Morphologic Complete Remission Research Articles

Akutna limfoblastna leukemija

Introduction. Acute lymphoblastic leukemia (ALL) is a heterogeneous disease distinguished by clonal replication and piling of immature lymphoid cells in the bone marrow and lymph organs. The etiology is unknown but radiation and some chemical exposure, as well as genetics, might play a role. The disease onset is abrupt. Clinical presentation is characterized by a variety of general symptoms: fatigue, malaise, night sweats, weight loss, fever. The diagnosis is based on a patient's history, physical examination, blood tests, bone marrow biopsy, cytogenetic, and immunohistochemical tests. Core treatments are poly-chemotherapy, radiation therapy, hematopoietic stem cell transplantation. Case report. We presented a 20-year-old patient. On his first visit, he complained of neck pain. He was treated by a physiotherapist. After he finished with physical therapy we noticed pancytopenia in his lab work. His general practitioner (GP) referred him to a hematologist where further medical examinations were performed and ALL diagnosis was made. The treatment started with a chemotherapy regimen. An allogeneic hematopoietic stem cell transplantation was performed afterward. In the follow-up, regular haematologic assessments were done, as well as, the assessment of MRD (Minimal Residual Disease). Complete morphologic remission was maintained the whole time but with positive MRD findings. He received an antiCD22 monoclonal antibody therapy and the therapeutic response was good. Eight months later B-ALL relapse was confirmed. The patient's general condition got worse and in spite of an intensive therapy the patient died. Conclusion. Acute lymphoblastic leukemia is a disease whose outcome depends on many a factor. Due to atypical symptoms, it should be taken into consideration to shorten the time to diagnosis, provide timely treatment and thus influence the disease outcome.

Transplant in AML with measurable residual disease: proceed or defer?

Allogeneic stem cell transplantation plays a central role in the management of fit adults with high-risk acute myeloid leukemia (AML) in first complete morphologic remission (CR1). Advances in both donor selection and transplant technology have both dramatically increased accessibility of transplant and led to significant reductions in transplant-related mortality over the past 2 decades. There has, however, been no concomitant reduction in the risk of disease relapse, which remains the major cause of transplant failure. Pivotal to the design of innovative strategies with the potential to reduce relapse risk is accurate identification of patients at the highest risk of disease recurrence. Multiple retrospective studies have identified an increased risk of disease relapse in patients allografted for AML in CR1 with evidence of pretransplant measurable residual disease (MRD). The prognostic significance of pretransplant MRD has been confirmed recently in prospective analyses. The optimal management of patients with evidence of pretransplant MRD remains a matter of conjecture with regard to 2 key issues. First, should the presence of pretransplant MRD delay a decision to proceed to transplant, allowing time for delivery of additional MRD-directed therapy prior to transplant? Second, to what extent can the intensity of the conditioning regimen or the magnitude of the graft-vs-leukemia effect be manipulated to improve the outcome of such patients?

Long-Term Data Confirm the Superiority of Total Body Irradiation-Containing Conditioning Regimen in Comparison to a Chemotherapy-Based Preparation in Children with Acute Lymphoblastic Leukemia Above the Age of 4 Years Given an Unmanipulated Allograft. Results of the Forum Randomized Clinical Trial

Background Conditioning regimen plays a crucial role in determining the outcomes of patients with hematological malignancy treated with allogeneic hematopoietic stem cell transplantation (HSCT). In a multicenter, international, randomized, phase 3 clinical Trial (FORUM study), we documented (Peters C, et al. J Clin Oncol 2021) that children with acute lymphoblastic leukemia (ALL) given total body irradiation (TBI) in combination with etoposide had a superior probability of 2-year overall (OS) and event-free survival (EFS) in comparison to patients given either one of two myeloablative chemo-conditioning regimens. In this report, we assessed whether the benefit deriving from the use of TBI was sustained over time. Patients and methods Patients <18 years at diagnosis, 4-21 years at HSCT, in complete morphological remission (CR) pre-HSCT, and with an HLA-compatible related or unrelated donor (UD) were randomly assigned 1:1 to a myeloablative conditioning regimen with fractionated TBI and etoposide or fludarabine, thiotepa, and either busulfan (BU) or treosulfan (TREO). Children transplanted from a matched sibling donor (MSD) received cyclosporine A only as graft-versus-host disease (GvHD) prophylaxis, whereas recipients of UD HSCT also received short-term methotrexate and anti-thymocyte globulin (ATG). Further details on transplant procedure have been described elsewhere (Peters C, et al. J Clin Oncol 2021). Results Between 04/2013 and 12/2018, 413 patients were randomly assigned to receive either TBI (212) or a chemotherapy-based conditioning (201). Compliance with random assignment was 92%. Sixty-five percent of patients were male, 72% had B-cell precursor ALL, 73% were transplanted from a UD, and in 82% of patients bone marrow was the stem cell source employed. Fifty-four percent of patients were in CR1 at time of allograft, while 40% and 4% were transplanted in CR2 and CR3, respectively. Patient's outcomes were updated on February 1st, 2022. With a median follow-up of 3.7 years (range, 0.3-7.9), the 3-year probability of OS of patients allocated to TBI/etoposide or to the chemotherapy-based conditioning regimens was 90±2% and 71±3% (p<0.001), respectively, while that of 3-year EFS was 81±3% and 59±4% (p<0.001). OS and EFS remain comparable in patients receiving BU or TREO in combination with fludarabine and thiotepa. In detail, OS and EFS of the 102 patients given BU, fludarabine and thiotepa were 73±5% and 69±5% respectively, while those of the 99 patients treated with TREO, fludarabine and thiotepa were 62±5% and 61±5%. Multivariate analysis confirmed that the use of chemotherapy-based regimens correlated with worse OS [Hazard Ratio, HR, 2.63, 95% confidence interval, CI, (1.61- 4.31), p<0.001] and EFS [HR 2.30, 95%CI (1.58- 3.35) p<0.001]. Transplantation in >CR1 predicted a worse EFS in multivariate analysis, as well [HR 1.67, 95% CI (1.14- 2.45) p=0.009]. Patients given TBI benefited from a lower cumulative incidence of relapse (Figure 1A), as well as from a lower 3-year cumulative incidence of non-relapse mortality (2+1% vs. 10+2% in the chemo arm, p=0.007). The 3-year cumulative incidence of chronic GvHD was comparable between the 2 arms (15+3% vs. 12+2% in the TBI and chemo arm, respectively, p=n.s.). We also analyzed the probability of GvHD/relapse-free survival (GRFS) considering both relapse and chronic GvHD as events; children prepared with TBI had a better 3-year GRFS probability (Figure 1B) as compared to chemo-prepared patients. Five patients (all transplanted from an UD) developed secondary malignancies: 4 of them had received TBI. Conclusions With a significantly longer follow-up, we confirm that the use of TBI during the conditioning regimen is associated with better outcomes in children and adolescents with ALL above the age of 4 years transplanted in CR, mainly due to a lower risk of leukemia recurrence. The number of cases of secondary malignancies, particularly in patients offered radiotherapy, deserves particular attention and underlines the importance of continuous safety monitoring over time to comprehensively evaluate the long-term sequels observed in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relapse Is the Most Common Treatment Failure Post HSCT in Children with ALL below 4 Years of Age Given a Chemo-Based Conditioning Regimen. Results from the Prospective Multinational Forum-Trial

