ALBERT

Description: Study objectives We aim to evaluate disease characteristics and treatment practices of pediatric pts. with Immune thrombocytopenia (ITP) in Russia. Materials and methods The ITP Registry was a multicenter, prospective, observational cohort study. Inclusion criteria: diagnosis of primary ITP, informed consent of the patient/guardians. Exclusion criteria: secondary or congenital thrombocytopenia. Data from medical records were registered in the e-CRF in average every 3 months. Descriptive statistics were used. Patients were registered since June 2011 till June 2014. Results Ninety-three pediatric pts, 46 male (49.5%) and 47 female (50.5%) with a median age 8.4 yrs (range 0.5-17.8) from 5 centers in various regions of Russia were included. The mean observation period reached 17.1 ± 6.5 mo (range1.4 to 28.6 months). Seventy (75.3%) pts had acute and 24.7% pts had insidious disease onset. The presence of trigger factors for ITP development was found in more than half of the cases (in 61.3% of patients), they are listed in Table 1. Table 1. Triggers N % No triggers 36 38.7% Infection 46 49.5% Vaccination 8 8.6% Other 3 3.2% Total 93 100% Median disease duration at enrollment was 1.07 years (range 0 to 16.7 yrs). ITP duration shorter than 5 years at the enrollment was reported in 89.2% pts, up to 1 year - in 43 (46.2%), 1- 5 years - in 40 (43%), 5-10 years - in 8 (8.6%), 〉10 years - in 2 (2.2%) pts. Newly diagnosed ITP was reported in 35 (37.6 %) pts, persistent ITP - in 12 (12.9 %), chronic ITP - in 46 (49.5 %) pts.Median platelets count was 12,0 x 109/L (range 0.0 - 72.0 109/L). Ninety-two (98%) pts experienced hemorrhagic manifestations during the course of ITP: skin hemorrhages - in 98.9%, oral bleeding - in 15.1%, epistaxis - in 36.6%, gastrointestinal bleeding - in 1.1%, intracranial bleeding - in 1.1%, hematuria - in 1.1%, and other hemorrhages - in 9.7% of pts. Relationship between hemorrhagic syndrome and platelet count at the enrollment is provided in table 2. Table 2. Relationship between hemorrhagic syndrome and platelet count (at enrollment) Hemorrhage highest grade according to WHO Platelet count (visit 1) Total pts / % 〈 30,000 30,000 -50,000 〉50,000 0 3 5.5% 3 5.5% 49 89.1% 55 100% 1 11 40.7% 5 18.5% 11 40.7% 27 100% 2 5 62.5% 0 0% 3 37.5% 8 100% 3 2 66.7% 1 33.3% 0 0% 3 100% Total 21 22,6% 9 9.7% 63 67.7% 93 100% Severe course of ITP after enrollment was observed in 12 (13%) pts (of whose 6 (6.5%) had clinically significant hemorrhage at the disease onset and 6 (6.5%) had new clinically significant hemorrhages during follow-up period. Refractory ITP at enrollment was reported in 9 (9.7%) pts and was associated with the resistance to the first-, second- and subsequent lines of therapy. At enrollment 42 (45.2%) pts received specific treatment for ITP. Before enrollment, splenectomy was reported in only 1 (1.1%) 14-years old patient who had a complete response. During the study, splenectomy was performed in 6 (6.6%) pts with chronic ITP; the duration of the disease at the time of splenectomy varied from 2 to 10 years, with average duration of 4.69 years (median - 4.5 years). Complete response to splenectomy was observed in 3 (50%) pts, a partial response - in 2 (33.3%), no response - in 1 (16.7%) patient. Loss of response to splenectomy was not reported. During the study, severe ITP was reported in 8 (8.7%) pts, 41 (44.6%) pt had various hemorrhagic manifestations of ITP at least at 1 visit, grade IV hemorrhagic syndrome was not reported. Thirty-eight (41%) pts received 1-st line treatment: glucocorticosteroids (GCS) - 23 (60.5%) pts, IVIG - 5 (13.2%), alfa-interferons -16 pts (42.1%). Twenty-three pts (24.7%) received second-line therapy: GCS - 1 (4.3%), IVIG -1 (4.3%), immunosupression - 1 (4.3%), rituximab - 2 (8.7%), romiplostim - 11 (47.8%), eltrombopag - 14 (60.9%). Conclusion For the first time new information on the features of the disease and patterns of management of pediatric pts with primary ITP in Russia was obtained in a prospective study. Interestingly, the preferred therapy for the 2nd or subsequent lines are TPO receptor agonists used outside the approved indications in research institutions, based on published clinical trial data. Splenectomy rate before and during the study was only 7.5% (7 pts) with chronic ITP; in 1 child (14.3%) splenectomy was ineffective. Low acceptance of splenectomy suggests TPO-mimetics as potential second-line therapy. In total, good disease control is achievable in the majority of pediatric pts with ITP. Disclosures Off Label Use: use of TPO-mimetics in children.

