RU2802131C1 - Method of assessing minimal residual disease (mrd) by multicolor flow cytometry in patients with acute myeloid leukemia (aml) in the post-induction phase of treatment - Google Patents

Abstract

FIELD: medicine; hematology.

SUBSTANCE: invention can be used to determine the prognosis of relapse in patients with acute myeloid leukemia (AML) based on the results of the assessment of minimal residual disease (MRD) in the post-induction phase of treatment after the 1st checkpoint — in the period from the 35th to the 42nd day after the 1st course of induction chemotherapy and the 2nd checkpoint — in the period from the 70th to the 84th day after the 2nd course of induction chemotherapy. Residual tumor cells are detected by multicolor flow cytometry in patients with acute myeloid leukemia at two control points. At the first stage, all CD34-positive cells are isolated and sequentially analyzed for the expression of all antigens, isolating all CD34-positive cells normally present in healthy donors. At the second stage, all CD117-positive cells are isolated and sequentially analyzed for the expression of all antigens, isolating all CD117-positive cells normally present in healthy donors. At the third stage, all CD117-CD34-cells are isolated and sequentially analyzed for the expression of all antigens, isolating all CD117-CD34 cells normally present in healthy donors. When diagnosing the presence of MRD after the 1st course and/or the 2nd course, a relapse is predicted in patients with AML. Bone marrow sample preparation includes lysis of erythrocytes and staining of leukocytes with a panel of monoclonal antibodies.

EFFECT: method provides the possibility of increasing the efficiency of assessing MRD status in patients with AML, selecting a threshold value for establishing MRD-positive status, identifying the risk of relapse in patients with AML by determining the patient's MRD status by multicolor flow cytometry using gating and staining of leukocytes with a panel of monoclonal antibodies.

4 cl, 6 dwg, 6 tbl, 6 ex

Description

The invention relates to medicine, clinical and laboratory practice in the field of hematology, and in particular to a method for assessing minimal residual disease (MRD) by multicolor flow cytometry in patients with acute myeloid leukemia (AML) in the post-induction phase of treatment at clinically significant points after the first (from 35 to 42 days of therapy) and after the second course (from 70 to 84 days of induction chemotherapy) to determine the possibility of a relapse of the disease.

Acute leukemias are tumor diseases of the hematopoietic tissue associated with clonal proliferation of progenitor cells in the bone marrow (BM). Depending on the cell in which genetic disorders occurred, leading to uncontrolled proliferation of the tumor clone, acute leukemias are divided into lymphoblastic (ALL), that is, represented by cells of the precursors of lymphocytes, and myeloid (AML), in which the tumor mass is represented by cells of the myeloid series.

The introduction of new approaches to the treatment of acute leukemia into practice has led to the fact that most patients can achieve complete remission (CR) at the early stages of treatment. But despite the achievement of CR, at different times after the end of treatment, 30-40% of patients develop a relapse, which may be associated with the persistence of MRD [2]. MRD refers to a population of tumor cells that can be detected by sensitive methods such as polymerase chain reaction (PCR), multicolor flow cytometry (MPC), and next generation sequencing in PD patients. The value of the MOB can be given in relative terms or as a percentage. For example, an MRR of 10 ^-3 (or 0.1%) means that there is one tumor cell per thousand normal cells. The persistence of MRD causes the development of relapses in acute leukemia. Changes in the size of the minimum population of leukemic cells after chemotherapeutic exposure characterize the chemosensitivity of the tumor: rapid and deep clearance of the tumor mass is associated with a more favorable prognosis of the disease. The detection of MRD (MOD-positive status) during the PR period is an independent prognostic factor that is used for the final stratification of patients into risk groups and making decisions on therapy modification [11].

The study of MRD is integrated into various protocols of therapy and is carried out at control points that differ in different protocols. The protocol for the treatment of patients with AML "AML-17" provided for the 1st induction course of the classic program "7 + 3" (daunorubicin 60 mg/m ² 1 time per day on days 1-3 and cytarabine 200 mg/m ² per day as a continuous infusion on days 1-7). As the 2nd induction course, the FLARIDA program was used, compiled on the basis of the FLA-G-Ida program (cytarabine at medium doses (1.5 g/m ² ) 2 hours after the administration of fludarabine (25 mg/m ² in 1 -5th days) and idarubicin (8 mg / m ² , 1, 3rd days). This course was repeated as a consolidation. In the modified protocol "mOML-17" as 2 induction courses, 2 courses of "7+3", and as a consolidation - 2 courses of FLA-G. There are no differences in the effectiveness of induction therapy according to the program "AML-17" and "mOML-17". consolidation, maintenance therapy was performed: depending on the randomization of 6 courses "5 + 5" or permanent 2-year maintenance therapy with 6-mercaptopurine and methotrexate All patients from the groups of intermediate and unfavorable prognosis in the period of the 1st PR were provided with allogeneic hematopoietic stem cell transplantation (allo-HSCT) as a planned stage of program therapy [2].