Background: Relapse and non-relapse mortality (NRM) are the major causes of treatment failure in infants and young children with high-risk ALL undergoing HSCT. Conditioning regimens for this high-risk population usually omit total body irradiation (TBI) to avoid acute and long-term side effects that are more pronounced in this age group. The optimal chemotherapeutic approach aiming improved event-free survival (EFS) and low NRM is not yet defined. Methods: The prospective FORUM trial used two myeloablative chemo-conditioning regimens for children <4 years (yrs) of age: fludarabine (FLU), thiotepa (THIO) and either intravenous busulfan (BU) or treosulfan (TREO) as described previously (Peters et al., JCO 2021). This report includes 194/202 patients (pts) (107 male, 87 female) from 26 countries enrolled in FORUM who received HSCT (5 pts received TBI-based conditioning and 3 pts with unknown conditioning were excluded). Median age at HSCT was 2.2 yrs (6 months - 4 yrs; 38 pts 0-1yrs (20%), 53 pts 1-2yrs (27%), 53 pts 2-3yrs (27%), 50 pts 3-4yrs (26%)). Stem cell source was bone marrow in 132 (68%) pts, peripheral blood stem cells in 37 (19%), and cord blood in 24 (13%). Donors were HLA-matched siblings (MSD) in 39 (20%) pts and 9 or 10/10 HLA allele-matched unrelated donors (MUD) in 155 (80%) pts.Clonal genetic abnormalities reported at diagnosis included 9 pts with BCR-ABL, 13 pts with TEL-AML, and 53 pts with KMT2A r-rearrangements; all pts underwent HSCT in complete morphological remission (CR) (142 pts CR1; 50 pts CR2, 2 pts CR3). FLU/THIO/BU and FLU/THIO/TREO were used in 101, and 93 HSCTs, respectively. Graft-versus-host-disease (GvHD)-prophylaxis was Cyclosporin-A-based in 90%; recipients of MUD-grafts also received ATG and 3 doses of methotrexate. At data cut-off, median follow-up (FU) was 3 yrs (range, 3 months - 7.2 yrs). Results: 3-yrs OS was 0.69±0.04, 3-yrs EFS was 0.52±0.04; 3-yrs Cumulative incidence of relapse (CIR) was 0.44+0.04, and 3-yrs NRM was 0.05 ± 0.02. OS and EFS did not differ between pts < 2 and 2-4 yrs of age at HSCT (Table 1). Pts transplanted in CR 1 had significantly better EFS (0.58±0.04) as compared to >CR1pts (0.36±0.07, p=0.01); due to a higher CIR. OS was worse for the 53 pts with KMT2A-rearrangements (3-yrs OS 0.56±0.08 vs. 0.73±0.04, p=0.040). EFS for pts < 1 yr of age at diagnosis was significantly inferior as compared to older pts (3-yrs EFS 0.40±0.05 vs. 0.63±0.05, p=0.002). OS/EFS were not significantly different when donor type, stem cell source, and immunophenotype were compared (Table 1). At 3-yrs, OS was 0.63±0.05 and 0.76±0.05 and EFS was 0.52±0.05 and 0.51±0.06 (p=0.794) for the FLU/THIO/BU and FLU/THIO/TREO-group, respectively (p=0.075) with no significant difference in CIR and TRM. However, pts who relapsed post-HSCT had a better 3-yr post-relapse OS after FLU/THIO/TREO compared to FLU/THIO/BU (0.38±0.11; 0.16±0.07. p = 0.012) respectively. Most relapses occurred prior to 1 yr after HSCT. Acute GVHD occurred in 32 pts (20 pts experienced grade III/IV) with no significant difference between the two conditioning regimen). The 3-yr incidence of chronic GVHD (cGVHD) was comparable between the BU- and -TREO-group (p = 0.943). cGVHD/relapse-free survival at 3 yrs for FLU/THIO/BU was 0.44±0.05 and for FLU/THIO/TREO 0.41±0.06 (p=0.943). In multivariate analysis, remission > CR1 and the presence of KMT2A, negatively influence OS while EFS was worse for patients who underwent HSCT > CR1 or had ALL-diagnosis below 1 yr of age. Donor type, stem cell source, age between 1 and 4 yrs at HSCT, conditioning regimen, and PCR-MRD positivity pre-HSCT did not significantly influence outcome. 1-yr TRM was low for both conditioning regimens (0.06±0.02 for FLU/THIO/BU and 0.03±0.02 after FLU/THIO/TREO (n.s.) Conclusion: Within the FORUM-trial, infants and young children receiving HSCT after conditioning with either BU- or TREO containing regimens have a lower OS and EFS as compared to children above the age of 4 yrs due to a higher CIR. Because of decreased NRM, these results represent an improvement as compared to previously reported series; however, KMT2A-rearrangement continues to be an obstacle to successful HSCT. Pre- and post-transplant immunotherapeutic strategies aimed at reducing relapse-incidence without increasing chemotherapy-associated complications are needed. Figure 1: 3-yrs OS according to Conditioning Flu: fludarabine, Thio: thiotepa, Treo: treosulfan, Bu: busulfan Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Influence of Conditioning Regimen Intensity on Outcomes Post-Allogeneic Transplantation for AML in Complete Morphological Remission