Description: Introduction The outcome hematopoietic stem cell transplantation (HSCT) in a cohort of children with chemorefractory leukemia is poor. The incidence of relapse exceeds 50% and survival varies from 10 to 40%. Additional attempts at remission induction with various combinations of chemotherapy are unlikely to improve the outcome and will contribute to toxicity. We hypothesized that personalized targeted therapy combined with high-dose chemotherapy may improve the outcome of allogeneic hematopoietic stem cell transplantation in a cohort of pediatric patients with refractory leukemia. Bcl-2 and CD38 were chosen as potential targets due to frequent expression in pediatric acute leukemias, availability of marketed targeted therapies, venetoclax and daratumumab, and expected non-overlapping toxicity profile of these agents and the conditioning regimen. Materials and methods A total of 16 pts with chemorefractory disease (T-ALL - 2, AML - 8, JMML - 6, 12 male, 4 female, median age 5,7 years), underwent HSCT between November 2017 and June 2018, median follow-up - 3 months (1,6-7). All pts were transplanted from haploidentical donors, had active disease (AD) at the moment of SCT, for 12 (75%) pts it was the first allogenic HSCT, for 4 pts it was the second HSCT. Median bone marrow leukemia burden before cytoreduction was 22% (3-75). Bcl-2 expression on the tumor cells was detected in all pts (100%) with the median expression of 69% (0,7-100), CD38 expression was detected in 10 pts (AML=7, ALL=2, JMML-1) with the median expression of 96% (71-100). Ten pts received treosulfan-based conditioning, 3 - busulfan-based and 3 -TBI-based. GVHD prophylaxis included tocilizumab at 8 mg/kg on day -1, post-transplant bortezomib and abatacept at 10 mg/kg on day -1, +7, +14, +28. Three pts received thymoglobulin 5mgkg. According to the expression of Bcl-2 and CD38 on tumor cells, 9 patients (56%) received Daratumumab (anti-CD38 monoclonal antibody) on day -6, 15 patients (94%) received venetoclax at 300 mg/m2/day on days -7 to -2. TCRαβ+/CD19+ depletion of PBSC with CliniMACS technology was implemented in all cases. The median dose of CD34+ cells in transplant was 11 x106/kg (range 7-18), α/β T cells - 40x103/kg (range 11- 139). Modified (CD45RA-depleted) donor lymphocyte infusions (DLI) were administered to 15 pts, 9 pts received modified DLI on day 0. Result Primary engraftment was achieved in 13 (81%) of 16 pts. The median time to ANC and platelets recovery was 14 days (11-22). Engraftment was 100% (10 of 10) among patients with acute leukemia and 50% (3 of 6) among patients with JMML. Three patients with JMML had early disease progression. There were no significant toxic effects after HSCT and no cases of transplant-related mortality. The median NK- cells count by the day +30 was 0,185 x 106/ml (range 0,019- 0,472), the median levels of αβ T cells and gd T cells were 0,045 x 106 /ml (range 0 - 0,364) and 0,07 x 106 /ml (range 0 - 0,349, respectively. Acute GVHD grade 1-2 was developed in 2 pts (15%), none of them required systemic immunosuppressive therapy. There were no cases of chronic GVHD. One (7,6%) patient with AML relapsed on day +61. Three pts (1 with AML and 2 with JMML) died from disease progression, 1 patient with JMML died from complications after the second HSCT. At the moment of reporting 12 pts (9 of 10 with acute leukemia and 3 of 6 with JMML) are alive, in complete remission with a median follow up of 3 months (1,5-7m). Conclusion We suggest that addition of venetoclax and datatumumab to the backbone of myeloablative haploidentical HSCT with αβ T cell depletion is not associated with increased toxicity and may lead to improved early outcomes in a cohort of pediatric patients with chemorefractory acute leukemia. This approach can be further tested in a prospective trial with the goal to increase the anti-leukemic efficacy of HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Inhibition of terminal complement components