The researchers were faced with the task of determining whether the study of MRD after the 1st induction course and after the 2nd induction course of chemotherapy provides prognostically significant information, and to determine which of the two points is optimal and appropriate for measuring MRD in adult patients (under 60 years of age) AML.

Each protocol for the treatment of acute leukemia provides for its own MRD threshold value, depending on the control point of the study and the method used (MPC or PCR). These threshold values allow patients to be stratified into risk groups for relapse. MRD detection is integrated into various treatment protocols, with researchers suggesting the use of MRD thresholds set at various breakpoints. However, it is unacceptable to transfer the MRD threshold values obtained using one protocol to another, since different rates of tumor mass clearance will be observed under different regimens of chemotherapeutic exposure. Therefore, the number of aberrant cells taken as a threshold for patient stratification may differ, especially in the early stages of therapy. When developing a new protocol or regimen of chemotherapy (CT), it is necessary to conduct prospective studies and determine cut-off values and breakpoints, and not borrow them from other protocols.

It should be noted that the treatment of patients with acute myeloid leukemia in most regions of the Russian Federation is carried out in accordance with the Russian protocols "AML-10" and "AML-17", which provide for the determination of MRD at control points: after the first/second induction for all patients, after the second consolidation, before maintenance therapy, after completion of the treatment program for patients at intermediate risk, before allo-HSCT. However, not all of these time points provide prognostically significant information, and the optimal points for an expedient and informative determination of MRD were not fixed for domestic protocols, as well as a significant MRD threshold, that is, the level of MRD that would allow dividing patients into two groups that are significantly different from each other.

The following analogues are known from the prior art:

In the recommendations of the European LeukemiaNet (ELN) of 2017, PCR and MPC are recognized as the recommended methods for determining MRD, and the points after induction and after consolidation are chosen as control points for monitoring MRD to establish remission and the kinetics of response to treatment and after consolidation to detect possible morphological recurrence. However, the specific terms for monitoring and their significance are not specified, and it is impossible to apply them to Russian protocols [6]. The 2021 ELN recommendations indicate more precise timing suitable for MRD monitoring using the MCM method [9]. If a specific leukemia-associated immunophenotype is detected at the onset of the disease, then MRD is examined by the MPC method after 2 cycles of therapy and before the end of therapy. But the MRD monitoring plan proposed by ELN in 2021 cannot be extrapolated to all studies and is advisory in nature, therefore, it is necessary to allocate own points for monitoring MRD in AML, including for use in the AML-17 protocol.

In 2018, the National Research Center for Hematology of the Ministry of Health of the Russian Federation published data on the diagnostic and clinical significance of detecting MRD in patients with AML using the Russian protocols AML-10 and AML-17. In this study, the determination of MRD-negative status after the 1st course of induction, but not after the 2nd course of induction, was significantly associated with better overall survival (OS) and disease-free survival (RFS). Therefore, the point after the 1st course of induction was taken as the key point for monitoring MRD to determine the long-term outcomes of patients with AML [1, 3].

The essential difference of the proposed method is that, in contrast to the prototype in the developed method, the key points for monitoring the MRD, when the results will be of prognostic value, are the points after the 1st induction and after the 2nd induction.

During the study, the main task was to develop an alternative accessible method for assessing MRD in AML and to select the most effective monitoring points for MRD to assess the risks of relapse in AML patients receiving treatment according to the AML-17 protocol. Our study showed that not only the MRD after 1 induction is important, but also after the second one, and the study of MRD only after the first course of chemotherapy is not enough.

The present invention is aimed at achieving the following technical result: increasing the efficiency of assessing MRD status in patients with AML, choosing a threshold value for establishing MRD-positive status, identifying the risk of relapse in patients with AML depending on MRD status, which will subsequently lead to the appointment timely and adequate treatment, reducing the number of studies in the observation of patients and, as a result, reducing the financial costs of such studies. The proposed method is relatively accessible and cost-effective, which may allow it to be used in routine practice.

The developed method is implemented as follows:

The determination of MRD in patients with AML is performed by the MPC method using standardized panels of monoclonal antibodies, selected taking into account the recommendations of international and European groups of researchers [5, 11, 13].

Detection of tumor cells is carried out at 2 (two) control points: in the period from 35 to 42 days after the 1st course of induction chemotherapy and in the period from 70 to 84 days after the 2nd course of induction chemotherapy with the restoration of peripheral blood parameters and the achievement of PR according to the myelogram .

The material for the study is a bone marrow aspirate. During the study, BM leukocytes are incubated with monoclonal antibodies labeled with various fluorochrome dyes. Monoclonal antibodies are specific to the so-called clusters of differentiation, or CD (CD is short for "clusters of differentiation") - surface or intracellular molecules - antigens (receptors or ligands) that are used for cell immunophenotyping [7].

