Background. Melanoma of the skin is characterized by a rapid progression and early metastasis. It has been shown the disseminated tumor cells, which are often found in the bone marrow, has an important prognostic value. The study of disseminated tumor cells in melanoma might be one of the possible additional sources of information about the nature of the disease and potential application points for drug therapy. Aim. To study the frequency of detection of disseminated tumor cells in the bone marrow in melanoma, depending on the clinical and morphological characteristics of the tumor. Materials and methods. The study included 67 patients with a verified diagnosis of melanoma who were examined and treated at the Blokhin National Medical Research Center of Oncology from 2014 to 2019 years. Male patients accounted for 50.7% (n=34), female patients 49.3% (n=33). The average age of patients: 50.11.6 years. Immunological and morphological examination of the bone marrow were perfomed. Morphological examination was performed by two independent morphologists. Disseminated tumor cells were evaluated by flow cytometry among all nucleated cells (Syto41+) based on the expression of the HMB-45 antigen and the absence of expression of the CD45 panleukocyte antigen (FACS Canto II, USA, Kaluza Analysis v2.1). Statistical data processing was performed using the IBM-SPSS Statistics v.21 Results. Morphologically bone marrow damage was not detected in any case. Disseminated tumor cells (CD45-HMB-45+) in the bone marrow of melanoma patients were detected in 62.7% (n=42) of cases by flow cytometry. The frequency of bone marrow damage in the early stages is not lower than in advanced ones (p=0.029). This is clearly seen in the enlarged analysis. The percentage of DTC detection. At stages I and II was 60.0% (6/10) and 84.6% (11/13), respectively, at stages III and IV 44.4% (8/18) and 65.4% (17/26). In addition, the frequency of detection of disseminated tumor cells in the bone marrow was higher in young patients (p=0.02). There was no correlation between the frequency of bone marrow damage depending on BRAF status. Conclusion. The connection of disseminated tumor cells with the clinical and morphological characteristics of the melanoma has been established. Melanoma is characterized by frequent bone marrow damage, even in the early stages, in young patients.

The possibilities of using artificial intelligence technologies based on computer microscopy in the diagnosis of acute lymphoblastic leukemia and minimal residual disease have been investigated. The diagnosis “acute lymphoblastic leukemia” is made on the basis of a set of diagnostic studies. Among them, microscopy of bone marrow preparations (morphology) is mandatory. Laser flow cytofluorometry is also among the main techniques used to diagnose acute lymphoblastic leukemia. We propose a procedure for microscopy of bone marrow preparations based on the use of artificial intelligence technologies. The composition of the antibody panel for laser flow cyto-fluorometry is determined from the results of bone marrow preparation microscopy. The system for recognizing bone marrow cells in a microscopic image of a bone marrow preparation is based on the description of the texture features of cells and classification using the support vector method. A reference knowledge base required to analyze the capabilities of the bone marrow cell recognition system has been compiled. The knowledge base includes two sets of images of bone marrow cells. The first is used to establish the possibilities of classifying bone marrow cells in the diagnosis of acute lymphoblastic leukemia. The second is used for differential (clarifying) diagnosis of T- and B-cell acute lymphoblastic leukemias. The conducted experiments have confirmed the high efficiency of the computer microscopy method based on the use of artificial intelligence technologies. The proposed approach can be used as a means of supporting medical decision-making in the diagnosis of acute lymphoblastic leukemia and minimal residual disease.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is associated with poor prognosis for all patients except children with t(1;22) or Down syndrome. The frequency of complete remission in case of AMKL is comparable to the frequency of complete remission in other variants of AML, and the median survival is much lower. This determines the necessity to update criteria for assessment of the effect of treatment using flow cytometry definition of the level of minimal residual disease (MRD). Nowadays, there are no unified and standardized approaches for the measurement of MRD in case of myeloid leukemia, including AMKL, which prohibits adequate assessment of the therapy effect and in some cases determination of the indications for allogeneic hematopoietic stem cells transplantation. The article identifies diagnostic features and describes approaches for the measurement of the level of MRD in case of AMKL. Aim. The aim is to demonstrate the algorithms for diagnosing and measuring MRD in case of AML-M7 in children. Materials and methods. The article analyzes the clinical and immunological profile of 10 boys and 4 girls with the initial diagnosis of AMKL between the ages of 3 months 12 years old, 13 of them have received treatment in the FSBI N.N. Blokhin National Medical Research Center of Oncology and one in the GBUZ Morozovsky DGKB between 1995 and 2020, The measurement of MRD was carried out in 6 patients. The measurement of MRD was carried out using both morphocytochemical method and multiparameter flow cytometry with megakaryocyte markers (CD61, CD42, CD41) in combination with other myeloid markers (CD13, CD33), CD34, CD117 and aberrant markers (mainly CD7). Results. We showed that adequate measurement of the level of MRD had required detailed immunophenotyping during diagnosis to determine the aberration of megakaryoblasts. CD9 marker (100%), CD33 myeloid marker (69.2%), stem cell antigen CD34 (46.2%), CD13 (38.2%) in addition to megakaryocyte markers (100%) were most often expressed on blast cells in case of AMKL. The CD117 antigen was present on the blasts in 33.3% of cases. The expression of the T-cell-associated CD7 antigen (46.2%) was frequent. The measurement of MRD was carried out during the treatment (usually after an induction course) on the basis of the markers of megakaryocytic cell line (CD61, CD41, CD42a, CD42b), weak CD45 expression, as well as the immunophenotype characteristics during initial diagnosis. The level of MRD ranged from completely negative (0%; 0.006%) to evident (1.05%). Conclusion. The detection of residual tumor megakaryoblasts in case of AML-M7 using flow cytometry is a promising method to evaluate the effect of therapy. The adequate measurement of the level of MRD requires detailed immunophenotyping during the diagnosis to determine the aberration of megakaryoblasts.