provides effective control of severe hemolytic PNH, but remains prohibitively expensive in countries with limited resources. Allogeneic hematopoietic stem cell transplantation offers a chance of permanent cure at the expense of increased risk of transplant-related morbidity and mortality in severe PNH. We report two patients with hemolytic PNH, associated with severe aplastic anemia, who were treated with limited eculizumab course to reduce the risk of complications of matched unrelated donor (MUD) transplantation. Patient #1 is a 22 y.o. young man. Severe aplastic anemia (SAA) was diagnosed at the age of 13 y.o. He received combined IST with ATG and cyclosporine A (CsA) and remained transfusion–free with complete hematologic response until 17 y.o., when SAA relapsed. CsA was re-administered as monotherapy due to severe anaphylaxis to ATG. At the age of 20 y.o. episodic hemolysis and severe abdominal pain bouts developed. PNH was diagnosed with standard flow cytometric assay. Two years later 10/10 matched unrelated donor was found and a decision was made to proceed to allogeneic transplantation. The preparative regimen included total doses of fludarabine - 150mg/m2, cyclophosphamide - 100mg/m2, thoracoabdominal irradiation - 6Gy and alemtuzumab 30 mg/m2. Eculizumab was administered weekly since day -14 till +14 at 600mg x2 and 300mg x3 resulting in prompt resolution of hemolysis. PBSC graft processing included TCR alpha/beta and CD19 depletion on CliniMACS instrument according to manufacturer's instructions. The final graft contained 6,4*106 per kg NC, 6,6*106 per kg CD34+, 15*104 per kg TCR alpha/beta+ lymphocytes. GVHD prophylaxis included tacrolimus till day +30 and Mtx at +1,3,6,10. Engraftment was stable with Plt and WBC on day + 18. PNH clone in granulocytes and monocytes remained around detection limit. A peculiar feature of engraftment was stable split chimerism with all hematopoietic lineages except CD3 originating from the donor. T cells remained almost completely of recipient origin until thymic function recovered with dominant production of donor T cells (Fig. 1). Independent of this T-cell chimerism dynamics, the hematopoiesis remains stable and of donor origin for one year from transplant. No manifestations of acute or chronic GVHD were observed. Transplant toxicity included CMV reactivation controlled with standard antiviral treatment. Patient#2 is a 15 y.o. girl. The diagnosis of SAA was established at the age of 8 y.o. Combined IST with ATG and CsA induced partial hematologic response. Three years later she developed SAA relapse and hemolytic PNH complicated by cerebral sinus thrombosis and severe ischemic attack. She recovered upon endovascular thrombectomy and systemic thrombolysis eculizumab was started at 600 mg/biweekly leading to resolution of hemolysis. Four months later 10/10 MUD was identified and the patient proceeded to transplantation. The preparative regimen included fludarabine - 150mg/m2, cyclophosphamide - 100mg/m2, thoracoabdominal irradiation - 6Gy and ATG(horse) - 90 mg/m2. Eculizumab was administered weekly till day +21 at 300mg/week. Graft processing included TCR alpha/beta and CD19 depletion. The graft contained 13,8*106 per kg NC, 13,5*106 per kg CD34+, 31*104 per kg TCR alpha/beta+ lymphocytes. GVHD prophylaxis included tacrolimus till day +30 and Mtx at +1,3,6. Engraftment was rapid with Plt and WBC on day + 12 and + 15 respectively. Graft function is stable for 100 days post-transplant and PNH clone in granulocytes and monocytes is at the limits of detection. T-cell chimerism remains mixed with 10% of recipient cells. Grade 1-2 skin aGVHD was observed and controlled by short steroid course. No other early transplant-associated toxicity was observed. Conclusion Combined use of peri-transplant eculizumab and TCR alpha/beta depletion of the unrelated graft potentially protects patients with severe PNH from transplant-related toxicity. Disclosures: Boyakova: Alexion: Research Funding.