The work is carried out on flow cytometers equipped with at least two lasers - devices for a full-fledged multiparametric analysis that allows you to combine from 6 to 13 fluorescent labels with minimal restrictions on the choice of combinations of fluorochromes.

Examples of monoclonal antibody panels used for MRD monitoring are shown in Tables 1 and 2. Table 1 illustrates the use of the 6-color antibody panel, Table 2 shows the use of the 11-color monoclonal antibody panel.

Table 1 - Panels of monoclonal antibodies for monitoring MRD in patients with acute leukemia for a 6-color flow cytometer Laser, nm Blue, 488 Red, 633 Filters, nm 530/30 585/42 670> 780/60 660/20 780/60 fluorochrome FITC PE PerCP/
PerCP-Cy5.5 PE-Cy7 APC APC-Cy7 / APC-H7 Tubes for the determination of MRD in patients with AML (set 1) Tube numbers Antigen (clone) 1 CD7 (8H8.1) or CD56 (NCAM16.2) CD19(SJ25C1) or CD4 (RPA-T4) HLA-DR (L243) CD34 (8G12) CD33 (P67.6) CD45 (2D1) 2 CD65 (VIM8) CD15 (HI98) CD14 (MϕP9) CD34 (8G12) CD33 (P67.6) CD45 (2D1) 3 CD66b (G10F5) CD11b (D12) CD16 (3G8) CD34 (8G12) CD33 (P67.6) CD45 (2D1) 4 CD99 (3B2/TA8) CD13 (L138) CD117 (104D2) CD34 (8G12) CD33 (P67.6) CD45 (2D1) Tubes for the determination of MRD in patients with AML (set 2) Tube numbers Antigen (clone) 1 CD38 (HIT2) CD7 (8H8.1) or CD56 (NCAM16.2)
or CD4 (RPA-T4) HLA-DR (L243) CD117 (104D2) CD34 (8G12) CD45 (2D1) 2 CD38 (HIT2) CD133
(clone 7) CD19 (SJ25C1) CD117 (104D2) CD34 (8G12) CD45 (2D1) 3 CD99 (3B2/TA8) CD33 (WM53) CD13 (WM15) CD117 (104D2) CD34 (8G12) CD45 (2D1) 4 CD65 (88H7) CD33 (WM53) CD13 (WM15) CD56 (NCAM16.2) CD123 (7G3) CD45 (2D1) 5 CD66b (G10F5) CD33 (WM53) CD14 (MϕP9) HLA-DR (L243) CD36 (CB38) CD11b (ICRF44) 6 CD15 (MMA) CD33 (WM53) HLA-DR (L243) CD117
(104D2) CD34 (8G12) CD11b (ICRF44)

Note: FITC - fluorescein isothiocyanate (fluorescein isothiocyanate), PE - phycoerythrin (phycoerythrin), PerCP - peridinin-chlorophyll protein, PerCP-Cy5.5 - peridinin-chlorophyll protein-cyanine 5.5 (peridinin chlorophyll protein complex - cyanine 5.5), PE- Cy7 - phycoerythrin-cyanine 7 (phycoerythrin-cyanine 7), APC - alophycocyanine (allophycocyanine), APC-Cy7 - alophycocyanine-cyanine 7 (allophycocyanine-cyanine 7), nm - nanometers

For the isolation of MRD in AML, gating is used, that is, the separation of populations of interest from other cells using special software for further analysis, in which already analyzed cells are sequentially studied and excluded.

In the first step, all CD34-positive cells are isolated and compared by immunophenotype analysis with those found in healthy donors. When implementing the proposed invention, the detection of MRD is carried out using the analysis of the nature of cell clustering and the surface expression of a number of markers (CD45, CD33, CD34, HLA-DR, CD123, CD38), as the expression correlates with normal analogs of CD34-positive cells. Then the same actions are carried out with CD117- and CD33-positive populations (an example of gating is shown in Figure 1) [4, 8,10, 12]. Monitoring MRD only in the analysis of CD34-positive cells does not provide reliable and complete results, so it is imperative to consider all subpopulations of cells.

By sequentially decomposing all cells of the sample into different subpopulations present in healthy donors, it becomes possible to detect a cell population that has a different immunophenotype, i.e. such an immunophenotype and such a combination of antigens on the surface, which are not found in healthy donors. If such cells are detected, they are quantified - the proportion (%) of all cells in the sample is calculated and a conclusion is made about the presence / absence of MRD.

If MRD >0, then a conclusion is made about the MRD-positive status of the patient, if MRD = 0, then a conclusion is made about the MRD-negative status of the patient, indicating the sensitivity of the method.