Введение. Детекция диссеминированных опухолевых клеток (ДОК) при солидных опухолях является важной составляющей оценки прогноза заболевания. Поражение костного мозга наблюдается часто. Существуют данные, указывающие на важную роль субпопуляций лимфоцитов костного мозга в процессах гематогенного метастазирования. Цель. Оценить частоту поражения костного мозга у больных немелкоклеточным раком легкого (НМРЛ) на основании обнаружения ДОК методом проточной цитометрии, а также их влияние на популяции лимфоцитов костного мозга. Материалы и методы. Исследовали 62 образца костного мозга больных с верифицированным диагнозом НМРЛ: аденокарцинома (33), плоскоклеточный рак (27), другие виды (2). Методы: морфологический, многоцветная проточная цитометрия. Изучены ДОК, популяции лимфоцитов CD3, CD4, CD8, CD19/CD20, CD16, CD27. Сбор и анализ: FACS Canto II, США, программа Kaluza Analysis v2.1. Результаты. В костном мозге ДОК (EPCAM+CD45-) были обнаружены у 43,5% пациентов c НМРЛ (в качестве порогового значения приняли 1 клетку на 10 млн миелокариоцитов). Наличие ДОК не коррелировало с размером опухоли, статусом лимфоузлов, стадией опухолевого процесса. ДОК чаще наблюдались при более дифференцированных опухолях ( р =0,023). Выявлено достоверное повышение уровня субпопуляций CD16+CD4-NK-клеток ( р =0,002), CD27+CD3+Т-клеток ( р =0,015) при поражении костного мозга. Заключение. Установлена возможность детекции ДОК в костном мозге больных НМРЛ, у 43,5% больных НМРЛ в костном мозге выявлены ДОК, при этом их наличие установлено даже при локализованном опухолевом процессе. Более частое поражение костного мозга наблюдалось при высокодифференцированных опухолях. Выявлена взаимосвязь между ДОК и костномозговыми популяциями лимфоцитов: субпопуляции CD16+CD4-, CD27+CD3+.