Description: Introduction Relapse, graft-versus-host disease (GvHD) and GvHD-associated mortality are major obstacles to success of transplantation from unrelated and haploidentical donors in children with acute lymphoblastic leukemia. Future directions will focus on optimizing conditioning regimens and enhancing graft-versus-leukemia effect. Negative depletion of α/β(+) T cells and CD19+ B lymphocytes, which permits to maintain mature donor-derived natural killer cells and γδ(+) T cells in the graft may improve GvHD control, immune reconstitution and prevent the relapse. Patients and methods : A total of 67 pediatric patients with acute lymphoblastic leukemia (T-ALL- 26, B-ALL-41, 29 female, 38 male, median age 9,4 years, range 0,15-20) underwent allogeneic HSCT between May 2012 and May 2016. Forty two patients received haploidentical graft, 25 - a graft from matched unrelated donor. Disease status at transplant was CR1 in 19pts., CR2 in 35 pts. and CR〉2 13 pts. Transplantation in CR1 was performed according to risk stratification scheme in the current institutional ALL protocol.Fifty patients recieved treosulfan-based myeloablative preparative regimen (74%), TBI-based regimen was used in 17 patients (26%). Two regimens of GvHD prophylaxis were used: regimen 1 (n=28): ATG (horse, ATGAM) 50mg/kg, post-grafting immunosuppression consisted of short course Tacro/MTX (n=20) before day +30 or no post-transplant prophylaxis (n=8[ММ1] ); regimen2 (n=39): ATG (rabbit, thymoglobuline) 5mg/kg, rituximab 200mg/m2 (n=30), bortezomib (n=24) and post- transplant bortezomib (n= 19) or Tacro (n=19). All patients with TBI- based regimen received rabbit ATG. TCRαβ+/CD19+ depletion of HSCT with CliniMACS technology was implemented in all cases according to manufacturer's recommendations. The median dose of CD34+ cells in transplant was 10 x106/kg (range 3,9-18,8), TCRα/β - 19x103/kg (range 0,2-300). Results Primary engraftment was achieved in 64 of 67 pts. (two patients died before engraftment), the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. All evaluable patients achieved sustained complete donor T cell and myeloid chimerism by day +30. Early (100 day) mortality was pTRM was 7,5% (95% CI: 0,3-17),2-year pTRM - 17% (95%CI: 9-30). The 3 early deaths included bacterial sepsis (n=2) and viral infections (n=1), seven late: viral infection in four pts. (ADV=2, ADV+CMV=1, CMV=1), bacterial sepsis in two pts. and rhinocerebral mucormycosis in 1 pt., all late deaths were associatedwith chronic GvHD and prolonged corticosteroid therapy. Cumulative incidence of acute graft-versus-host disease (GvHD) grades II - IV and III - IV was 23,9% (95% CI: 16-36), and 7,5% (95% CI: 3-17) respectively. CI of cGvHD was 22,9% (95% CI: 14-36). Regimen 2 was more effective in prevention of aGvHD II-IV: CI at 2 year after HSCT was 12,8% vs 35,7% in regimen 1, p=0,05 and in cGvHD 8,8% vs 35,7%, p=0,028. No correlation between graft composition, donor type in aGvHD and cGvHD was noted Cumulative incidence of relapse at 2 years was 32% (95%CI: 22-47). Two years pEFS (event=death or relapse) was 49,6% (95%CI: 36-63), 2-year pOS - 50% (95%CI: 40-67). In patients, who received TBI-based conditioning pEFS was 62% (95%CI: 37-86), as compared with treosulfan-based 46,5% (95%CI: 31-62), p=0,65. There was no significant difference in survival and relapse rate according leukemia subtype and donor type. Median time of follow-up for survivors was 2 years (range, 0,3 - 4). Discussion: We confirm that the depletion of TCR-alpha/beta and CD19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric ALL patients. Viral infections and leukemia relapse await further improvement of control. All major outcomes were equivalent between transplantation from unrelated and haploidentical donor. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic transplantation of hematopoietic cells (HSCT) is an established method to treat different hematologic malignancies and disorders of hematopoietic and lymphoid system. Graft-versus-host-disease is one of the main risk factor for success of the procedure. Simultaneous depletion of alpha-beta T-cells and CD19+ cells in graft is the promising way to reduce the risk. The approach was recently introduced in clinical practice and many aspects of immune system reconstitution are still unknown. We applied improved technology for T cell receptor (TCR) repertoire sequencing to study origin and dynamics of T cell clones during 1 year follow-up period after allogeneic TCRαβ/CD19-depleted HSCT in children. We performed TCR repertoire sequencing for peripheral blood samples of patients before HSCT, at 2, 6 and 12 months after HSCT (n=21, 21, 17, 16 respectively), and for respective donor blood apheresis samples before abT/CD19 depletion. Twelve of the patients were diagnosed with acute leukemia and the others with