Introduction: Acute myelogenous leukemia (AML) is a major indication for allogeneic hematopoietic cell transplantation (alloHCT). Myeloablative (MAC) regimens are preferred for their intensity, however, reduced intensity conditioning regimens (RIC) have been increasingly used to permit older patients and patients with comorbidities to undergo transplant. The literature is conflicting regarding the comparison of outcomes between RIC and MAC patients. While MAC regimens are associated with less relapse, non-relapse mortality (NRM) is higher. RIC regimens on the other hand, demonstrate less NRM but higher relapse rates. We sought to compare RIC vs MAC regimens at our centre for alloHCT of AML in complete remission (CR). Methods: We retrospectively analyzed the outcomes of 451 patients that underwent alloHCT for AML at the Princess Margaret Cancer Centre (PMCC) with either RIC or MAC using various donors between the years 2015-2020. Primary endpoint was 2-year overall survival (OS), secondary endpoints included cumulative incidence of relapse (CIR), cumulative NRM, and GVHD free/relapse free survival (GRFS). Conditioning regimen intensity was determined as defined by Bacigalupo et al (BBMT 2009). Of the total cohort 331 patients received RIC and 121 MAC. Among MAC regimens, 59% received FLU(4)Bu(4) (Fludarabine 140mg/m2 + Busulfan 12.8mg/m2). Among RIC patients 71% received Flu(4)Bu(2)TBI(200) (Fludarabine 140mg/m2+ Busulfan 6.4 mg/m2 + TBI 200cGy). Results: Median age at transplant was 48 years (range: 19-67) for MAC recipients and 61 (range: 18-75) for RIC recipients (p<0.001). Other patient characteristics are described in Table 1. For the entire cohort at 2 years, the incidence of NRM was 19.1% for MAC (95% CI: 12.4-26.8) and 22.5% for RIC (95% CI: 18.1-27.1) (p=0.44). Two-year CIR was 19.8% (95% CI: 12.9-27.8) for MAC recipients and 24.5% (95% CI: 20.0-29.4) for RIC recipients (p=0.15). Two-year OS was 61% and 53% for MAC and RIC, respectively (p=0.02). Acute GVHD occurred in 58.3% of MAC recipients versus 46.8% of RIC recipients. Chronic GVHD occurred in 43.3% of the MAC recipients versus 38% of the RIC recipients. At two years GRFS was 40.8% (95% CI: 35.4-46.1) for MAC and 33.7% (95% CI: 25.0-42.6) for RIC, p=0.30. When examining the Disease Risk Index (DRI) for the entire cohort, OS for high risk was 43.3% (95% CI: 32.5-53.5) vs 66.6% (95% CI: 61-71) and 64% (95% CI: 38-80) for intermediate risk and low risk respectively, p<0.001. A propensity score (PS) matched analysis was done matching patients for age, HLA matching (mismatched vs full match), DRI (low, intermediate, high risk) and in vivo T cell depletion (yes vs no). Two-year OS was 67% for the MAC recipients (95% CI: 57-76) and 66% for the RIC recipients (95% CI: 56-75), p=0.95 (Figure 1a). NRM for MAC was 22% (95% CI: 14-30) versus 14% (95% CI: 8-22) for RIC, p=0.19. CIR at 2 years was 18.5% for MAC (95% CI 11-27) and 24.3% for RIC (95% CI: 16-33), p=0.22. GRFS at 2 years was 36% (95% CI: 26.4-45.7) for MAC and 48.3% (95% CI: 38-58) for RIC recipients, p=0.17. In the subgroup analysis examining matched related donor (MRD) recipients in particular, the OS at 2 years was for MAC 83.6% (95% CI: 66.8-91.3) vs 57.3% (95% CI: 45.1-67.7) for RIC, p=0.006 (Figure 1b) Two-year NRM was 10.3% for MAC vs 23.4% for the RIC group, p=0.14. CIR at 2 years was 12.2% for MAC vs 23.4% for RIC in the MRD setting, p=0.11. Conclusions: Our study shows that in the MRD setting there is a statistically significant improved survival, especially in patients with low and intermediate DRI scores. In other donor settings, there is no significant difference in outcomes between patients that undergo alloHCT with MAC vs RIC regimens. MAC conditioning regimens should continue to be the preferred choice of intensity for patients that may tolerate it. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative Impact of Mutated NPM1 MRD in Patients with AML Undergoing Allogeneic Stem Cell Transplantation

