ALBERT

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

Description: Background. New insight into pathogenesis of aplastic anemia (AA) in conjunction with effectiveness of Eltrombopag allowes to recommend it for initial treatment of de novo AA. However, eltrombopag was an attractive option for patients at different stages of the therapy. Aim. In our study we present various application points of eltrombopag as a therapeutic option in patients (pts) at different stages of the disease. Materials and patients. We are presenting our single-center experience, that include 11 pts with acquired aplastic anemia (SAA/NSAA = 10/1) on different stages of the disease. All pts were divided into four groups. In the 1st group we included refractory pts after two (n=3) or three (n=2) lines of antithymocyte globulin (ATG) therapy. The 2nd group included two pts who received the second ATG course in combination with eltrombopag, after faild first ATG. In the 3rd group included 2 pts with refractoriness after 1 course of ATG, who couldn't receive the second ATG/CsA because of severe infections. The 4th group included two pts with one lineage thrombocytopenia but transfusion independent. The median time from the start of immunosuppressive therapy (IST) to beginning of eltrombopag was Me(min-max): 56(12-115), 51(8-94), 1(1-1), 69(37-100) months respectively. The daily dose of eltrombopag for pts was 100-150 mg. Results. Hematological response are present in 3 out of 5 pts in the 1st group. Median duration of eltrombopag treatment was 8,5 (3-12) months. It is necessary to note that responding pts were refractory to 2nd (n=1) or 3rd (n=2) ATG and transfusion dependence. All pts in the 2nd group achieve PR (Me duration eltrombopag treatment was 12 months). All pts from 3rd group achieved granulocytic response, that allowed to recover get out from infections and to hold the 2nd ATG. However in 1 pt monosomy 7 was detected by FISH in bone marrow after therapy. It was noted that before eltrombopag administration there were not cytogenetic aberrancies but there were myelodysplastic features by flow cytometry (low index of granularity in granulocytes, low HLA-DR expression of monocytes and high CD56 expression in both granulocytes and monocytes) and PNH-clone. After eltrombopag treatment and monosomy 7 detection we performed a FACS sorting and FISH-study for PNH+ and PNH- granulocytes. Monosomy 7 was found in PNH- but not in PNH+ granulocytes. From the 4th group thrombocytopenia resolved in 1 pt (11 months eltrombopag duration), in over case present threelineage relapse subsequently. Conclusion. Eltrombopag can be used in pts with long-term ineffective IST and as a bridge to the next step of IST. It is necessary to remain cautious about the earlier development of clonal complications, even in patients from the so-called favorable risk group with the PNH clone. Disclosures No relevant conflicts of interest to declare.

Description: The stromal microenvironment regulating hematopoiesis in patients with hematologic malignancies undergoes significant alterations. The changes in the concentration of colony-forming fibroblast units (CFU-F) in the bone marrow (BM) and disruption in the functioning of multipotent mesenchymal stromal cells (MSCs) are shown in many studies for patients with acute leukemia. Often it is not possible to distinguish the cause of changes in stromal progenitor cells after treatment: interaction with tumor cells or the effects of therapy. Most of the patients with diffuse large B-cell lymphoma (DLBCL) do not have BM involvement. It was assumed that the properties of MSCs in these patients were not changed, and so this could be an attractive model for investigation the effect of antitumor drugs on human BM stromal microenvironment. The aim of the study was to compare the properties of MSCs in patients with DLBCL in the onset of the disease and a month after the end of therapy. Methods The study included 20 patients with DLBCL (11 male, 9 female) aged 42-60 years in the onset of the disease and a month after the end of treatment. 3-5 ml of BM were collected during diagnostic punctures after informed consent. MSCs and CFU-F were cultured by standard methods. The total MSCs production, the doubling-population level per day, the concentration of CFU-F, the relative gene expression level (REL) in MSC by real-time PCR and the mean fluorescence intensity (MFI) by flow cytometry were analyzed. The control group included 31 donors of the corresponding age. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Results The total cell production for 4 passages in primary patients' MSCs was significantly higher than in donors (11.4 ± 2 x 106 per flask versus 6.9 ± 1.1, p = 0.04). It remained elevated after therapy (10.2 ± 1.5). At the same time, the MSCs population-doubling level per day was significantly decreased in patients in comparison with donors (0.6 ± 0.03 vs. 0.4 ± 0.04, p

