "Becton Dickinson", "Beckman-Coulter", "Sigma", on the cytofluorimeter "FACSCalibur" -USA), characterizing the development of antigen-specific effect. DC cultures for active specific autoimmunotherapy were obtained by original technology (Patent BY12361, LP Titov, AE Goncharov) from the monocyte-macrophage fraction of peripheral blood of patients. Control was carried out on the number of DC, their viability, morphology, im-munophenotype, microbiological purity. Primers of the DC were short-chain peptides (synthesized in the Research Institute of Highly Pure Biological Preparations, St. Petersburg), similar to the loci most susceptible to mutations in the DNA-binding domain of the suppressor protein p 53 (LLGRNSFEV, KLCPVQLWV, YLGSYGFRL, GLAPPQHLIRV), which was vosoko expressed by cells of distant malignant tumors.
To monitor the efficacy and safety of dendritic therapy, the immune status and patients were monitored after the initial complex antitumor therapy of BC before the initiation of immunotherapy with primed DC, during and after immunotherapy. The total number of leukocytes, monocytes; immu-nophenotype of peripheral blood lymphocytes (CD3, CD 19, CD27, CD4, CD8, CD25, CD69, HLA-DR, CD28, CD16, CD56, TCR-ap, TCR-yS, CD95); Intracellular cytokines of peripheral blood lymphocytes (INF-y, TNF, IL-17) after 6 hours of
stimulation with FMA, ionomycin and antigenic complex; antigen-specific T-lymphocytes were detected.
2) Clinical trials of anti-relapse DC-autoimmu-notherapy approved by the Commission under the Ministry of Health of the RB are conducted in 22 patients aged 27 to 58 years after the initial complex treatment for aggressive BC II stage. The selection of patients for anti-relapse autoimmunotherapy was performed among 127 patients in accordance with the results of molecular-biological typing of tumors removed from them. According to the study protocol, tumors should be characterized by high expression of the mutant p53 protein chosen as an antigen target for DC vaccine therapy, overexpression of the Ki-67 proliferation marker, expression of the major histocompatibility complex molecules HLA-A2. DC-immunotherapy was carried out on
an individual schedule and consisted of 5 courses of vaccinations (3 and 2 courses) with a three-month break. For each participant in the clinical trials, an individual temporary treatment plan has been developed that is consistent with the collection of blood and the manufacture of an antitumor vaccine. It was considered that possible cases of tumor progression after radical treatment may occur in 2-3 years of the postoperative period. Taking into account this fact and the duration of development of immunologi-cal effect, the most acceptable time to initiate antirelapse DC-immunotherapy was 4 months after the end of 1 systemic treatment.
Results. DC in all the examined samples on the 7th day of cultivation were morphologically homogeneous, characterized by stellate cellular forms with the presence of cytoplasmic processes typical for D C (Figure 2).
Figure 2. Micrograph of primary cultures
In the culture of MDC, as a rule, there are single lymphocytes (small rounded cells), fibrocytes (ob-
Figure 3. Immunophenotype of DC cultivation, increase of 400
In order to control the immunophenotype of MDC from patients with breast cancer, the expres-
long spindle-shaped cells) that make up no more sion of the following molecules was studied: the tothan 25% of the number of DCs. tal myeloid marker - the molecule of the integrins
family - CD11c, involved in intercellular adhesion;
a highly specific marker of mature DC - CD83 molecule; Costimulatory molecule of CD86. All cultures of DC were characterized by a high density of expression of the studied molecules CD11c, CD86 and CD83, which is confirmed by high rates of their fluorescence intensity (Fig. 3).
The DC culture was considered to have passed immunophenotypic control in the presence of a relative number of DCs expressing the CD11c molecule - more than 90%, CD86 - more than 90% and CD83 - more than 50%. To each culture DC attached analytical passport. In the process of immunotherapy, there was no significant change in the quantitative indices of T-lymphocyte subpopulations, although there is a tendency to decrease the T-lymphocyte count by decreasing the fraction of CD3 + TCRap + cells (p < 0.1). The ratio of CD4 / CD8 cells, which makes it possible to characterize the state of cellular immunity, did not change during the immunotherapy (before immunotherapy - 1.4 (1.0-1.7), after therapy - 1.4 (1.0-1.7), P = 0.897 The B-lymphocyte content increased after immu-
notherapy (p = 0.0003). A significant general group decrease in the content of T-regulatory cells was revealed after immunotherapy (up to - 4.71 (2.91--6.68)%, after - 2.51 (1.89-3.37)%, p = 0.0004), which indicates the lack of development of immunological tolerance.