Introduction: In NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) the detection of measurable residual disease (MRD) using quantitative PCR methods for NPM1mut allows for risk stratification and therapy adjustments during disease course. Even though it is well understood that a persistent NPM1mut burden at allogeneic stem cell transplantation (HSCT) is associated with relapse and shorter survival, the quantitative impact of NPM1mut at HSCT has not been investigated in detail. Methods: We analyzed 81 NPM1mut AML patients (pts) who received their first allogeneic peripheral blood HSCT at a median age of 61 (range 33-76) years in morphologic complete remission (CR) or CR with incomplete peripheral recovery (CRi). Conditioning regimens were non-myeloablative (70%), reduced-intensity (11%), or myeloablative (19%) according to EBMT guidelines. At diagnosis, cytogenetics and co-mutational status of FLT3, IDH1, IDH2, DNMT3A, and CEBPA were assessed. NPM1 MRD up to 28 days prior to HSCT in CR/CRi was measured by digital droplet PCR using a competitive probe approach, and results were normalized to ABL1 (limit of detection 0.001%NPM1mut/ABL1). For patients with a positive NPM1 MRD test result, receiver operator characteristics (ROC) curves were used to analyze distinct NPM1/ABL1 MRD threshold. Presence of chronic graft-versus-host disease (GvHD) was assessed according to Glucksberg criteria. Median follow up after HSCT was 4.4 years. Results: ROC curves identified 0.01% NPM1mut/ABL1 as the most informative cut-off to define MRDpos and MRDnegpts in both, bone marrow and blood, and irrespective of conditioning intensity. Using this cut-off 31 (38%) pts were NPM1mut MRDpos at HSCT and had a significantly higher cumulative incidence of relapse (CIR, P<.001, after 2 years 4% vs 66%) and shorter overall survival (OS, P<.001, after 2 years 77% vs 38%). Patients transplanted NPM1mut MRDpos had a 20.8-times higher risk of relapse than those transplanted MRDneg. While there were no differences regarding the gene mutation profile at diagnosis in pts with or without NPM1mut MRD at HSCT (Figure 1A), pts with NPM1mut MRD at HSCT had a significantly higher diagnostic WBC (49.6 vs 17.9; P=.03), LDH (12.9 vs 6.7; P=.02), and more CD34+ cells in bone marrow at diagnosis (1.4% vs 0.5%; P=.02). We observed a negative correlation of the NPM1mut /ABL1 MRD levels before HSCT and the time to relapse; the higher the NPM1mut MRD burden at HSCT the earlier the relapse occurred (R= -0.5, P=.03). This correlation was strongest in pts with a FLT3-ITD present at diagnosis (R= -0.82, P=.002). When utilizing two cut-offs for low (≥ 0.01% but < 1%) and high (≥ 1%) NPM1mut /ABL1 MRD levels there was a significantly shorter time to relapse in relapsing AML pts with high NPM1mut MRD levels (median time to relapse: 73 days) before HSCT compared to pts with low MRD levels (median time to relapse: 149 days). In addition, compared to patients with low NPM1mut MRD levels, pts with high NPM1mut MRD levels showed a trend for shorter OS (P=.10; OS after 2 years: 77% for NPM1mut MRDnegpatients vs 48% for low NPM1mut MRDpos patients vs 21% for high NPM1mut MRDpos patients; Figure 1B), indicating that pts transplanted with higher NPM1mut MRD may be harder to salvage. Presence of a FLT3-ITD and age at HSCT did not alter the negative impact on outcomes of low or high NPM1mut MRD at HSCT. We also evaluated the prognostic impact of the presence of chronic GvHD as a surrogate marker for graft-versus-leukemia effects with respect to the NPM1mut MRD status at HSCT. In a landmark analysis for pts surviving longer than 100 days, NPM1 MRDneg (< 0.01% NPM1mut/ABL1) pts had the lowest CIR (4% after 2 years), followed by NPM1mut MRDpos pts developing a chronic GvHD (25% after 2 years) and by NPM1 MRDpos pts who did not develop a chronic GvHD and had a very high CIR (92% after 2 years, P<.001). Conclusion: AML patients with > 0.01% NPM1mut/ABL1 MRD before allogeneic HSCT had a significant higher risk of relapse and shorter OS. High WBC, LDH, and CD34+ cells in bone marrow at diagnosis associated with persisting NPM1mut MRD at HSCT. The higher the NPM1mut MRD burden at HSCT, the earlier the relapse, and the lower the chances of successful salvage therapy after relapse. Development of a chronic GvHD after HSCT was associated with a reduced risk of relapse in NPM1mut MRDpos pts, indicating that bolstering immunologic effects post-HSCT for NPM1mut MRDpos AML pts may be a promising option to improve outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relapse Prophylaxis Post-Haploidentical Bone Marrow Transplantation and Cyclophosphamide (Haplo/Cy) By Infusion of Donor-Derived Expanded/Activated Gd T Cells: A Phase I Trial

Background: Effector γδ T cells immediately recognize and kill malignant cells in a broad-based non-MHC restricted manner. Increases in circulating donor-derived γδ T cells during post-bone marrow transplant (BMT) recovery have been significantly associated with improved disease-free survival (DFS). Relapse post Haplo/Cy BMT occurs in approximately 45% of patients. We sought to mitigate relapse in this context by expanding, activating, and infusing donor-derived haploidentical γδ T cells. We now report preliminary clinical and biologic correlative findings from the first cohort of patients who have been treated with ex vivo expanded and activated donor γδ T cells (EAGD). This single-center Phase I clinical trial represents the first systemic infusion of allogeneic EAGD cells in the post-BMT setting Methods: Standard of care reduced-intensity flu/cy/TBI conditioning was followed by an unmanipulated bone marrow graft and 50mg/m2 Cy on days +3 and +4 post-transplant. EAGD were manufactured using the Miltenyi Prodigy bioreactor and cryopreserved. The product was infused intravenously within 5 days of neutrophil engraftment (ANC >500/µL X 3d). Peripheral blood was collected at EAGD infusion and monthly thereafter through day +90, with additional collections every 6 months through 1 year. Biologic parameters included multiparameter flow cytometric immunophenotyping and single cell cytokine analysis of the EAGD graft. Peripheral blood analysis includes leukocyte count and differential, immunophenotyping, and serum Th1/Th2/Th17 cytokine analysis. Primary endpoints include dose-limiting toxicities (DLT) and grade 3-4 adverse events while secondary endpoints include incidence of acute and chronic GvHD, relapse, and overall survival. Results: Three patients have received the first dose level of 1 x 106 EAGD/kg. All three patients remain in morphologic complete remission at 26.5, 24.2, and 12.5 months post-BMT. One patient is receiving ongoing hypomethylating therapy for the occurrence of recipient chimerism. Grade 1-2 toxicities include constipation, CMV reactivation, emesis, fatigue, and hypomagnesaemia. Steroid-responsive cutaneous acute Grade I-II GVHD has been observed in all patients with one patient experiencing Grade II intestinal GvHD. No chronic GVHD, DLTs, treatment-related ≥ grade 3 adverse events, or cytokine release syndrome has occurred. EAGD grafts contained 88.7% - 99.2% Vγ9Vδ2+ γδ T cells with small populations of NK cells and <1.0 x 10 αβ T cells/kg. EAGD principally expressed Granzyme B, MIP1α, MIP1β, and IL-2. Significant peripheral lymphodepletion persisted through the first 100 days post-BMT followed by slow recovery of CD4+, CD8+, γδ T and B cells. NK cells remained within the low normal range throughout. T cells transitioned from a CD45+CD27- effector phenotype to CD45RA CD27 central to effector memory phenotype as recovery progressed. CD3 CD4 CD25 FoxP3 Treg cells remained <3% of circulating T cells. Preliminary serum cytokine and chemokine analysis revealed predominant expression of IFNγ, IL-12p70, IP-10, RANTES and TNFα. Conclusions: Early indications suggest that EAGD transfusion with the initial dose level of 1 x 106 EAGD/kg has manageable toxicity and an appropriate immune recovery profile with 3 of 3 patients alive and progression-free. Clinical Trial Registry: https://clinicaltrials.gov/ct2/show/NCT03533816