Description: Background. The recent ELN recommendations (2017) have introduced new criteria of response in AML - complete remission with MRD-negativity (CR/MRD-neg). It's a well-known fact that AML with different cytogenetics has different chemosensitivity, and so - CR rate in patients with favorable, intermediate or poor cytogenetic markers substantially differs. MRD negativity corresponds to more favorable long-term results in CR patients, but it's not clear whether the MRD-negativity rate is the same in favorable, intermediate or poor risk AML pts. As it is still poorly understood whether the high risk AML patients need classical «7+3» induction, we introduced a new approach for this cohort of patients - prolonged low-dose cytostatic exposure after hypomethylating priming. Aim. To evaluate the MRD-negativity in CR AML patients from different cytogenetic subgroups after the first classical non-intensive induction: «7+3», «Aza+7+3», «Aza-Ida-Ara-C», and the second induction differed by intensity. Materials and patients. 83 AML patients with a median age of 37 y.o. (17-59), m/f 31/52, after achieving CR (69 pts) were included in the MRD study in the National Research Center for Hematology. From March 2016 till Dec 2017, 49 pts disregarding cytogenetic risk were treated with «7+3» as the first induction course (Dauno 60 mg/m2 1-3 days, Ara-C 100 mg/m2 bid 1-7 days). The second induction course for these pts was «7+3» with continuous Ara-C infusion (200 mg/m2 1-7 days). Since Dec 2017 till July 2018 in all patients epigenetic priming with 3 days of Azacitidine (75 mg/m2) was applied additionally in order to obtain the cytogenetic results prior to cytostatic exposure. All patients were tested by molecular markers (FLT3, CEBPa, mNPM1) but this data did not influence the treatment choice. 8 pts from favorable/intermediate cytogenetic risk group after Aza-prephase have got «7+3» (Dauno 60 mg/m2 4-6 days, Ara-C 200 mg/m2 by continuous infusion, 4-10 days), and 13 pts from poor cytogenetic risk group (complex, -7,-5, inv3, monosomy) after Aza-priming received prolonged low-dose schedule (Ida 3 mg/m2 4-10 days, Ara-C 10 mg/m2 bid sc 4-17 days). The second induction course for favorable/intermediate risk group was FLAG-Ida, for poor risk group - the same as the 1st induction. MRD testing was performed by 6-color flow cуtometry with FACS Canto II (BD) machine after 1st and 2nd chemotherapy courses. The rate of MRD-negativity was calculated among CR patients. Results. Totally among 83 patients CR was achieved in 83%, early death was registered in 6% and refractory AML was diagnosed in 11%. The induction and MRD testing results after the 1st and the 2nd induction course according to prognostic group and treatment arm are demonstrated in the Table.1. Morphological CR rate differs substantially in patients from favorable/intermediate and poor risk by any treatment approach (p=0,04). The prolonged low-dose induction with Aza-prephase induced the same numbers of CRs as the classical «7+3»: 69% vs 46% (p=0,42). Epigenetic priming provided identical MRD-negative CRs in all treatment and cytogenetic groups: 75% vs 62% in favorable/intermediate risk group (p=0,29), 67% vs 50% in poor risk (p=0,62). The second intensive induction with Flag-Ida in favorable/intermediate risk group did not increase the portion of MRD-neg CR pts in comparison with «7+3»: 88% vs 71% (р=0,14). Early relapse rate through all groups showed comparable results. Conclusion. Though the morphological CR achievement is highly influenced by cytogenetic subgroup in AML, the MRD-negativity rate among CR pts by flow cytometry after the 1st and the 2nd induction course is similar. The de-intensification of induction for poor risk group did not lead to the deterioration of therapy efficacy and provided the same rate of MRD-negativity. The 2nd intensified induction after «7+3» did not increase the MRD-negativity in CR pts. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (T-regs) influence on T-effectors cells. Despite the well-known fact that T-regs participate in pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence, data about functional status of T-regs and aGVHD is still limited. Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30 after allo-HSCT. We use PBMC from 29 patients with hematological malignancies obtained by density gradient media. This method was used due to lymphopenia in this group of patients. Group with no aGVHD consist of 22 patients (AML=12, ALL n=5, LPD=1, CML=2, AA=1, MDS=1) after allo-HSCT n=17 from MUD, n=5 from mismatch unrelated donor. MAC conditioning regimen was used in 17 cases, 5 patients receive RIC. The group of aGVHD after day +30 include 7 patients. (AML=4, ALL n=3) after allo-HSCT from MUD (n=5), mismatch unrelated donor (n=1) and n=1 from sibling HLA-identical donor. Two patients receive MAC and 5 patients receive RIC conditioning regimen. All patients in both groups receive standard immunosuppression (MMF+CSA+ATG). All patients developed II-IV grade aGVHD (II (n=1); III (n=4); IV (n=2)) with a median time onset on day +50 (34-150). The anti-CD4-APC-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population. A Bland-Altman plot (difference plot) was used in analyzing the agreement between the two different methods of T-regs assay (CD4+CD25high and CD4+CD25highCD127low) - differences were not found (p=0,942). Due to this fact in our experiments CD4+CD25high cells were identified as T-reg cells (Gregg et al., 2005). 