non-malignant inherited and acquired blood disorders. For each patient data on recipient's T cell chimerism and counts of CD3+, naïve CD3+, alpha-beta T-cells and recent thymic emigrants (RTE) have been collected during 1 year follow-up period. Barcoding of each original TCR mRNA molecule passed to massive parallel sequencing allowed us to: (1) reduce sample preparation biases and quantitatively reconstruct of TCR repertoires; (2) equalize repertoire data analysis depth which is absolutely necessary for correct comparison of samples; (3) prevent risk of cross-contamination between samples and increase confidence of T clone origin determination. Two months after TCRαβ/CD19-depleted HSCT T cell repertoire mostly consists of several hundreds highly abundant clones. For patients with low recipient T cell chimerism from 13 to 504 largest T-cell clones (median 255, IQR 219, n=9, T cell chimerism 2% (IQR = 46.5 or 724.75, n = 10)). In addition CD4+ RTE count was higher for patients with high T cell chimerism. This observation was additionally confirmed by analysis of flow cytometry data for the expanded cohort of 105 patients at d60 after αβT-cell depleted HSCT (Wilcoxon rank sum test p-value = 0.002). Our results demonstrate that early after αβT-cell depleted HSCT repertoire of T cells are extremely skewed and unlikely able protect recipient efficiently. Observed recovery of T cell count mostly results from expansion of a few clones that have to divide intensely for the whole 60 days period in order to achieve the observed counts. Early reconstitution of TCR diversity and RTE counts in patients with substantial recipient T cell chimerism is mostly explained by surviving recipient T cells and intrathymic T cell progenitors, respectively. This work was supported by the Russian Science Foundation project №14-35-00105. Zvyagin I. is supported by grant MK-4583.2015.4. Disclosures No relevant conflicts of interest to declare.

Description: Study objectives The aim of the study was to evaluate disease characteristics and treatment practices of ITP in Russia. Materials and methods The ITP Registry was a multicenter, prospective, observational cohort study. The observation period for each patient in the Registry was not less than 12 months. Inclusion criteria: diagnosis of primary ITP, informed consent of the patient. Exclusion criteria: secondary thrombocytopenia. Data from medical records were registered in the e-CRF in average every 3 months. Descriptive statistics was used. Patients were registered since June 2011 till June 2014. Results Five hundred and seven adult 394female (77.7%)/113 male(22.3%) pts from 26 hematology centers in various regions of the Russian Federation were included. Observation period ranged from 1.4 to 29.8 months with an average of 18.6 ± 6.2 mo. Median age was 50.2 years (range 18.6-89.1). Median disease duration was 1.83 years (range 0-56.07). History of ITP of lasting 〈 1 year was reported in 186 (36.7%) pts, 1-5 years - in 142 (28%), 5-10 years - in 56 (11%), over 10 years - in 87 (17.2%), and was considered as unknown - in 36 (7.1%). Newly diagnosed ITP was reported in 19.5% of adult pts; persistent - in 16.6% and chronic ITP - in 63.9% of pts, respectively. Median platelets count was 14,0 x 109/L (range 0.0- 119.0 x 109/L). Hemorrhagic manifestations in the history of ITP were reported in 92.5% of pts: skin hemorrhages - in 89.5% of pts, oral bleeding - in 50.3% , epistaxis - in 37.3% , gastrointestinal bleeding - in 7.7%, intracranial bleeding - in 0.4%, hematuria - in 4.5%, and other hemorrhages - in 20.9% of pts. Severe ITP at the time of enrollment was observed in 158 (31.2%) pts (104 pts (20.5%) had a clinically significant bleeding at the disease onset, and 54 (10.7%) pts developed new clinically significant hemorrhages during the treatment. Refractory ITP at the time of enrollment was reported in 100 (19.7%) pts (resistance to the first, second and subsequent lines of therapy in 62 (12.2%) pts); 38 (7.5%) pts did not respond to splenectomy. At the time of enrollment, 250 (49.3%) pts received medical treatment for ITP. Severe ITP after enrollment was observed in 124 (24.8%) adult pts. Throughout the study, various hemorrhagic manifestations of ITP were reported in 48.0% of pts, severe hemorrhagic syndrome was reported in 10.0% of pts; Before enrollment, splenectomy was reported in 94 pts (18.5%); complete response (CR) was maintained in 34 (36.2%) pts, partial response - in 20 (21.3%), and no response - in 7 (7.4%). Thirty-two (34.0%) pts had lost the response after initial success. During the study, splenectomy was performed in 44 (10.8%) pts, of those - in 7 pts (15.9%) with newly diagnosed ITP; in 6 pts (13.6%) - with persistent ITP, and in 31 pts (70.5%) - with chronic ITP. The duration of the disease at the time of splenectomy varied from 0 to 21 yrs; with a median