Full Spectrum 40-Parameter Single-Tube Flow Cytometry Analysis for Longitudinal Immunophenotyping and Tracing of Clonal Heterogeneity in AML

Acute Myeloid Leukemia (AML) remains notoriously difficult to treat. AMLs manifest themselves within individual patients with remarkable heterogeneity. Heterogeneity can be observed within the tumor itself, whereby multiple genetically distinct subclones can co-exist. We have shown that such differences in genetics translate into differences at the cell biological level as well (epigenome, transcriptome, proteome, metabolome and drug sensitivities) (de Boer et al. 2018) (Erdem et al. 2022). More recently, we began to dissect the heterogeneity within the tumor immune microenvironment. For instance, we identified a subgroup of AML patients that harbor a large population of tumor supportive M2 polarized macrophages in the bone marrow and these patients have the poorest prognosis. Reversely, patients with large populations of tumor suppressive M1 polarized macrophages display a much better prognosis. We also uncovered that patients with high levels of CD4+ T cells display a relatively favorable outcome. Little is known about clonal dynamics and microenvironmental changes over time or as a consequence of treatment. With the rise of new mutation-specific treatments targeting specific genetic subclones, it is important to examine the clonal composition of residual cells, especially at Measurable Residual Disease (MRD) timepoints. This can pose challenges as patients in morphologic complete remission have relatively low blast counts and limited material is available. Full spectrum flow cytometry is a novel technique that allows the use of 40 fluorescent markers in a single panel, enabling extensive immunophenotyping requiring relatively low cell numbers. We set up two antibody panels, one for immunophenotyping based on the OMIP-069 panel (39 colors) (Park et al. 2020) and one for the detection of subclones and macrophages (37 colors). We ran both panels on AML bone marrow samples at diagnosis (n=20), after initial induction therapy (n=4) and on normal bone marrow (NBM) (n=5). Full spectrum flow cytometry showed similar results to conventional flow cytometry, although with a higher resolution. With both panels combined we could identify and quantify 105 distinct cell types. UMAP landscapes were created as a template by merging AML samples and NBM samples, on top of which activation markers and aberrant AML-specific plasma membrane (PM) protein expression profiles could be projected. Additionally, density plots of individual patient samples were generated to compare longitudinal alterations in clonal dynamics and the tumor immune microenvironment before and after treatment. Additional univariate and multivariate survival analyses were performed to link observed changes to disease progression. At diagnosis, considerable heterogeneity amongst individual patients was observed, as expected. Patients either had a tumor supportive M2 macrophage phenotype or a tumor suppressive M1 macrophage phenotype. The lymphoid compartment was overall strongly reduced in AML compared to NBM, but was highly heterogeneous between individual patients whereby the strongest heterogeneity was noted in CD4+ and TCR gamma delta (TCRγδ) T cell subsets. Low levels of either CD4+ T cells and TCRγδ showed trends towards poorer prognosis. After induction therapy, the immune landscapes recovered and started to resemble those of NBM, most notably for T cell subsets but less so for B cells that took longer to recover. AMLs with an M2 phenotype at diagnosis shifted towards an M1 phenotype and non-classical monocytes shifted to more classical monocytes. In general, AML-specific PM markers were lost upon treatment reaching complete remission, although in some cases markers remained detectable within CD45+ blast populations. One patient containing NPMcyt (VAF=0.4) and FLT3-ITD (VAF=0.04) mutations was treated with gilteritinib in addition to cytarabine, reached morphologic complete remission whereby the FLT3-ITD mutation was lost, and the immune landscape restored. However, NPM1 droplet PCR analyses revealed the presence of residual mutant cells, which corresponded with residual cell numbers expressing aberrant CKIT/IL1RAP expression. Whether these cells will give rise to relapsed disease is currently under close investigation. We conclude that full spectrum flow cytometry is a powerful approach that allows extensive longitudinal immunophenotyping and subclone detection requiring low cell numbers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Analysis on the Outcomes of Children with Acute Leukemia (AL) Receiving an Alpha/Beta T and B-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (TBdepl-haploHSCT)