30000 of CD4+ cells were analyzed on a BD FACSCanto II to achieve sufficient statistical power (Becton Dickinson, USA). Results. As we can see on chart 1 level of Granzyme B was higher in patients who never developed aGVHD. In accordance with chart 2 percentage of Granzyme B positive T-regs in group of patients who never developed aGVHD was 7,26±1,89% in comparison with 2,04±0,93% in group who developed aGVHD after day +30 (p=0,02*). We should note that according to our previous experiments bacterial or viral infection (e.g CMV) and HLA-disparity does not affect the level of granzyme B expression in T-regs. Using ROC- curve analysis (see Chart 3) we obtained area under curve (AUC) 0,74. Conclusion. Our data shows that Granzyme B in T-reg cells after allo-HSCT in patients with standard IST may predict aGVHD onset after day +30 with satisfactory test results (AUC=0,74, «cut-off» - 4,15%; sensitivity - 85,71%; specificity - 45,45%).). According to data of our transplant center (for 2006-2016), "granzyme B-based strategy" can help us to predict up to 47,3% of all aGVHD. Disclosures: No relevant conflicts of interest to declare. Chart 1: Example of Granzyme B expression (right; shown on horizontal axis) in T-regs (left; upper right quadrant) on day +30 after allo-HSCT in group with standard immunosuppression Chart 2: Percentage of Granzyme B positive T-reg cells in patient without aGVHD and patients who develop aGVHD after day +30. Chart 3: ROC curve analysis of Granzyme B in T-regs as predictor of aGVHD. Figure Figure. Figure Figure. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Presence of measurable by flow cytometry (FC) residual disease (MRD) is a well-known risk factor in AML patients (pts), which is associated with adverse prognosis. The main idea in MRD by FC is searching for leukemia-associated immunophenotype (LAIP) which is based on «different from normal (DfN) approach». DfN approach allows to capture any immuphenotypic (IP) shifts during the therapy. LAIP may change in 16-91% AML cases. There are some ELN recommendations for monoclonal antibodies (mAbs) which allows todetect "mandatory (most frequent)" aberrations. But each laboratory uses its own tactics for MRD detection, taking into account the capabilities and equipment, and approved standardized panel still does not yet exist. Aim. To find the most common and adverse IP aberrations of blast cells in the onset of AML, and to determine the frequency of IP shifts during treatment and in relapse. Patients and methods. From March 2016 to February 2018 71 newly diagnosed AML (18-79 y.o.) pts were included in the initial LAIP (before any treatment) detection in hematology department of National Research Center for hematology, Moscow, Russia. MRD was detected in bone marrows by FC with BD FACS Canto II. The standard panel included mAbs against CD19, CD7, CD2, CD38, CD65, CD66b, CD99 (FITC); CD56, CD15, CD11b, CD13 (PE); CD33 (APC); HLA-DR, CD14, CD16, CD117 (PerCP-Cy 5.5); CD45 (APC-H7) and CD34 (PE-Cy7). MRD was determined after 1st and 2nd treatment course and in relapse. Pts received different treatment strategy due to age, comorbid status, severe infectious complications and type of AML. Most common for 80% of pts was induction course «7+3». Next cycles differed by intensity. The threshold value of 0.01% was estimated for long-term prognosis in our research. Results. The distribution of aberrations of blasts in the AML onset is shown in pic. 1. The most common aberration was "reduced /absence expression": in 60% of pts it was observed in the combination CD34+/HLA-DRlow (HLA-DR-). The appearance of this aberration and its preservation during the course of therapy were also more common (10% - 15%) than others (pic 1). When analyzing the results of long-term survival, it was shown that low expression of CD38 and/or CD117, as well as increased expression of CD13 in combination with CD34+ on blast cells in AML onset, is associated with bad prognosis (pic.2). CD117 and CD38low are the earliest markers of progenitor cells and show the region for identifying stem cells and leukemic stem cell. When analyzing the remaining groups of aberrant combinations (overexpression, asynchronous and non-linear expression), no correlation with the long-term prognosis was