The essential features of the developed method for determining MRD in AML in the post-induction phase of treatment are:

- detection of MRD is also carried out after the first induction course of chemotherapy in the period from 35 to 42 days after the 1st course of induction chemotherapy (1st checkpoint) and in the period from 70 to 84 days after the 2nd course of induction chemotherapy (2- i checkpoint);

- determination of the prognosis of the probability of recurrence in patients with AML is performed on the basis of the results of the MRD assessment: after the 1st control point and the 2nd control point;

- detection of tumor cells after the 1st and after the 2nd induction courses of chemotherapy by the MPC method occurs using a uniform structured logical cell gating;

- detection of tumor cells is carried out by multicolor flow cytometry.

The present invention provides an alternative clinically and prognostically meaningful method for measuring minimal residual disease in patients with acute myeloid leukemia by multicolor flow cytometry.

The technical result of the invention is its simplicity, accuracy, speed, and versatility, which allow a laboratory equipped with a flow cytometer to determine the minimum residual disease.

An additional technical result is that this method is available for research in routine laboratory practice, allows you to compare the results of different research groups and, in the future, apply it in clinical trials.

The implementation of the claimed invention is aimed at further prescribing timely and adequate treatment, reducing costs and the number of studies in the observation of patients.

The present invention is confirmed by the study, the description of which is given below.

The study included 73 patients who were treated at the National Medical Research Center for Hematology from February 2017 to January 2021. Inclusion criteria for the study were: newly diagnosed AML, age under 60 years, absence of acute promyelocytic leukemia and changes characteristic of myelodysplasia. From February 2017 to December 2018, therapy was performed according to the AML-17 protocol (n=37), where the 1st induction course was carried out according to the 7 + 3 program, and in the 2nd course and in consolidation, FLARIDA program based on FLA-G-Ida. Since January 2019, a modified mAML-17 protocol (n = 36) was used, where the first 2 courses were carried out according to the 7+3 program, and the consolidation included 2 courses of FLA-G. Since there are no differences in the effectiveness of induction therapy under the AML-17 and mAML-17 programs, for analysis, the groups were combined into one - AML-17. At the diagnostic stage, standard cytogenetic and FISH studies, molecular genetic tests (mutations of the FLT3 , NPM-1 and CEBPα genes (ССААЕ/enhancer-binding protein α)) were performed and risk groups were formed according to the ELN-2017 classification (Table 3). The determination of MRD was performed on days 35-42 (after the 1st phase of induction) and 70-84 days (after the 2nd phase of induction) from the start of therapy, provided that the peripheral blood parameters were restored when PR was achieved according to the results of the myelogram.

Table 3 - Clinical and laboratory characteristics of patients with AML Parameter Values Number of patients, n 73 Median age (range), years 37 (17-58) Male/female ratio, n 28/45 Leukocytosis ≥100×10 ⁹ /l, n 17 Median LDH (spread), U/l 1029 (205-21224) CNS involvement, n 12 Cytogenetic risk group:
Favorable, n
Intermediate, n
Unfavorable, n 15
53
5 Risk group according to ELN-2017: Favorable, n:
t(8;21),n
inv(16) or t(16/16), n
CEBPα mutations, n
NPM1 mutations, n
NPM1 mutations with low allelic load FLT3-ITD, n 34
8
7
3
18
5 Intermediate, n:
Normal karyotype without mutations, n
Low allelic load FLT3-ITD, n
NPM1 mutations with high allelic load FLT3-ITD, n
Trisomy 8 chromosomes, n 26
13
3
2
3 t(9;11),n 1 Unfavorable, n:
Translocation involving the MLL gene locus (11q23), n
High allelic load FLT3-ITD, n
t(6;9),n 13
2
8
1

In the group with a favorable prognosis, 6 patients had an MRD-positive status after the 1st course, 2 of these patients achieved an MRD-negative status after the 2nd course, in one patient the MRD-negative status, which was established after the 1st course, changed on MRD-positive after the 2nd course. In the group of poor prognosis in 2 patients, MRD-negative status after the 1st course changed to MRD-positive status after the 2nd course of therapy, and 3 patients with MRD-positive status after the 1st course MRD after the 2nd course was not detected . MRD status was assessed after the 1st course of induction in 57 out of 58 patients with AML in PR after the 1st course, and in 38 patients (67%) MRD-negative status was confirmed. After the 2nd course, MRD status was assessed in 59 out of 65 patients in PR, while MRD-negative status was determined in 44 (75%) patients with AML.

The change in the number of residual tumor cells, determined after the 1st and 2nd courses of induction, is shown in Figure 2. The MRD values after the 1st course of induction ranged from 0.009% to 8% (median - 0.43%), and after the 2nd course - from 0.006% to 4% (median - 0.18%). Data on the effectiveness of therapy and complete remission depending on the prognosis according to the ELN-2017 classification are described in Table 4.