Background: We previously reported (Locatelli et al., Blood 2017) that TBdepl-haploHSCT is a suitable and effective option for children with AL lacking an HLA-compatible donor and/or needing an urgent allograft. This type of transplant is characterized by a low incidence of both acute and chronic graft-versus-host disease (GvHD) and by low non-relapse mortality (NRM), translating into survival outcomes comparable to those of patients transplanted from an HLA-compatible donor. Here we report an update on a cohort of 213 children with AL of our study, with a minimum follow-up of 180 days after the allograft. Patients and methods: Between October 2010 and January 2022, 213 children with AL received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. All patients were in morphological complete remission (CR) and were prepared to TBdepl-haploHSCT using a fully-myeloablative conditioning regimen including a combination of total body irradiation (TBI) and/or cytotoxic drugs. All patients received also anti-T-lymphocyte globulin (ATLG) before transplantation (12 mg/kg total dose, from days -5 to day -3) to modulate bi-directional donor/recipient alloreactivity and rituximab (200 mg/sqm) on day -1 to prevent post-transplantation EBV-induced lymphoproliferative disorders (PTLD). No patient received any post-transplant pharmacological GvHD prophylaxis. Data were analyzed as of July 15, 2022. Results: Table 1 reports details on characteristics of patients included in the analysis. The median follow-up of surviving patients is 47.6 months (range: 6 months - 11.7 years). Two hundred and eight patients (98% of the total number) engrafted with a median time to neutrophil and platelet recovery of 13 (range 9-24) and 11 (range 7-23) days, respectively. Only 5 patients experienced graft failure; all were rescued with a second allograft. Eleven out of the 213 patients (5%) died of transplant-related complications (4 because of idiopathic pneumonia, 2 each because of disseminated adenovirus infection and thrombotic microangiopathy and 1 each of cardiac insufficiency, combined CMV/rhinovirus pneumonia and sepsis from Pseudomonas aeruginosa), the 5-year cumulative incidence of NRM being 5.2% (95% CI, 2.8-8.8). Forty-four patients relapsed at a median time of 192 days (range 56-1711) after HSCT, the 5-year cumulative incidence of relapse being 22.7% (95% CI, 16.9-29.2). Cumulative incidence of grade II-III acute GvHD was 14.7% (95% CI 10.6-20.3). Three patients and 1 developed gut and liver GvHD, respectively, while for all other patients skin was the sole organ involved. Fourteen out of the 193 patients at risk developed chronic GvHD, in all cases of limited severity, the cumulative incidence of this complication being 8.1% (95% CI 4.8-13.3). The 10-year probability of overall and leukemia-free survival (LFS) were 75.4 (95% CI 68.6-80.9) and 71.6% (95% CI 64.4-77.6), respectively. In univariate analysis, disease status at transplantation (CR1 vs CR2 vs CR>3), use of TBI during the preparative regimen and age at transplant above the median value (9.5 years) were associated with better patient's outcome, mainly because of a reduced incidence of relapse. Notably, among TBI-based regimens, the use of thiotepa and fludarabine was associated with an improved DFS as compared to other combinations (82.4% vs 63.9%, p=0.01). In a multivariable model for LFS including also gender and type of disease, only TBI (hazard ratio (HR) 0.38 (95% CI, 0.17-0.85, p=0.01) and disease status at HSCT (HR 1.72 (95% CI, 1.05-2.79, p=0.02) remained statistically significant. The 10-year GvHD/relapse-free survival was 65.1% (95% CI 57.7-71.4). The median CD3+ cell count on day +30, +90, +180 and +360 were 140, 185, 533 and 1058 /mcl, respectively. Conclusions: TBdepl-haploHSCT is becoming a widespread haplo platform for pediatric patients affected by both malignant and non-malignant disorders. This cohort of homogeneously treated patients, the largest reported so far with a median follow-up of almost 4 years, confirm once more the effectiveness and safety of this approach. Furthermore, this analysis identified subgroups of patients (e.g., those in CR>3) for whom post-transplant strategies aimed at reducing the risk of relapse (the main cause of treatment failure) are desirable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fast-Track Measurable Residual Disease Detection By Multiparametric Flow Cytometry in Acute Myeloid Leukemia before Allogeneic Hematopoietic Cell Transplantation

Introduction In acute myeloid leukemia (AML), measurable residual disease (MRD) becomes more and more important for individualized treatment decisions. The MRD status at the time of allogeneic hematopoietic cell transplantation (alloHCT) provides prognostic information beyond baseline risk features. Peri-transplant treatment strategies and conditioning intensity can be stratified according MRD-levels. However, therapeutic interventions (e.g. conditioning intensity) usually require the prompt availability of reliable information on the MRD status. We assessed the prognostic value of a standardized, reproducible, and fast turnover multiparametric flow cytometry (MFC) approach that has been validated at the end of intensive induction therapy to monitor MRD before alloHCT (Röhnert, Leukemia 2022). This approach allows for the simultaneous detection of MRD by the leukemia-associated immunophenotype (LAIP) and the different from normal (DfN) method. Methods Between October 2016 and March 2020, bone marrow (BM) aspirates of patients (pts) with AML were analyzed for the presence of MRD immediately before the start of conditioning. therapy with the aim of analyzing to predictive value of this fast-track parameter. Pts receiving sequential regimens (re-induction chemotherapy followed by reduced-intensity conditioning) were excluded. Briefly, the LAIP-based DfN analysis is based on a hierarchical gating strategy with fixed gates and encompasses the antigens CD34, CD117, HLA-DR (backbone markers), CD13, CD33 (myeloid markers), CD7 and CD56 (lymphoid markers). Within less than 5 minutes, the analysis checks for the presence of the aberrant categories i) deficiency of myeloid markers and ii) cross-lineage expression of lymphoid markers. Reference values for 32 immunophenotypically defined populations were established on BM aspirates of 90 leukemia free controls. An aberrant category already detectable at diagnosis on ≥10% of myeloid progenitors/ monocytes was termed aberrant LAIP (aLAIP) and a de-novo appearance (after chemotherapy) post-treatment DfN (ptDfN). In conclusion this approach is very fast and provides high reproducibility. Results We identified 58 pts with a median follow-up of 33.3 months after alloHCT (Table 1 with pts characteristics). Response assessment was performed in median 8 days before alloHCT. Most of the patients were in morphologic complete remission (CR n=37). A subset of 21 patients had refractory disease with a median BM blast count of 6.5%. Samples at AML diagnosis were available for 48/58 (83%) pts, of whom 34/48 (71%) showed at least one aLAIP at initial diagnosis. Before start of conditioning, 44/58 (76%) patients were MRDpos. The proportion of MRDpos in responders was lower compared to non-responders: 24/37 (65%) vs. 20/21 (95%) (p=0.0106). MRDpos was designated by aLAIPonly, ptDfNonly and aLAIP+ptDfN in 18%, 48% and 34% of cases, respectively. Noteworthy, 8 of 21 pts classified MRDpos by ptDfNonly had no sample available at diagnosis and therefore could not be assessed for aLAIP persistence. Amongst 37 pts in CR before alloHCT, 12/24 (50%) of MRDpos pts relapsed compared to 3/13 (23%) of MRDneg pts. TRM in these patients did not differ (13% vs. 8%). In a univariable Cox regression analysis, the 1-year Overall survival (OS) rate of MRDpos cohort was significantly shorter compared to MRDneg pts (64% vs. 93%, Hazard ratio (HR) 10.5, p=0.004). MRD was detected by ptDfNonly in 23/44 (52%) of MRDpos pts. The OS of these pts was comparable to MRDpos by aLAIP with or without ptDfN (Figure 1). As expected, the MRDpos cohort was enriched for non-responders. However, when the analysis was restricted to pts in CR (n=37), MRD status preserved its prognostic significance (HR 7.9, p=0.019). And even in the subpopulation of pts in first CR, MRDpos was associated with a significantly reduced OS compared to MRDneg (HR 6.3, p=0.043). Interestingly, a single non-responder was classified as MRDneg and showed a long-term OS. Conclusions Our fast-track LAIP-based DfN approach provides relevant prognostic information prior to conditioning for allo HCT. On top of the classical LAIP approach, the ptDfN analysis gives additional prognostic information. The LAIP-based DfN approach might be a suitable tool for fast-track and reliable MRD assessment before starting the preparative regimen for alloHCT and might help to guide potential treatment modifications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of Dexamethasone Versus Prednisolone Prephase in Induction Therapy of Pediatric Acute Lymphoblastic Leukemia: A Single-Center, Randomized Controlled Study from India