revealed. From 15 relapses of AML, the change of the primary LAIP was detected in 40%. Of the 7 refractory AML pts, the change of primary LAIP was detected in 57%. Complete replacement of the primary LAIP - in 18% from those with relapse and refractoriness (pic 3). Conclusion. The immunophenotyping shifts in AML pts is a very common phenomenon. In our study 60% of LAIP in relapse remained the same and 43% was the identical in refractory pts. LAIP shifts during chemotherapy reflects the biology of the tumor and its low sensitivity to therapy, but eventually leads to relapse development. To cover all potential changes in the IP and early suspect the relapse development to change the tactics of therapy, it is necessary to use the complex standardized mAbs panel, which allows determining as many aberrancies, as possible. Figure Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Minimal residual disease (MRD) detection by multicolor flow cytometry (MFC) after early induction or consolidation AML therapy is a well known factor of adverse prognosis, especially in intermediate risk group. New assessment of response in AML is complete remission with MRD-negative status. However, there is still no standardization of MFC method for AML patients (pts). Optimal time point for MRD status and its threshold value vary in different studies. Aim. To study different consolidation treatment regimens in AML pts by measuring of MRD reduction, to determine optimal time point and threshold level for additional risk stratification. Patients and methods. From March 2016 to February 2018 42 pts (younger than 60 y.o.) with newly diagnosed AML were included in prospective MRD study in hematology department of National Research Center for hematology. Initial leukemia-associated immunophenotype was analysed before any treatment, and bone marrow assessment for MRD detection was performed in CR pts after 1st and 2nd chemotherapy courses. The standard antibody panel included CD19, CD7, CD2, CD38, CD65, CD66b, CD99 (FITC); CD56, CD15, CD11b, CD13 (PE); CD33 (APC); HLA-DR, CD14, CD16, CD117 (PerCP-Cy 5.5); CD45 (APC-H7) and CD34(PE-Cy7). For results record BD FACSCanto II (Becton Dickinson) machine was used. The first course for all pts was the standard «7+3». Before March 2017 the second course was also «7+3» and included 25 pts, after March 2017 - 17 pts received intensive «Flarida» regimen (Fludarabine 30mg/m2 1-4, Ara-C 1g/m2 1-4, Idarubicin 8mg/m2 1,3). The 2nd MRD test was compared in these two groups. There was no difference between the two groups by pts in CR after the first «7+3», ELN status and age. Results. By using of special mathematical and statistical method the threshold value of 0,01% was estimated for long-term prognosis. Two years relapse-free survival (RFS) in MRD-positive (MRD+) and MRD-negative (MRD-) groups after the 1st induction was 23% and 87%, respectively (p

Description: Introduction. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be curative for acute myeloid leukemia (AML) patients. However, the disease relapse after allo-HSCT lead to poor outcomes almost in all cases. Minimal residual disease (MRD) is a strong prognostic marker of increased risk of relapse and reflects overall (OS) and relapse-free survival (RFS) in patients with AML. Immunophenotypic evaluation using multiparameter flow cytometry (MFC) is an important strategy for detecting MRD, because of its simplicity and wide availability. Aim. Determine the impact of MRD detected by MFC before allo-HSCT on clinical outcomes. Patients and methods. To assess the impact of MRD status before transplantation we include 56 AML patients who underwent allo-HSCT in National Research Center for Hematology between July 2016 and June 2019. Patient's characteristics are shown in table 1. All these patients were in first compete morphologic remission at the time of allo-HSCT. MRD was evaluated by MFC. Bone marrow samples were obtained before allo-HSCT and were analyzed by 6-color MFC (BD FACS Canto II, USA). Positive MRD was identified as a cell population deviating from the normal patterns of antigen expression on specific cell lineages at specific stages of maturation for all patients. In addition, for patients with LAIP at diagnosis, positive MRD was also defined as a cell population carrying LAIP markers at diagnosis. The probabilities of OS and RFS were estimated using the Kaplan-Meier method. Results. Among 56 AML patients before allo-HSCT (all were in CR at the time of MRD analysis). Median of follow-up was 9.3 months.11 patients (19,6%) were identified as MRD+ (0,011%-5,08%, mean 1%). And 5 of them relapsed (45,5%). Out of 45 MRD negative patients before HSCT relapse was registered in one (relapse rate 2,2%). Statistical analysis revealed significant differences between MRD- and MRD+ patients: MRD+ had much worse OS (22,9% vs. 85.5%, p=0.0047, Fig.1A) and RFS (26% vs. 97.4%, p