Table 4 - Efficacy of therapy and events during remission in patients with AML, depending on the prognosis according to the ELN-2017 classification, who were treated according to the protocol of the AML-2017 protocol Parameter Forecast according to ELN 2017 classification Total favorable intermediate adverse Number of patients, n 34 26 13 73 Achievement of PR, n (%) 33 (97.1%) 23 (88.5%) 9 (69.2%) 65 (89.1%) Achievement of PR after the 1st course of induction, n (%) 31 (91.2%) 18 (69.2%) 9 (69.2%) 58 (79.5%) Achievement of PR after the 2nd course of induction, n (%) 2 (5.9%) 5 (19.2%) 0 7 (9.6%) Refractory, n (%) 0 3 (11.5%) 3 (23.1%) 6 (8.2%) early lethality,
n(%) 1 (2.9%) 0 1 (7.7%) 2 (2.7%) MRD-negative status after the 1st course of induction, n (%) 24 (80%) out of 30 10 (55.6%) of 18 4 (44.9%) out of 9 38 (66.7%) out of 57 MRD-negative status after the 2nd course of induction, n (%) 22 (81.5%) out of 27 17 (73.9%) of 23 5 (55.6%) out of 9 44 (74.6%) out of 59 Allo-HSCT in the 1st PR, n (%) 11 (33.3%) of 33 12 (52.2%) of 23 6 (66.7%) out of 9 29 (44.6%) of 65

A univariate analysis of OS, time to recurrence (RTD) and DFS in AML patients was performed depending on the following clinical and laboratory parameters: gender, age, molecular genetic risk according to ELN-2017, lactate dehydrogenase (LDH) activity, MRD status after 1 -th and 2nd induction courses (Table 5). OS significantly depends on gender and the presence of MRD after the 1st course and at the time of PR. The presence of MRD either after the 1st or 2nd course of induction, or in the PR) significantly affected the VRR and DFS. To evaluate the results, a point of 20 months from the start of therapy was taken, since at this point 50% of the patients included in the study remained under observation and survival estimates were quite reliable.

Table 5 - Indicators of OS, RRR, RFS of the presence of MRD (results of one-way analysis) Parameter OS for 20 months BRR for 20 months. BRV for 20 months. MRD after course 1 (not detected vs detected) 95.8% vs 51.8%
(p=0.0007) 16.0% vs 45.3%
(p=0.0008) 84.0% vs 47.4%
(p=0.0001) MRD after course 2 (not detected vs detected) 85.2% vs 58.3%
(p=0.09) 14.1% vs 55.0%
(p=0.002) 83.9% vs 40.0%
(p=0.001) MRD at PR (detected vs not detected) 53.1% vs 94.4%
(p < 0.0001) 48.7% vs 17.1%
(p=0.0004) 45.5% vs 83.0%
(p < 0.0001)

As a result of the multivariate analysis with stepwise selection, the only significant sign influencing OS and DFS was the MRD status at the time of PR. The analysis included the following factors as initial factors: MRD status at the time of remission, gender, age over 40 years, the presence of neuroleukemia, the proportion of blast cells at the onset of the disease (more than 60%), LDH more than 800 U/ml, leukocytosis more than 100×10 ⁹ / l, ELN-2017 risk, cytogenetic risk, presence of FLT3-ITD mutation and FLT3-ITD allelic load over 0.5. Achieving MRD-negativity in PR significantly (10-12 times) reduces the risk of death and relapse (for OS - relative risk 0.09; p = 0.001), for DFS - 0.16; p = 0.0002).

Next, a study was conducted of factors affecting the long-term prognosis of the disease. According to the literature data, it is known that the most informative prognostic factor is the ELN-2017 risk group, therefore, based on the Cox regression model, the combined effect of key factors on DFS was studied: the ELN-2017 risk group, the time to reach MRD-negativity (after the 1st course). The graph showing the hypothetical (model) estimates of DFS depending on combinations of the values of these factors shows that the achievement of MRD-negativity actually levels out the value of the ELN-2017 risk factor at the start of therapy (high risk of MRD-positive status, RR = 5, 5 (CI 2-14.7), p=0.0007) (Figure 3).

Thus, it is concluded that the detection of MRD, i.e. positive MRD status after induction courses is associated with worse OS, DFS, VRR in patients and increases the risk of relapses, and MRD-negative status after the 1st course of induction eliminates the value of the ELN-17 risk factor at the start of therapy.

As a first step in assessing the predictive value of MRD status at the 1st and 2nd breakpoint to predict the risks of adverse events after the 2nd breakpoint, we compared OS and DFS scores in 4 groups.

The combination of MRD statuses at two control points defines the following four groups of patients with AML:

1) patients in whom the MRD-negative status was confirmed both after the 1st and after the 2nd induction courses (MRD1,2 = 0 group), n = 30;

2) patients with MRD-positive status after both the 1st and 2nd courses (group "MOD1.2 > 0"), n = 11;

3) patients in whom MRD was detected after the 1st course, but was not detected after the 2nd course (MRD1 > 0 group), n = 8;

4) patients in whom MRD was detected after the 2nd course, but was not detected after the 1st course (group "MOD2 > 0"), n = 4.