Evaluation of minimal residual disease (MRD) on the 15th day of treatment of acute lymphoblastic leukemia from B-linear precursors (B-ALL) in children is of key importance in the prognosis of the disease. When evaluating the MRD, it is necessary to take into account the features of the primary immunophenotype of tumor B-lymphoblasts. To assess the MRD on the 15th day of treatment several immunological approaches have been proposed that have a general concept, but differ in fundamentally important details. The purpose of this work was to analyze the established flow cytometry (FC) protocols of the main research groups (BerlinFrankfurt-Munster Group, St. Jude Hospital, Children’s Oncology Group) and to compare the results evaluated according to those approaches. This study was approved by the Independent Ethical Committee N.N. Blokhin National Medical Cancer Research Center. The study included 131 patients with B-ALL aged 1 to 17 years (median 5.53). Pre-Pre-B immunosubvariant prevailed (92.4%). A morphological (myelogram count) and immunological (MRD assessment) study of the BM was performed in all patients on the 15th day. Comparing the FC protocols of the MRD on the 15th day, it was shown that CD10 was a more reliable criterion for the detection of B-LP in comparison with CD34. The expression of CD45 may serve as an additional criterion for the detection of B-LP. The recalculation of the mononuclear cells is a more stringent criterion for determining the MRD. The scientific novelty is that for the first time on the 15th day, a detailed comparison of flow cytometry data with a cytological picture of the bone marrow was carried out. It was shown for the first time that not all B-LP detected on the basis of CD10+ /CD19+ /CD34+ /CD45low are aberrant according to CD58/CD38.

Introduction.Detection of disseminated tumor cells (DTC) in solid tumors is an important component of the assessment of disease prognosis. Bone marrow damage is common. There is evidence indicating an important role for bone marrow lymphocyte subpopulations in hematogenous metastasis. Aim.To evaluate the frequency of bone marrow damage in patients with non-small cell lung cancer (NSCLC) based on the detection of DTC by flow cytometry, as well as their effect on the population of bone marrow lymphocytes. Materials and methods.62 bone marrow samples of patients with a verified diagnosis of NSCLC: adenocarcinoma (33), squamous cell carcinoma (27), other types (2). Methods: morphological, multicolor flow cytometry. Studied DTC, lymphocyte populations CD3, CD4, CD8, CD19, CD20, CD16, CD27. Collection and analysis: FACS Canto II, USA, Kaluza Analysis v2.1. Results.In bone marrow, DTC (EPCAM+CD45-) were found in 43.5% of patients with NSCLC (1 cell per 10 million myelocariоcytes was taken as the threshold value). The presence of DTC did not correlate with the size of the tumor, the status of the lymph nodes, and the stage of the tumor process. DTC was more often observed in more differentiated tumors (p=0.023). A significant increase in the level of subpopulations of CD16+CD4-NK-cells (p=0.002), CD27+CD3+T-cells (p=0.015) with bone marrow damage was revealed. Conclusion.The possibility of detecting DTC in the bone marrow of patients with NSCLC was established, in 43.5% of patients with NSCLC in the bone marrow DTC was detected, and their presence was established even with a localized tumor process. More frequent bone marrow damage was observed with well-differentiated tumors. The relationship between DTC and bone marrow lymphocyte populations was revealed: subpopulations of CD16+CD4-, CD27+CD3+.

