ALBERT

Description: The Journal of Physical Chemistry B DOI: 10.1021/jp402430w

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 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: Introduction Relapse, graft-versus-host disease (GvHD) and associated non-relapse mortality are the main obstacles to successful hematopoietic stem cell transplantation in children with leukemia. αβ T cell depletion was developed to prevent GvHD and improve immune reconstitution in recipients of mismatched grafts. Most current protocols use rabbit anti-thymocyte globulin (ATG) as an essential component of preparative regimen to secure engraftment and GVHD control. In order to avoid damaging effects of circulating ATG on graft NK and gd T cells, we have replaced ATG with pharmacologic blockade of IL-6 and CD80/CD28 co-stimulation axis in our ongoing study. Patients and methods Major transplantation outcomes were compared between participants of the current prospective trial (ATG-) and a retrospective control group (ATG+). A total of 165 children with acute leukemia (67 AML, 98 ALL, 68 female, 97 male, median age 8,7 y) underwent allo HSCT between 01.11.2013 and 01.03.2018. Of them 134 - from haploidentical donor and 31 from unrelated donor. All pts were in complete remission (CR1=80, CR2=67, CR〉2=18). Ninety-two pts received treosulfan-based conditioning, 73 - TBI-based (all ALL). Either melphalan (n=46) or thiophosphamide (n=98) or etoposide (n=21) were added as a second agent. Fludarabine was used in all pts. Two types of GVHD prophylaxis were used: type 1 (ATG+), (n=98): thymoglobulin 5mg/kg, rituximab 200mg/m2 with either bortezomib on days +2, +5 (n=72) or tacro (n=9) or without any additional agents (n=17); Type 2 (ATG-) (n=67): tocilizumab at 8 mg/kg on day -1, bortezomib on day +2, +5 with abatacept at 10 mg/kg on day -1, +7, +14, +28 (n=63) or without added agent (n=4). αβ T cell depletion with CliniMACS was used in all cases. The median dose of CD34+ cells was 9x106/kg, αβ T cells - 16 x103/kg. Modified (CD45RA-depleted) donor lymphocyte infusions (DLI) were administered to 113 pts. Twenty-five patients received DLI on day 0 and 88 pts received DLI after engraftment. Median time of follow-up for survivors was 2 years (range, 0,3 - 4,5). Results Three patients died before engraftment due to septic event. Primary engraftment was achieved in 161 of 162 evaluable pts (99,3%), the median time to neutrophil and platelet recovery was 16 and 15 days. Among the whole cohort the cumulative incidence of acute graft-versus-host disease (GvHD) grades II - IV and III - IV was 11,5% (95% CI: 7,5-17,6) and 4,8% (95% CI: 2,5-9,5) respectively. The cumulative incidence of cGvHD was 10 % (95% CI: 6,3-15,9). The incidence of aGvHD and cGvHD was not different between ATG (+) and ATG (-). Among the whole cohort 2-year pTRM was 8% (95%CI: 4,8-13,5). pTRM was significantly lower among ATG (-) group - 1,5% (95%CI:0,2-10,4) versus 12,2% (95%CI:7,2-20,8) among ATG (+) group, p = 0,015. The cumulative incidence of relapse at 2 years was 21% (95%CI: 15,5-29), 24% (95%CI: 16-35), among ATG (+) and 19% (95%CI: 11-32), among ATG (-), p = 0,8. Two-year pEFS was 70% (95%CI: 62-77), 2-year pOS - 78% (95%CI: 71-85). Among patients, who received ATG (-) regimen, pEFS was 76% (95%CI: 68-89), as compared to 65% (95%CI: 56-75) among ATG (+), p=0,1 and pOS was 89% (95%CI: 81-97) versus 72% (95%CI: 63-81), p=0,032, respectively. αβ T cell recovery at day +30 was associated with a trend to decreased incidence of relapse, CI of relapse was 32% (95% CI:22 - 47) in those with αβ-T cell count 〈 median vs 18 % (95% CI: 11-32) in those with αβ-cell count 〉median, p=0,08. EFS among αβ T" high" was 81% (+/-10) vs 56% (+/-14) among αβ T"low", p=0,002. Discussion We confirm that the depletion of αβ T cells from the unrelated and haploidentical graft in combination with intensive conditioning regimen ensures high engraftment rate and low transplant-related mortality. Our analysis suggests that polyclonal ATG serotherapy is not an essential part of the transplant regimen in αβ T-depleted transplantation. Combined administration of tocilizumab and abatacept after αβ T cell-depleted grafting effectively prevents GVHD, does not compromise engraftment, appears to decrease non-relapse mortality and improve survival. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Hematopoietic stem cell transplantation from non-sibling donors remains the only curative option for severe aplastic anemia patients refractory to ATG/CsA immunosuppression. Although the results of MUD and haploidentical transplantation in SAA have improved significantly, graft-versus host disease (GVHD) remains a serious problem, associated with significant morbidity and mortality. We investigated the role of new method of graft processing - TCR alpha/beta depletion as a way to improve the results of MUD and haploidentical transplants in SAA. Patients and methods Forty two patients with SAA were treated since November 2012 till February 2016. Median age at HSCT was 11(3-22) years, 27 male/15 female. All pts. were refractory/relapsed (36/6) after at least two courses of ATG/CsA, 3 pts. had concurrent severe hemolytic PNH. Time from diagnosis to transplant was 17(143-8,6)/15(99-5,5) months. Donors were unrelated volunteers in 32 cases, haploidentical parents in 10 cases. Preparative regimen included cyclophosphamide 100-150 mg/kg, fludarabine 150mg/kg, ATG and 2-6Gy thoraco-abdominal irradiation, in haplo transplants patients additionaly received thiophosphamide at 10mg/kg. Two patients recieved alemtuzumab instead of ATG because of anaphylaxis. Post-transplant GVHD prophylaxis included Tacro and Mtx on days +1, +3, +6. PBSC grafts were depleted of TCR alpha/beta cells and CD19 cells with CliniMACS device as recommended by the manufacturer. Patients received a median of 10(6,0-23) x106 CD34 per kg, 8(1-39) x104 TCR alpha/beta per kg. Results All patients engrafted with a median of 15 days for WBC and 13,5 days for platelets. In 4 patients after MUD transplantation secondary graft failure (rejection) developed, two of them were successfully retransplanted. Cumulative incidence of aGVHD 2-3 was 9% (95% CI: 3-27%) and 40%(95% CI: 18-85%) in MUD and haplo respectively, 90% patients with aGVHD had only skin involvement. No grade 4 aGVHD detected. Cumulative incidence of grade 3 aGVHD was 3%(95% CI: 0,5-21%) and 10%(95% CI: 2-64%) in MUD and haplo, respectively. Cumulative incidence of cGVHD was 12%(95% CI: 0,5-58%) and 30%(95% CI: 11-77%) in MUD and haplo respectively. A median follow up is 2 years. Seven patients died of viral infection - CMV (2 pts.), viral infection - CMV plus GVHD (2 pts.), microangiopathy (1 pt), 2 pts. died after second transplantation (1 - disseminated toxoplasmosis, 1 - ADV and CMV and GVHD). Event and GVHD-free survival is 73%(95% CI: 57-89%) and 60% (95% CI: 30-90%) in MUD and haplo respectively (pic.1) Overall survival is 86%(95% CI: 74-99%) and 78%(95% CI: 50-100%) respectively for MUD and haploidentical (pic.2). Prolonged stable mixed chimerism in T-cells was detected in recipients of MUD grafts in contrast to haploidentical grafts (pic/3). Conclusion TCR alpha/beta depletion is a robust platform for allogeneic transplantation from MUD and haploidentical donors in severe aplastic anemia. Results should be further improved by additional measures to control viral infections and prevent rejection in MUD transplants. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Description: Introduction The outcome HSCT in a cohort of children with chemorefractory acute leukemia (AL) 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 may contribute to toxicity. We hypothesized that personalized targeted therapy combined with high-dose preparative regimen may improve the outcome of alloHSCT in a cohort of pediatric patients with refractory AL. Bcl-2 and CD38 were chosen as potential targets due to frequent expression in pediatric acute leukemia, 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 26 pts with chemorefractory acute leukemia (AML-18, T-ALL-6, ABL-2, primary refractory - 9, refractory relapse - 17, 18 male, 8 female, median age 11.3 years), underwent HSCT between November 2017 and May 2019. All patients were transplanted from haploidentical donors, all patients had active disease (AD) at the moment of SCT. For 19 (73%) pts it was the first alloHSCT, for 7 (27%) pts it was the second HSCT. Median bone marrow leukemia burden before cytoreduction was 11% (3-90). Bcl-2 expression on the tumor cells was detected in 23 pts with the median expression of 58% (1.5-99), CD38 expression was detected in 24 pts with the median expression of 88% (11-100). Thirteen pts received treosulfan-based conditioning and 13 - TBI-based (12 Gy). TBI was used among patients with second HSCT (n=7), patients with T-ALL (n=4) and patients with massive extramedullary disease (n=2). GvHD prophylaxis included tocilizumab at 8 mg/kg on day -1 and abatacept at 10 mg/kg on day -1, +7, +14, +28. According to the expression of Bcl-2 and CD38 on tumor cells, 24pts (92%) received daratumumab (anti-CD38 monoclonal antibody) on day -7 at 10-16 mg/kg, 21 pts (81%) received venetoclax at 300 mg/m2/day on days -7 to -2 and 12 pts additionally received plerixafor at 240 mcg/kg x 3 on days -7 to -5. TCRαβ+/CD19+ depletion of PBSC with CliniMACS technology was implemented in all cases. The median dose of CD34+ cells in the graft was 9.5 x106/kg (range 4.9-13.3), αβ T cells - 26.5x103/kg (range 5.6- 84). Modified (CD45RA-depleted) donor lymphocyte infusions (DLI) were administered prophylactically in all pts. Results Two patients died before engraftment due to septic event. Primary engraftment was achieved in 24 of 24 evaluable pts (100%), the median time to neutrophil and platelet recovery was 12 and 13 days. All pts achieved complete remission on day +30. There were no significant toxic effects after targeted therapy, 4 pts (21%) had daratumumab-related infusion reactions, one patient with T-ALL died after engraftment due to septic event. aGVHD grade I-II developed in 5 pts (20%), two of them required systemic immunosuppressive therapy. There were no cases of aGVHD 〉 grade II, neither cases of cGVHD. The median NK- cells count by the day +30 was 0.07 x 106/ml (range 0.01- 0.4), the median levels of αβ T cells and gd T cells were 0.018 x 106 /ml (range 0 - 1.1) and 0.015 x 106 /ml (range 0,01 - 1.3), respectively. Nine (37.5%) pts (5 with T-ALL, 1 with ABL, 3 with AML) relapsed, median interval to relapse was 103 days (55-345). At the moment of reporting 14 (53%) patients are alive in complete remission with a median follow up of 12.7 months (1.9-23m). Event-free and overall survival at 1 year for AML are 76% (SE13) and 76% (SE14), all patients with T-ALL and one with ABL died of disease progression. Conclusion We suggest that addition of venetoclax and daratumomab to the backbone of myeloablative haploidentical HSCT with αβ T cell depletion is not associated with increased toxicity and may lead to improved outcome in a cohort of pediatric patients with chemorefractory AML. In a subgroup of patients with T-ALL this approach does not seem to produce visible benefit. This approach can be further tested in a prospective trial with the goal to increase the anti-leukemic efficacy of HSCT. Figure Disclosures Maschan: Miltenyi Biotec: Other: lecture fee. Off Label Disclosure: venetoclax daratumomab plerixafor