In this analysis, the main basic hypothesis was tested: for events after the 2nd checkpoint, does the previous MRD status have residual information content, i.e. status at the previous point, or only the current status reached by the 2nd point is important.

Estimates of OS, DFS and VRR of these groups of patients are shown in Figure 4, the reference point of time intervals (landmark) - at the time of recovery of peripheral blood after the 2nd course. To assess survival and recurrence, a point of 30 months was taken, since 2/3 of the AML patients included in the study were still under observation, and survival estimates already differed significantly.

The most favorable prognosis was observed in patients with AML, in whom MRD was not determined either after the 1st or after the 2nd induction courses. OS by 30 months of this group of patients was 100%, DFS - 91%, and VRR - 9%. When MRD was detected, the clinical outcome characteristics were significantly worse regardless of whether MRD was detected after the 1st course, after the 2nd course, or after both courses of induction (Figure 5). So, RRR for 30 months. after the start of the 2nd course in patients from the group "MOD1,2 > 0" was 58%, in the group "MOD1 > 0" HRR was 43%, in the group "MOD2 > 0" - 50%. OS in patients of the "MOD1,2 > 0" group was 45%, in "MOD1 > 0" OS was 47%, in "MOD2 > 0" - 67%. So, BRV for 30 months. after the start of the 2nd course in patients from the group "MOD1,2 > 0" was 36%, from "MOD1 > 0" MRR was 50%, and "MOD2 > 0" - 50%.

Based on the analysis of the scores shown in Figure 5, it can be concluded that the early achievement and maintenance of MRD-negativity significantly improves the prognosis. Not achieving or late achieving MRD-negativity does not differ much in predicting the risk of adverse events.

The study of the comparative prognostic significance of the 1st and 2nd MRD measurements was continued on a two-factor Cox model. Additionally, it also studied the question of the possibility of clarifying the threshold for the conclusion about MRD-positivity. Table 6 shows the results of a two-factor Cox regression model with the inclusion of measurements of MRD values after the 1st and 2nd courses in patients with AML. It was shown that MRD-positive status at the first checkpoint increases the risk of an adverse event by 1.68 times, and MRD-positive status after the 2nd course of induction increases the risk of an adverse event by 1.63 times.

Table 6 - Results of the two-factor Cox regression model with the inclusion of measurements of the MRD level after the 1st and 2nd courses in patients with AML Factor Relative risk* p 1 logarithm of MOB after 1st course 1.68 0.013 2 logarithm of MOB after the 2nd course 1.63 0.014 *Relative risk shows how many times the likelihood of an adverse event increases, subject to the presence of this factor

As can be seen from Table 6, the relative risk, showing the weight of the factors included in the prognostic model for MRD after the 1st induction and MRD after the 2nd induction, is approximately the same. This means that although the probabilities of adverse outcomes after the 2nd checkpoint are predicted, the information about MRD at the first point is just as important. This confirms the results of the analysis based on Kaplan-Meier survival estimates in 4 groups according to MRD status. Using the Kaplan-Meier estimates and the Cox model, in our case, we solved one problem of two-factor analysis, during which we checked and evaluated the contribution to the prognostic model of measuring MRD at the 1st and 2nd points of therapy at the induction stage. And the hypothesis that the measurement of MRD at the 2nd point levels out the prognostic value of the measurement at the 1st point was not confirmed.

To determine the threshold values of MRD in AML, special methods of ROC analysis were also used - "ROC-survival", which is an output from the Cox analysis.

In our case, for the "ROC-survival" as a starting point for calculating the time interval to an adverse event (relapse or death), the date of restoration of peripheral blood parameters after the 2nd course was taken, and the control time point for constructing ROC dependencies after 18 months after the 2nd course. The factors included in the ROC-logistics model are the logarithms of the MRD values after the 1st and 2nd courses. The results of the analysis are presented in Figure 6.

In Figure 6a, up to 0.1% (log(MOB%) = -1.0), sensitivity and specificity change little, but with an increase in the threshold above 0.1% (log(MB%) = -1.0), sensitivity starts to drop quite sharply. In Figure 6b, up to the level of 0.03% (lg(MOB%) = -1.5), the sensitivity and specificity change little, but with an increase in the threshold above 0.03%, the sensitivity begins to fall quite sharply. This indicates that the optimal MRD threshold is in the range of 0% to 0.1% or even 0 to 0.03% for the second MRD measurement. After the 1st course of MRD induction, less than 0.1% was determined only in three patients, and after the 2nd course - in 4 patients. Therefore, precise determination of the optimal MRD cut-off value that will be clinically relevant for a confident conclusion of MRD-positivity requires continued data collection. But we can confidently say that it should not be higher than 0.1%.