T-regulatory cells (T-reg.) having the pheno-type CD4 + CD25highCD127- / lowFoxP3 + are the main cells of the immunity system with immu-nosuppressive activity. In the course of the studies, the median values of T-regulatory cells were 0.062 (0.04-0.087) x 106 / ml before immunotherapy and 0.047 (0.037-0.059) x 106 / ml (p = 0.03) after DC-IT [10].
To determine the dynamics of the formation of a specific immune response to DC therapy, a determination by flow cytometry of INF-y-producing CD3 + T cells (antigen-specific cells - ASC) was used after co-cultivation with a complex antigen. The number of ASCs was 0.22 (0.06-0.47)% at the beginning of treatment and 1.11 (0.66-1.58)% after the course of immunotherapy (p = 0.0001, Fig. 4).
A
c
f 0,03
B
ê 0,01
101 102 103 1t FLI-Height
D
0,02
10 10 10
Figure 4. Analysis of antigen-specific CD3 + INF-y
A - a cytogram in the coordinates of CD3 / SSC, FL1 /FL2 coordinates, expression of INF-y under shows the region of CD3 + cells; B - cytogram in the action of FMA (positive control); C - T cells
spontaneously producing INF-y (negative control); D - T cells producing INF-y in response to stimulation with peptides of mutant p 53.
The increase in ASC during immunotherapy was revealed in 80% of cases [11].
The average number of DCs administered to patients as a single injection was 5.3 ± 3.1 x 106. The median amount of DC administered to patients for the entire course of immunotherapy was 27.0 (21.7-32.3) x 106. The amount of DC varied, which is due to different levels of monocytes in the blood of patients.
The assessment of tolerability and safety of the applied immunotherapy was carried out based on the examination of patients and the study of laboratory indicators before the start of therapy, during and after it. A thorough control of the parameters of a general analysis of blood, urine and a biochemical blood test was performed to exclude hepato-, nephro- and hematotoxic effects of autovaccine.
After immunotherapy, 8 out of 22 patients (36.4%) noted low and medium intensity skin itching at the site of DC injection, in 5 patients -mild weakness in the healthy mammary gland a day after the injection, which did not require medical care.
Monitoring of clinical and laboratory status of patients testified to the absence of negative effects of multi-stage DC-immunotherapy. There was no need for additional drug therapy and changing the schedule of vaccinations, which confirms the safety of treatment.
Thus, the analysis of clinical observations of patients in conjunction with the results of a dynamic laboratory examination showed satisfactory toler-ability and safety of using autologous monocyte DC in patients during the initial complex treatment of aggressive BC early clinical stages.
Analyzing the duration of disease-free postoperative period in patients included in the clinical trials of the developed method, it is taken into account that this group is stratified by the criterion of high biolog-
ical aggressiveness of cells of distant tumors, as well as the T2 criterion (tumor size 2.0-5.0 cm), which prognostically has a higher risk of disease progression compared with the retrospective control group with an average degree of histological malignancy (BC T2 N0_j G2).
The duration of the disease free period was monitored for 60 months after initial systemic treatment with the additional application of DC-IT. In this period, two patients recorded a return of the disease 39 and 44 months after the initial treatment, and one patient was diagnosed with BC in the second breast after 41 months after the initial treatment. Analysis of the immunograms in two patients did not detect an increase in ASC, indicating that there was no immune response to DC-IM. This fact indicates that the absence of an immune response in patients after DC-IT (the absence of proliferation of ASC with intracellular cytokine production) can predict the futility of immunotherapy.
At the same time, a comparative analysis of the duration of the disease-free period in the study and control groups showed the effectiveness of the method used[12].
The results indicate a clear trend of prolongation of the disease-free period and, thus, an increase in the overall 3-year and 5-year disease-free survival of patients with active specific DC-IT (3-year disease-free survival in the group with DC-IT - 0.954 +0.003%, in the retrospective group - 0.789 + 0.019%. 5-year disease -free survival (0.840 + 0.021%) in the group with DC-IT [13; 14].
Conclusion. The study allowed to specify the details of the developed method of active antigen-specific DC-autoimmunotherapy for use in clinical practice.
The method is considered as an additional element of an anti-relapse strategy that enhances a specific antitumor immune response in the treatment of patients with biologically aggressive early BC. DC-IT extends the disease-free period in patients at high risk of disease progression.
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