of 1.03 year. CR to splenectomy was observed in 31 (70.5%) pts, partial response - in 10 (22.7%), and no response in 1 (2.3%), while 2 (4.5%) pts lost response. Since the response to splenectomy might change during the observation in the study, the best response was registered. Table 1. Distribution of the best response to splenectomy variable (before study entry and in the course of the study). Number of Pts (n) % Best response to splenectomy Missing data 1 0.7% Complete response 65 47.1% A response 30 21.7% No response 8 5.8% Loss of response 34 24.6% Total 138 100% Two hundred and forty-three (47.9%) pts received their first-line treatment during the study; glucocorticosteroids (GCS) - 222 (91.3%) pts, immunoglobulins (IVIG) - 2 pts (0.8%), other drugs - 26 (10.7 %) pts. A second-line therapy was administered to 133 pts (26.23%), of which 27 (20.3%) received GCS, IVIG - 23 (17.3%), alfa-interferons - 6 (4.5%), immunosuppressants - 8 (6%), rituximab - 18 (13.53%), romiplostim - 39 (29.3%), eltrombopag - 37 (27.8%), other drugs - 2 (1.5%) pts. Conclusion For the first time in Russia, information regarding the clinical presentation and the "real life" management practice of adults with primary ITP was obtained in a large cohort of pts in a prospective study. The Registry showed a variability of ITP clinical course. One fifth of pts were refractory to therapy. The main therapy options for the ≥ 2nd line in a cohort of adult pts were splenectomy and TPO receptor agonists. However, large proportion of pts still received GCSs in the 2nd and even 3rd line of therapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction CD19 CAR-T cell products were recently approved as therapy for advanced B-cell lineage malignancies. The predominant manufacturing model for this type of therapy is a centralized industrial-type production process. An attractive model of CAR-T cell production and delivery to the patient is via a point-of care-manufacturing process. We report on first cases of implementation of this approach in a setting of compassionate use program. Patients and methods Four patients with relapsed/refractory B-cell lineage malignancies were eligible for compassionate use of CD19 CAR-T cell therapy. Three patients, median age 15 years, had relapsed B-cell lineage acute lymphoblastic leukemia (B-ALL) after haploidentical hematopoietic stem cell transplantation (HSCT). Bone marrow disease burden at therapy start was 12%, 74% and 0,159. The patient with low minimal residual disease (MRD) in the bone marrow had skeletal involvement with multiple lymphomatous mass lesions. One patient had refractory primary mediastinal B-cell lymphoma (PMBCL). The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells from patient-derived leukapheresis product. Automatic production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct, transduction with lentiviral (second generation CD19.4-1BB zeta) vector (Lentigen, Miltenyi Biotec company) and expansion over 12 days in the presence of TexMACS GMP Medium supplemented with MACS GMP IL-7/IL-15 combination. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab 1 hour before CAR-T cell infusion at 8mg/kg. Results All production cycles were successful. Median transduction efficacy achieved was 57%(52-63). Median expansion of T cells was x26(24-43). CD4/CD8 ratio in the final product was 0,750,22-6). All final products passed the in-process and quality controls. The cell products were administered at a dose of 1*106/kg of CAR-T cells. No immediate infusion reactions were reported. In 2 cases mild cytokine release syndrome (CRS) was diagnosed. In one case mild CAR-T cell related encephalopathy developed. In one case additional dose of tocilizumab and one dose of dexamethasone were administered to control CRS and encephalopathy. All patients survived to the point of response evaluation. In 3 cases CAR-T cell expansion was detected to a maximum 25%. MRD-negative responses detected by flow cytometry and PCR were achieved in 2 cases with bone marrow involvement. In two cases with prominent mass lesions an objective response was documented. In the patient with 74% blast in bone marrow at start of therapy, neither CAR-T cell expansion nor leukemia response were documented. Details of therapy and response are summarized in table 1. Conclusion Production of CAR-T cells with the CliniMACS Prodigy TCT process is a feasible and an attractive option that provides a point-of-care manufacturing approach to enable rapid delivery of CAR-T cells to patients in need. Robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy. Table 1. Table 1. Disclosures Dropulic: Lentigen, A Miltenyi Biotec company: Employment. Shneider:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen, A Miltenyi Biotec company: Employment. Alex:Miltenyi Biotec: Employment. Essl:Miltenyi Biotec: Employment.