The essence of the invention has also been verified by specific clinical cases.

Clinical case No. 1. Patient born in 1991 In April 2019, acute myeloid leukemia was diagnosed. The standard cytogenetic study revealed no chromosomal aberrations. Molecular genetic testing did not reveal any mutations. The patient was assigned to the group of intermediate genetic risk according to ELN. An immunophenotypic study after the 1st induction (on the 35th day) revealed 0.51% MRD. After the 2nd course (on the 70th day), the MRD was 0.1%. At the same time, as a result of the end of induction therapy, bone marrow remission was stated. After 4 more courses of chemotherapy, the patient was recommended to undergo maintenance therapy. In the future, in case of successful selection of an unrelated donor, it was planned to perform allo-HSCT. However, in September 2019, a 40% blastosis was detected in the myelogram, the patient was again hospitalized, and an early relapse of AML with a resistant course was noted in the department of the National Research Center for Hematology. Despite the measures taken, on 02.2020, the patient's death was ascertained from infectious complications refractory to ongoing antibacterial and antifungal therapy against the background of progression of therapy-resistant acute myeloid leukemia. This clinical case confirms that MRD detected after the first or second course of induction is an unfavorable prognosis factor and is associated with the development of a relapse of the disease.

Clinical case No. 2 Patient born in 1998 In October 2018, acute myelomonoblastic leukemia was diagnosed. Molecular genetic testing revealed the FLT3/ITD mutation. No chromosomal abnormalities were found. The ELN risk is set to high. After the first and second inductions according to the AML-17 protocol, the patient remained in morphological remission, but MRD persisted (0.3% after 1 induction on day 38 and 0.18% after 2nd induction on day 78) . Given the unfavorable prognosis of the underlying disease, young age, and the presence of a related HLA-identical donor, it was decided to include allo-HSCT in the treatment regimen, which was performed in March 2019. cells in the amount of 46%. In the myelogram, blast cells accounted for 83.6%, in the molecular genetic study, the admixture of the recipient's DNA in the sample was 93%, and cells with the FLT3/ITD mutation were detected (the frequency of the mutant allele was 33%), an early clinical and hematological relapse was diagnosed. This clinical case confirms that MRD detected after the first or second course of induction is a factor of poor prognosis and is associated with the development of disease recurrence.

Clinical Case #3

Patient born in 1990 In June 2018, he was diagnosed with acute myelomonoblastic leukemia. Molecular genetic testing revealed the FLT3/ITD mutation. No chromosomal abnormalities were found. Intermediate ELN risk has been established. The patient underwent the 1st induction course according to the AML-17 protocol, after which he was stated to be in clinical and hematological remission, however, on the 36th day of therapy, MRD remained in the study using the MPC method (0.365%). After the 2nd induction course (on the 70th day) of the AML-17 protocol, clinical and morphological remission was maintained, MRD status was negative. Taking into account the unfavorable prognosis for the underlying disease, young age and intact somatic status, as well as the absence of HLA-identical related donors, it was decided to perform allo-HSCT from an unrelated HLA-identical donor. Two months after transplantation, the proportion of blast cells in the myelogram increased (8.8%), the FLT3/ITD mutation was detected in the molecular genetic study, and the MRD by the MPC method was 11%. In a molecular genetic study of hematopoiesis chimerism after unrelated transplantation, the admixture of own hematopoiesis was 12%. A week later, 60% of blast cells were found in the myelogram, and the admixture of own hematopoiesis in the BM sample was 54%. Thus, this clinical example confirms that MRD detected after the first or second course of induction is a factor of poor prognosis and is associated with the development of early relapse of acute AML.

Clinical case #4

Patient born in 1984 AML was diagnosed in August 2016. Cytogenetic examination revealed t(8.21), and molecular genetic examination revealed FLT3/ITD and CEBPA mutations. Thus, the patient is assigned to a favorable ELN risk group. From August to September, two courses of induction "7 + 3" were held. After the first course, the morphological and molecular remission of the disease was stated, the MRD status after the first and second induction was negative. In November 2016, the first course of consolidation was conducted under the 7+3 program with idarubicin. In December 2016, the second course of consolidation was conducted under the 7+3 program with mitoxantrone. Considering the unfavorable prognosis of the disease and the absence of an HLA-identical sibling, it was decided to include hematopoietic stem cell transplantation from an unrelated donor in the treatment regimen, which was performed in February 2017. On day +16 after allo-HSCT, the hemogram showed restoration of peripheral blood parameters.

On day +25 after allo-HSCT, according to the morphological study, the disease remains in remission. In June 2017, the patient was diagnosed with aGVHD and specific treatment was started. Despite the development of aGVHD, the patient remains in complete clinical, morphological and molecular remission of AML until 2022. Thus, this clinical example confirms that the MRD-negative status after the first or second course of induction is a favorable prognosis factor and indicates a low probability of recurrence of the underlying disease.