Aim. To increase the accuracy of risk stratification of breast cancer Stage I–III patients based on circulating tumour cell (CTC) determination.Methods. Forty-seven breast cancer patients aged 28–77 years (median, 51 years) who were examined and treated at N.N. Blokhin National Medical Research Center of Oncology between 2015 and 2017 were enrolled in the study. The presence of CTCs was evaluated for all patients before treatment.Results. CTCs were detected in 85.1% (40 out of 47) of the patients. The CTC detection rate in patients with early and locally-advanced breast cancer was approximately the same, 88.5% (23 of 26) and 81% (17 of 21), respectively (p = 0.7). Another important prognostic factor, which tended to be reliably linked to the frequency of CTC detection, was the grade of malignancy. Grade 2 tumours were more often observed in CTC-positive patients (77.5%) than in CTC-negative patients (22.5%, p = 0.06). None of the remaining clinical and morphological parameters were statistically associated with CTC positivity.Conclusion. This study demonstrated that regardless of the clinical stage, CTCs were detected with approximately equal frequency in patients with early and locally-advanced breast cancer (88.5% and 81%, respectively). However, owing to the small number of patients studied, none of the differences were found to be statistically significant. Nevertheless, investigations on the relationship between the presence of CTCs, molecular-biological subtypes and grade of malignancy are important to evaluate their use as independent prognostic factors. Further studies are needed to assess the use of CTCs as an additional criterion or prognostic factor in breast cancer management.Received 17 September 2020. Accepted 25 September 2020.Funding: The study did not have sponsorship.Conflict of interest: Authors declare no conflict of interest.Author contributionsConception and design: S.B. Polikarpova, N.N. Tupitsyn, O.A. Chernyshova, V.E. Ponomarev, E.A. Bogush, I.V. Vysotskaya, V.Yu. KirsanovData collection: V.E. Ponomarev, O.A. ChernyshovaData analysis: V.E. Ponomarev, O.A. ChernyshovaDrafting the article: V.E. Ponomarev, O.A. ChernyshovaCritical revision of the article: S.B. Polikarpova, N.N. Tupitsyn, E.A. Bogush, I.V. Vysotskaya, V.Yu. KirsanovFinal approval of the version to be published: V.E. Ponomarev, O.A. Chernysheva, S.B. Polikarpova, E.A. Bogush, I.V. Vysotskaya, V.Yu. Kirsanov, N.N. Tupitsyn

Minimal residual disease (MRD) as a tool to monitor response to therapy is both a criterion for detailed risk stratification and an independent prognostic factor in childhood acute lymphoblastic leukemia (ALL). Immunological assays particularly flow cytometry (FC) are priority methods in MRD monitoring. Multicolor flow cytometry makes it possible to most fully characterize the immunophenotype of tumor B lymphoblasts and reveal leukemia-associated immunophenotypes not only according to the CD58 and CD38 antigens but also as an additional criterion of aberrancy. This allows you to identify and select individual criteria for further monitoring of minimal residual disease for each patient with ALL. The aim of this chapter is to compare immunophenotyping features of normal B-cell precursors and B-lymphoblasts in acute leukemia and to show possibilities of use of a leukemia-associated immunophenotype in monitoring of the MRD.

The study of methods and models for the analysis of images of bone marrow preparations in computer microscopy systems for the diagnosis of minimal residual disease is carried out. A sample of preparations for patients with acute lymphoblastic leukemia was formed for the study. Bone marrow cell images were obtained for this sample. As a result of experimental studies, the results of classification of bone marrow cells using different classifiers were obtained.

Introduction. Acute myeloid leukemia (AML) is a clonal disease of the blood system that occurs as a result of mutations in the genome of hematopoietic progenitor cells. As a result of mutations, the linear differentiation of hematopoietic cells is replaced by the proliferation of malignant myeloid progenitors. Currently, the risk group for AML in children is determined mainly by the presence of specific gene and chromosomal abnormalities and an increased level of peripheral blood leukocytes. The features of the immunophenotype of blast cells can also influence the course of the disease.The aim of the work is to assess the relationship between the immunophenotypic parameters of blast cells and the probability of achieving remission in children with AML.Materials and methods. The study included 109 patients aged 3 months to 17 years who received treatment according to the AML BFM 87, AML BFM 2004, NII DOG AML 2007 и NII DOG AML 2012 protocol in the period from 1991 to 2020.Results. The study showed the relationship between the probability of achieving remission and the presence of markers CD33, CD19 and CD14 on tumor cells. Expression of lymphoid antigen CD19 on blasts was associated with a higher rate of remission (73.0 % vs 95.5 %, p = 0.027). The absence of the linearly associated myeloid marker CD33 negatively correlated with the remission rate (61.1 % vs 87.7 %, p = 0.007). In the presence of monocyte antigen CD14 on blasts, the probability of achieving remission was low (95 % vs 50 %, p = 0.013).Conclusion. Characteristics of the immunophenotype of tumor cells in AML in children are associated with the probability of achieving remission.