Description: Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is indicated for patients with relapsed or refractory acute lymphoblastic leukemia. Patients with persistence of minimal residual disease (MRD) before HCT are at increased risk of disease relapse. Multiparameter flow cytometry (MFC) is the most commonly used method of MRD detection in clinical practice. This study aimed to evaluate MRD status before HCT on outcome of ALL patients receiving allogeneic HSCT from haploidentical donors with TCRαβ+/CD19+ depletion of the graft. Materials and methods A total of 120 pts with ALL (T-lineage ALL (T-ALL)- 37, B cell precursor (BCP)-ALL-83, 45 female, 75 male, median age 8.7 years (0.5-20) underwent allogeneic HSCT between June 2013 and June 2019. All pts received Haplo graft and were in morphologic remission. Disease status at transplant was CR1 in 35 pts, CR2 in 68 pts and CR〉2 in 17 pts. Transplantation in CR1 was performed according to risk stratification scheme in the current institutional ALL protocol (Moscow-Berlin 2008, 2015). MRD detection in the bone marrow prior to НSСТ was performed in all pts by MFC according the AIEOP BFM FLOW Network SOP. MRD negativity was defined as

Description: Objectives Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric and adult acute leukemias. Such rearrangements are more frequent in infants, in whom they are found in 60% - 80% of acute lymphoblastic leukemias (ALL). KMT2A-r group itself is genetically heterogeneous. KMT2A-r can occur with at least 94 partner genes previously described in «THE MLL RECOMBINOME IN 2017». Detection of KMT2A-r is an essential part of AL initial diagnostics. Also, the accurate detection of all KMT2A-r types is crucial in order to perform minimal residual disease (MRD) monitoring, as it is clear now that MRD-based therapy adjustment has a very strong impact on outcome. Here we report a case of novel KMT2A partner in pediatric ALL - NUT family member 2A (NUTM2A). Methods The patient is 8 y.o. girl with T-cell acute lymphoblastic leukemia with pleural effusion. Bone marrow and pleural fluid aspirates were analyzed by G-banded karyotyping and FISH with KMT2A breakapart probe. Pleural fluid aspirates were also analyzed by real-time RT-PCR for 8 most common KMT2A rearrangements screening, long-distance inversed PCR and targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA). Sanger sequencing was used for validation. Results Conventional cytogenetics and FISH showed 47,XX,t(10;11)(q22;q23),+mar[5] karyotype with 100% KMT2A-rearranged nuclei. Gene fusion analysis identified novel fusion KMT2A-NUTM2A with exon 11 - exon 1 breakpoint junction. NUTM2A is a gene at 10q23.2. This gene is not fully described in the literature. Rearrangements of this gene were identified in endometrial stromal sarcomas (ESS) (Cheng-Han Lee et al. 2012) and small round cell sarcoma (SRCS) (Sugita et al. 2017). In case of ESS, this rearrangement results in an in-frame fusion between YWHAE and NUTM2A (or highly homologous gene NUTM2B), and in case of SRCS it results in an in-frame fusion between NUTM2A and CIC. To our knowledge, KMT2A-NUTM2A fusion in our study is the first case demonstrating NUTM2A rearrangement in hematological malignancies. Conclusions Here we for the first time show the novel KMT2A-NUTM2A fusion transcript, which was found in pediatric T-cell acute lymphoblastic leukemia. Anchored multiplex PCR is one of the most sensitive way to detect rare variants of KMT2A rearrangements. It also allows selecting patient-specific primers for further PCR detection of MRD. Disclosures No relevant conflicts of interest to declare.

Description: Laberko et al report excellent survival outcomes for patients receiving TCRαβ/CD19-depleted hematopoietic stem cell transplantation (HSCT) for primary immunodeficiencies, demonstrating comparable results with mismatched related and matched unrelated donors.