Background: Acute lymphoblastic leukemia (ALL) is the commonest cancer in children. Despite advances in treatment, approximately 20% of pediatric ALL patients still relapse. Although the use of dexamethasone in induction regimen has been reported to significantly decrease CNS relapse risk & improve event-free survival (EFS) compared to prednisolone, there are concerns of significant toxicities like infection & osteonecrosis with dexamethasone. Objectives: The aim of this single-center randomized study was to compare the efficacy and safety of dexamethasone prephase versus prednisolone prephase during the initial seven days (days 1 - 7) of induction chemotherapy in newly diagnosed pediatric ALL patients treated with ALL IC-BFM 2009 protocol,in terms of day 8 steroid response, day 15 bone marrow minimal residual disease (MRD) response, and day 33 morphological complete remission (CR) rates. Methods: The study enrolled total 62 newly diagnosed pediatric ALL patients (age > 1 year & ≤ 18 years) between June 2019 and April 2021, after obtaining informed consent and with institutional ethics committee approval. Patients with lymphoblastic lymphoma (LBL) and infantile ALL (age < 1 year) were excluded. Patients who were randomized to dexamethasone prephase group received intravenous dexamethasone 10 mg/m2/day on days 1-7 of induction,& those in prednisolone prephase group received prednisolone 60 mg/m2/day p.o. in three divided doses on days 1-7 of induction therapy .All the patients subsequently received prednisolone 60 mg/m2/day p.o. in three divided doses on days 8 - 28 of induction, followed by prednisolone taper over next 7 days. Induction regimen of ALL IC-BFM 2009 protocol was given. The cut-off values for day 15 BM Flow-MRD were <0.1%, 0.1-10%, and >10% as per ALL IC-BFM 2009 protocol. Morphological complete remission on day 33 was defined as bone marrow blasts < 5% of total nucleated cells, with normocellular or mildly hypocellular marrow. Steroid-related toxicities in the two treatment groups were compared as per NCI-CTCAE version 5. Results: Out of total 62 patients, 33 patients (53%) received dexamethasone prephase and 29 patients (47%) received prednisolone prephase. Forty-six patients (75%) had B-cell ALL, and 25% had T-cell ALL. The distribution of patients in standard-risk, intermediate-risk & high-risk groups as per ALL IC-BFM 2009 protocol risk stratification were 18%, 34% and 48% respectively. Twenty-nine out of 33 patients (88%) in dexamethasone prephase group achieved good steroid response on day 8 (peripheral blood absolute blast count < 1000/µl) compared to 17/29 patients (59%) in prednisolone prephase group [p = 0.018]. Day 15 bone marrow MRD assessment by flowcytometry was available in 57 patients (31/33 in dexamethasone prephase group & 26/29 in prednisolone prephase group). Nineteen out of 31 patients (61%) in dexamethasone prephase group achieved day 15 MRD < 0.1% compared to 6/26 patients (23%) in prednisolone prephase group [p = 0.02]. Twenty-eight out of 30 patients (93%) in dexamethasone group achieved morphological CR on day 33 of induction compared to 18/24 patients (75%) in prednisolone prephase group [p = 0.135]. Day 33 bone marrow Flow-MRD < 0.1% in the dexamethasone & prednisolone prephase groups were 82% and 50% respectively [p = 0.12]. At the time of analysis, 3/30 patients (10%) in dexamethasone group who achieved CR has disease relapse, compared to 8/24 patients (33%) in prednisolone prephase group [p = 0.04]. The event-free survival (EFS) at the end of study (18 months) was found to be significantly higher in the dexamethasone prephase group compared to prednisolone prephase group (p = 0.002). The incidence of steroid-related toxicities of any grade was similar in the two treatment groups. Conclusion: In the present study, the replacement of prednisolone with dexamethasone for a relatively shorter duration in the 7-day steroid prephase of ALL IC-BFM 2009 induction regimen was associated with significantly improved day 8 steroid response, day 15 bone marrow Flow-MRD response, day 33 morphological CR rates, and EFS at 18 months, without significant increase in steroid-related toxicity in induction. To our knowledge, this is the only study to compare dexamethasone versus prednisolone prephase in pediatric ALL induction, in the context of ALL IC-BFM 2009 regimen. Long-term follow up and enrollment of larger number of patients is planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Measurable Residual Disease Conversion Rate with Consolidation Chemotherapy in Acute Myeloid Leukemia