Despite the fact that melanoma is a tumor of visual localization, mortality from skin melanoma remains one of the leading causes of mortality worldwide. Early metastasis of melanoma, even during the initial stages of the tumor process, indicates a high malignant potential of the tumor. Moreover, metastatic melanoma is resistant to current chemotherapy regimens, which is a significant problem today. Progression, resistance to drug therapy of skin melanoma is associated with a population of tumor stem cells, which are characterized by dysregulation of signaling pathways and aberrant phenotypes. The formation of tumor stem cells contributes to their microenvironment. Surface markers expressed by tumor stem cells include transporter proteins that mediate chemoresistance. The study of the microenvironment, the activity of the expressed antigenic determinants makes it possible to understand the processes of chemoresistance formation and to discover (develop) strategies for its overcoming. The article provides an analysis of the literature data on the significance of tumor stem cells in the development of drug resistance of melanoma.

Despite the fact that melanoma is a tumor of visual localization, mortality from skin melanoma remains one of the leading causes of mortality worldwide. Early metastasis of melanoma, even during the initial stages of the tumor process, indicates a high malignant potential of the tumor. Moreover, metastatic melanoma is resistant to current chemotherapy regimens, which is a significant problem today. Progression, resistance to drug therapy of skin melanoma is associated with a population of tumor stem cells, which are characterized by dysregulation of signaling pathways and aberrant phenotypes. The formation of tumor stem cells contributes to their microenvironment. Surface markers expressed by tumor stem cells include transporter proteins that mediate chemoresistance. The study of the microenvironment, the activity of the expressed antigenic determinants makes it possible to understand the processes of chemoresistance formation and to discover (develop) strategies for its overcoming. The article provides an analysis of the literature data on the significance of tumor stem cells in the development of drug resistance of melanoma.

Опухоль из бластных плазмоцитоидных дендритических клеток (ОБПДК) – крайне редкий вариант гемобластоза. Представления о его клеточном происхождении драматически менялись по мере появления новых данных о молекулярно-биологических и иммунологических характеристиках опухоли. В работе рассмотрены клинические особенности ОБПДК, цитологические, морфо-иммунологические и молекулярно-генетические критерии диагностики ОБПДК. С учетом редкой встречаемости ОБПДК, весьма сложной диагностики, требующей постановки расширенной диагностической панели антител, стандартные терапевтические подходы не разработаны. Используются протоколы химиотерапии острого лимфобластного и острого миелоидного лейкоза с/без последующей аутологичной/аллогенной трансплантации костного мозга, но результаты остаются неудовлетворительными. Впервые в отечественной литературе приводится описание ОБПДК у ребенка 14 лет. Представлена детальная клиническая характеристика этой редкой опухоли, результаты дерматоскопии, гистологические, иммунологические и ее молекулярные особенности с дифференциально-диагностических позиций. Родители пациентов дали согласие на использование информации о ребенке в научных исследованиях и публикациях.

Disseminated tumor cells (DTCs) are studied as a prognostic factor in many non-hematopoietic tumors. Melanoma is one of the most aggressive tumors. Forty percent of melanoma patients develop distant metastases at five or more years after curative surgery, and frequent manifestations of melanoma without an identified primary lesion may reflect the tendency of melanoma cells to spread from indolent sites such as bone marrow (BM). The purpose of this work was to evaluate the possibility of detecting melanoma DTCs in BM based on the expression of a cytoplasmatic premelanocytic glycoprotein HMB-45 using flow cytometry, to estimate the influence of DTCs’ persistence in BM on hematopoiesis, to identify the frequency of BM involvement in patients with melanoma, and to analyze DTC subset composition in melanoma. DTCs are found in 57.4% of skin melanoma cases and in as many as 28.6% of stage I cases, which confirms the aggressive course even of localized disease. Significant differences in the groups with the presence of disseminated tumor cells (DTCs+) and the lack thereof (DTC−) are noted for blast cells, the total content of granulocyte cells, and oxyphilic normoblasts of erythroid raw cells.

