ALBERT

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: Background. Translocation t(7;12)(q36;p13) leading to MNX1 (HLXB9)-ETV6 fusion gene formation is recurrent chromosomal rearrangement, predominantly found in children under 24 months of age with acute myeloid leukemia (AML) and associated with dismal outcome. In the majority of cases t(7;12) is cryptic and can be detected by fluorescent in situ hybridization (FISH) only. Purpose. To evaluate the incidence of t(7;12)(q36;p13) and initial clinical parametres and treatment outcome of infant with AML carrying t(7;12) using novel 3-color FISH approach. Methods. 81 infants with de novo AML who was diagnosed between June 1999 and December 2014 were included in the current study. Median age in the observed group of patients was 6.9 months (range 0.3-11.7). Bone marrow morphology was done in all cases flow cytometry in 67 cases. FAB variant was available in 72 patients. Chromosomal banding analysis (CBA) were performed in all cases, while RT-PCR for the most common fusion gene transcripts (MLL rearrangements, RUNX1-RUNX1T1, PML-RARa, CBFB-MYH11, BCR-ABL1) were done in 52 cases, FISH for MLL rearrangements in 49 cases. All MLL-negative cases were screened for t(7;12) by novel 3-color FISH approach consisted of a break-apart probe composed of an orange labeled probe centromeric to the ETV6 gene in 12p13, a green labeled probe telomeric to ETV6 together witha probe combination made of two loci flanking the HLXB9 gene, both labeled in blue. All probes were applied simultaneously and constituted the 3-color probe set used as a novel approach in this study. All probes was synthesized on our demand by MetaSystems (Germany) and applied partly retrospectively, partly -from March 2012 - prospectively to available patients material. Written informed consent was obtained at all cases. Results. Among 81 included patients there were 80 cases of AML and 1 case of Biphenotypic acute leukaemia (BAL). Various 11q23/MLL rearrangements were revealed in 45 cases (55.6%); inv16 in 3 cases (3.7%), including 1 case of simultaneous detection of inv16 and t(7;12); t(1;22)(p13;q13) was found 5 cases (6.2%); complex karyotype in 5 cases (6.2%); t(7;12)(q36;p13) in 6 cases (7.4%); other cytogenetic aberrations and normal karyotype in 9 cases (11.1%) each. Translocations t(8;21) and t(15;17) were not detected. Median age of t(7;12)-positive patients was similar to t(7;12)-negative ones (7.0 vs 6.9 mo). All 6 patients with t(7;12) referred to AML high-risk group. Median WBC count was 29.7*109/L (range 15.7-210). Five of 6 patients had initial CNS disease, in all cases extramedullary organ involvement was observed. Three out of 6 t(7;12)-positive patients had immature FAB variants (one AML M0 and 2 AML M1), 2 patients had AML M5a and 1 BAL. Translocation t(7;12) was associated with immature immunophenotype. CD34-positive/CD117-positive cells were revealed in all 6 cases, that was significantly more common in comparison to t(7;12)-negative cases (p=0.004 and p=0.038, respectively). Interestingly, co-expression of CD7 antigen was detected in 5 out of 6 t(7;12)-positive cases and this phenomenon was observed in t(7;12)-positive group more frequent than in t(7;12)-negative ones (p=0.001). In 5 out of 6 cases t(7;12) was cryptic and was not found by CBA. In 5 patients extra copy of chromosome 19 was revealed, that could serve as predictor of presence of t(7;12). Application of 3-color FISH showed 2 types of breakpoint at chromosome 7: in 5 cases breakpoints localized at 7q36; 1 patient had interstitial deletion 7q11q36 and blue fluorescent signal was observed at 7q11. Hematological remission after first induction course was achieved only in one half of t(7;12)-positive patients. Relapses occurred in 3 patients. Hematopoietic stem cell transplantation (HSCT) was performed in 1st remission in 4 cases and 2nd remission in 1 case. EFS and OS were similar in t(7;12)-positive and t(7;12)-negative groups (0.33±0.16 vs 0.39±0.07 p=0.573 and 0.60±0.21 vs 0.45±0.08 p=0.571) with median of follow-up time 29 months. Conclusions. Our results provide additional clinical, immunological and cytogenetic data of t(7;12)-positive patients. Application of novel 3-color FISH approach revealed t(7;12)(q36;p13) in 7.4% infants with AML. Among MLL-negative patients it was the most common cytogenetic aberration (19.4%). Application of HSCT in both 1st and 2nd remission could overcome unfavorable influence of this translocation and led to appropriate OS. Disclosures No relevant conflicts of interest to declare.

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.