Clinical Case #5

Patient born in 1961 The debut of the disease was diagnosed in March 2016. No cytogenetic abnormalities were found in the patient. There were also no mutations in the molecular genetic study. Thus, the patient is classified as an intermediate risk group according to ELN. From April to May 2016, the first induction course of chemotherapy "7 + 3" with daunorubicin was carried out. After the first course of induction (on the 37th day of therapy), morphological and molecular remission of the disease with MRD-negative status was noted. Taking into account the long-term cytopenia and infectious complications after a course of chemotherapy, a decision was made to conduct a course of chemotherapy with low doses of cytarabine (MDC). In total, the patient underwent 15 courses of maintenance chemotherapy in the amount of "small doses of cytarabine". Before the start of the 16th course, a control examination was performed, which found that the morphological remission of the disease persists, while the MRR was 0.44%. From February to June 2019, three more courses of maintenance chemotherapy for MDC were carried out. Until August 2022, the morphological and molecular remission of the disease remains. Thus, this clinical example confirms that MRD-negative status after the first or second course of induction is a favorable prognosis factor, even if a small MRD was detected at the later stages of treatment, and indicates a low likelihood of recurrence of the underlying disease.

Clinical case No. 6

Patient born in 1973 AML installed in April 2017. No molecular genetic abnormalities were found in the patient. Cytogenetic examination revealed inv16. Thus, the patient was assigned to a favorable risk group according to ELN 2017. In April-May 2017, the first and second induction courses "7+3" were carried out. After the first course of induction on the 32nd day, hematological and cytogenetic remission of the disease was noted. MRD status is negative both after the first and after the second induction. In June and July 2017, the first and second course of consolidation of remission "Flar-Ida" were carried out. During the control puncture of the bone marrow in July 2017, it was confirmed that the remission of the disease persisted. Given the persisting bone marrow and cytogenetic remission of the disease, it was decided to start maintenance chemotherapy (six cycles of "5 + 5"). By January 2021, the patient remained in complete remission of the disease based on the examination. Thus, this clinical example confirms that the MRD-negative status after the first or second course of induction is a favorable prognosis factor and indicates a low probability of recurrence of the underlying disease.

The essence of the invention is explained on the graphic materials:

Figure 1 - Cytometric analysis of the main cellular components of the bone marrow of a blood stem cell donor. Data obtained on a CytoFlex flow cytometer, 11-color analysis;

Figure 2 - Individual dynamics of MRD values in 73 AML patients included in the AML-2017 protocol;

Figure 3 - Model assessments of DFS of patients in PR with MRD-negative and MRD-positive status after the 1st course of induction from different ELN-17 risk groups;

Figure 4 - OS, VRR, DFS in patients with AML in PR, depending on the MRD status after the 1st and 2nd induction courses;

Figure 5 - OS, DFS, VRR in patients with AML in PR, depending on the combination of MRD statuses after the 1st and 2nd courses of induction, time - at the time of recovery of peripheral blood after the 2nd course;

Figure 6 - Results of "ROC-survival" analysis. Sensitivity and specificity of 18-month DFS prognosis depending on the threshold for determining MRD status, a) after the 1st course of induction; b) after the 2nd course of induction;

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Claims (4)

1. A method for determining the prognosis of relapse in patients with acute myeloid leukemia (AML) based on the results of the assessment of minimal residual disease (MRD) in the post-induction phase of treatment after the 1st checkpoint - in the period from 35 to 42 days after the 1st course of induction chemotherapy and the 2nd checkpoint - in the period from 70 to 84 days after the 2nd course of induction chemotherapy, including the detection of residual tumor cells by multicolor flow cytometry in patients with acute myeloid leukemia at two checkpoints, differing in that that at the first stage, all CD34-positive cells are isolated and sequentially analyzed for the expression of all antigens, isolating all CD34-positive cells normally present in healthy donors; at the second stage, all CD117-positive cells are isolated and sequentially analyzed by the expression of all antigens, all CD117-positive cells normally present in healthy donors are isolated; at the third stage, all CD117-CD34- cells are isolated and sequentially analyzed by the expression of all antigens, all CD117-CD34 cells normally present in healthy donors are isolated, and when diagnosing the presence of MRD after the 1st course and / or 2nd course, a relapse is predicted in patients with AML . 2. The method according to claim 1, characterized in that they search for and quantify the population of cells that differ from the cells present in the bone marrow of a healthy donor. 3. The method according to claim 1, characterized in that the sampling of the bone marrow is carried out in accordance with the requirements for the collection of material for cytometric examination. 4. The method according to claim 3, characterized in that the sample preparation of the bone marrow sample includes lysis of erythrocytes and staining of leukocytes with a panel of monoclonal antibodies from 6, 11 and 13 reagents, in accordance with tables 2 and 3 of the description.