ALBERT

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: Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.

Description: Background: Patients (pts) with hematologic disease are at increased risk of severe SARS-CoV-2 infection. Recent observations reported poor outcomes of COVID-19 in pts with various cancer types and higher mortality rates compared with the general population. However, currently available data on COVID-19 in pts with hematologic disease are limited. Methods: CHRONOS19 registry is an observational prospective cohort study with the primary objective to evaluate the treatment outcomes in adult pts (age 18 or older) with hematologic disease and COVID-19. Secondary objectives are to describe severity and complications of COVID-19 and course of hematologic disease in SARS-CoV-2 infected pts, and to explore importance of various factors for disease severity and mortality. Pts with laboratory-confirmed or suspected (based on clinical symptoms and/or CT) COVID-19 were eligible for enrollment. Data were collected on a web platform and managed in a de-identified manner. Physicians from 8 hematology clinical centers and hospitals from all over Russia (Moscow, Ulan-Ude, Saransk, Vladimir, Nizhniy Novgorod, Kazan) participate in this study. Pts are followed for 30 days (ds) after COVID-19 diagnosis and up to 6 months (mos) for hematologic disease outcomes and overall survival assessment. The results of the first follow-up are presented in this interim analysis. Results: As of July 30, 2020, 184 pts were enrolled (females/males [n(%)]: 80(44%)/104 (56%); median [range] age: 55 [18-83] years. Disease type (malignant/non-malignant [n(%)]): 164(89%)/20(11%), including AML 36(20%), ALL 16(9%), MDS 5(2%), APL 5(2%), MM 38(21%), HL 4(2%), NHL 38(21%), MPN 9(5%), CLL 13(7%), others 20(11%). Concomitant diseases were in 95(52%) pts: cardiovascular 56(59%), pulmonary 6(6%), hepatic 6(6%) or renal 5(5%), diabetes 17(18%), obesity 4(4%), other 16(17%). 176 patients were evaluable for the primary outcome assessment with a median follow-up of 41(1-125) ds. Thirty-day all-cause mortality was 23% (41 pts died). Death due to COVID-19 complications occurred in 34 (83%) pts, 7(17%) pts died due to progression of hematologic disease. Fifty (28%) pts experienced COVID-19 complications, the most common were pneumonia in 125 (71%) pts, respiratory failure in 82(47%) pts, ARDS in 11(6%) pts, cytokine release syndrome in 15(9%) pts, multiple organ failure in 10(6%) pts, sepsis in 6(3%) pts, and pulmonary bleeding in 1(0,6%). Specific anti-COVID-19 treatment was given to 117 pts(67%) pts: most common first-line treatment was hydroxichloroquine+azithromycin in 84(72%) pts, azithromycin monotherapy in 27(23%) pts, other drugs in 6(5%) pts; second-line treatment comprised lopinavir+ritonavir in 38 pts, tocilizumab in 29 pts, umifenovir in 5 pts, baricitinib in 5 pts, canakinumab in 1pt, sarilumab in 1 pt. The rate of ICU admissions was 27%(47 pts), among them only 11(23%) pts survived, 36(20%) pts required mechanical ventilation, only 2(5.5%) pts survived. Eighty-eight(50%) pts received anticoagulants. With regard to the blood disease, treatment delays occurred in 101(57%) pts with a median 4 weeks, 6(3%) pts required change of treatment. At the first follow-up (30 ds) the rate of relapse / progression of hematologic disease was 16 of 151 evaluable pts (10.6%). Thirty-day overall survival was 75%. At the data cutoff, median overall survival was not reached. Antibody detection was performed in 70 pts: 53(76%) pts had IgG SARS-CoV-2 antibodies. Among factors possibly associated with poor survival were: stage of COVID-19 1(n=41) - 91,8%/ 2(n=75) - 90%/ 3(n=36) - 56,5%/ 4(n=22) - 13,6% (p