Immunotherapy, which is seen as a major tool for cancer treatment, requires, in some cases, the presence of several agents to maximize its effects. Adjuvants can enhance the effect of other agents. However, despite their long-time use, only a few adjuvants are licensed today, and their use in cancer treatment is rare. Azoximer bromide, marketed under the trade name Polyoxidonium® (PO), is a copolymer of N-oxidized 1,4-ethylenepiperazine and (N-carboxyethyl)-1,4-ethylene piperazinium bromide. It has been described as an immune adjuvant and immunomodulator that is clinically used with excellent tolerance. PO is used in the treatment and prophylaxis of diseases connected with damage to the immune system, and there is interest in testing it in antitumor therapy. We show here that PO treatment for 1 week induced positive pathological changes in 6 out of 20 patients with breast cancer, including complete response in a triple-negative patient. This correlated with an increased tumor CD4+ T-lymphocyte infiltration. The immune effects of PO are associated with myeloid cell activation, and little is known about the action of PO on lymphocyte lineages, such as natural killer (NK) and T cells. We reveal that PO increases T-cell proliferation in vitro without negative effects on any activation marker. PO does not affect dendritic cell (DC) viability and increases the expansion of immature DC (iDC) and mature DC (mDC) at 100 μg/ml, and it stimulates expression of several DC co-stimulatory molecules, inducing the proliferation of allogeneic T cells. In contrast, PO decreases DC viability when added at day 5 post-expansion. PO is not toxic for NK cells at doses up to 100 μM and does not affect their activation, maturation, and cytotoxicity but tends to increase degranulation. This could be beneficial against target cells that show low sensitivity to NK cells, e.g., solid tumor cells. Finally, we have found great variability in PO response between donors. In summary, our in vitro results show that PO increases the number of costimulatory molecules on DC that prime T cells, favoring the production of effector T cells. This may support the future clinical development of PO in cancer treatment.

Background. Minimal residual disease (MRD) is an independent prognostic factor in acute lymphoblastic leukemia (ALL) in children. The immunological assessment of MRD cell count is based on aberrant immunophenotype of tumor lymphoblasts. However, in the case of ALL originating from T-lineage precursor cells (T-ALL) no clear aberrancy criteria have been defined, yet. Flow-cytometric MRD assessment in T-ALL can be based on characteristics of normal T-cell ontogenesis, i.e. the absence of normal T-lineage precursor cells (T-LP) in bone marrow. Aim. To assess the feasibility of immunological method of flow cytometry for MRD detection based on T-LP immunophenotype on Days 15 and 33 of treatment of T-ALL children. Materials & Methods. The analysis included the data on primary immunophenotype and MRD assessment on Days 15 and 33 of treatment of 31 T-ALL patients in the age of 2-17 years. In the majority of cases (61.3 %) the cortical/ thymic immuno-subvariant of ALL was detected, in the rest of cases (38.7 %) it was the pre-T-cell one. Diagnosis was based on cumulative results of morphocytochemical and immunological bone marrow analyses. Assessing the MRD state the morphological and immunological analyses of bone marrow aspirate were carried out in parallel with one and the same tube. All patients enrolled in the trial were treated at Scientific Research Institute of Pediatric Oncology and Hematology of NN Blokhin National Medical Cancer Research Center according to the ALL IC-BFM 2009 protocol. Results. Our study demonstrated that at all therapy stages MRD can be assessed by the unified immunological method based on detecting cyCD3+CD7+/++smCD3- (T-LP) immunophenotype cells. It is important to ensure that the correct clones of monoclonal antibodies are used for detecting CD3 cytoplasmic and membrane molecules (UCHT1 and SK7, respectively). Standard risk group included no patients. The majority of patients (76.2 %) treated according to ALL IC-BFM 2009 protocol were assigned to medium risk group on Day 15 of treatment. By Day 33 a quarter of them (25 %) was included into high risk group. Conclusion. The capabilities of multicolor flow cytometry allow for the most complete characterization of primary immu-nophenotype of tumor T-cell lymphoblasts for further search of leukemia-associated immunophenotypes. Specific ontogenesis features of normal T-cells enable unification of immunological approaches to MRD assessment at all stages of T-ALL therapy.