Background Patients with acute myeloid leukemia (AML) who achieve complete remission (CR) following induction therapy but continue to have measurable residual disease (MRD) have inferior outcomes following allogeneic hematopoietic cell transplant (alloHCT). Determining the historical rate of MRD clearance in response to standard consolidative therapy is needed as an initial step in developing a new therapeutic strategy to clear MRD prior to alloHCT. Methods A multi-institution retrospective analysis was performed on consecutively treated AML patients in morphologic CR with detectable MRD post-induction therapy who received standard chemotherapy consolidation from January 2016 to December 2021. MRD was measured by multiparametric flow cytometry (MPFC, sensitivity 0.01-0.1%), next generation sequencing (NGS, sensitivity 1-5%), real-time quantitative PCR (qPCR, sensitivity 1:100,000-0.5%), and/or fluorescence in situ hybridization (FISH)/karyotype (sensitivity 1-5%). Any detectable disease-related abnormalities were considered to be evidence of MRD for the purpose of this study. Results Eighty-three patients met inclusion criteria for this analysis, with a median age at diagnosis of 51 years (IQR 43-63). Adverse-risk disease by ELN 2017 criteria was observed in 36 patients (43%). The majority received induction therapy with "7+3" (cytarabine + idarubicin or daunorubicin, 77 patients, 93%), and most (71 patients, 86%) required only 1 line of therapy to achieve CR. At the time of CR following induction therapy, 35 patients (42%) had MRD detected by MPFC, 38 patients (46%) had MRD detected by NGS, 10 patients (12%) had MRD detected by qPCR, and 32 patients (39%) were found to have an abnormal FISH or karyotype. Twenty-eight patients had MRD detected by more than 1 method. Consolidation therapy consisted of intermediate-dose cytarabine (defined as <3 g/m2, 41 patients, 49%) or high-dose cytarabine (defined as 3 g/m2, 39 patients, 47%) in all patients, except for 3 who received daunorubicin and cytarabine (liposomal) consolidation. The number of cycles of cytarabine consolidation were 1 cycle (36 patients, 43%), 2 cycles (23 patients, 28%), 3 cycles (15 patients, 18%), and 4 cycles (9 patients, 11%), respectively. Sixteen patients received cytarabine consolidation in combination with another agent including sorafenib (1 patient), midostaurin (10 patients), gilteritinib (1 patient), enasidenib (1 patient), and gemtuzumab (3 patients). In response to consolidation therapy, 27 of the 83 patients with measurable disease converted to MRD-negative (33%) after all given cycles of consolidation cytarabine. Of the 35 patients whose AML had MRD detected by MPFC prior to consolidation, 17 converted to MRD-negative (49%). Of the 38 patients whose AML had MRD detected by NGS prior to consolidation, 6 converted to MRD-negative (16%). Of the 10 patients whose AML had MRD detected by qPCR prior to consolidation, 6 converted to MRD-negative (60%). Of the 32 patients whose AML had MRD detected by FISH or karyotype prior to consolidation, 13 converted to MRD-negative (41%). Of note, 22 patients developed progressive disease following consolidation therapy (and therefore also did not achieve MRD-negativity). Multivariable logistic regression was performed to assess potential associations with MRD conversion during consolidation chemotherapy. Older age decreased the odds of conversion of AML to MRD-negative (OR = 0.96, 95% CI 0.92-1.00, p = 0.04). Higher ELN risk category, higher dose of cytarabine, and more cycles of consolidation did not affect the likelihood of conversion of AML to MRD-negative. Fifty-five patients received alloHCT following consolidation therapy. Thirty-one of these patients had AML with MRD detected post-consolidation, of whom 14 had MRD measured by MPFC. With a median follow-up time of 437 days post-transplant (IQR 151-914), two-year progression-free survival post-transplant was 57% in AML patients with MRD detected post-consolidation compared to 71% in AML patients with no MRD (p = 0.28). Conclusions The rate of MRD eradication with standard consolidation chemotherapy in AML is suboptimal. Combined with the known poor outcomes after alloHCT in patients with AML and detectable MRD pre-transplant, this demonstrates an unmet need to develop a new therapeutic strategy for eradicating MRD after induction therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Consolidation of High-Risk Acute Promyelocytic Leukemia (APL) in Community Infusion Centers: A Single Center Experience

Intro: APL is one of the most curable forms of acute myeloid leukemia. Whereas all-trans retinoid acid (ATRA) and arsenic trioxide (ATO) are the standard of care for treatment of low/intermediate risk APL (APL0406 trial, LoCoco et al. NEJM 2013), the optimal treatment of high-risk APL remains unclear. In our institution, we use the APML4 regimen (Iland et al. Blood 2012) to treat high-risk APL patients, which uses idarubicin+ATRA followed by ATRA+ATO for induction. Then, there are two consolidation cycles of ATRA+ATO, the first of which is 28 consecutive days of treatment. Consolidation is followed by two years of ATRA + 6-mercaptopurine + methotrexate maintenance (PETHEMA LPA99, Sanz et al. Blood 2003). In the APML4 trial, the outcomes were excellent for all risk groups, particularly for high-risk patients. However, this regimen cannot be administered in community infusion centers (CIC) that are not open on weekends. In addition, the expertise required to give maintenance therapy makes it challenging to administer outside of an academic medical center. Here, we examine outcomes of high-risk APL patients who received induction at our institution to document the types of consolidation regimens they received and their outcomes. In particular, we focus on the outcomes of high-risk APL patients who received induction with idarubicin+ATRA+ATO (as per APML4) at our institution, and then for practical considerations received consolidation with ATRA+ATO (as per APL0406) in the community. Methods: We performed a retrospective analysis of all patients between the ages of 18-89 who were diagnosed with high-risk APL at the Knight Cancer Institute between January 2009 and July 2022. We identified 19 patients with the diagnosis of high-risk APL. One patient died soon after diagnosis due to intracranial bleed (excluded from final analysis). The remainder completed induction and consolidation. Result: Table 1 shows the main clinical features at diagnosis. The most common complications during induction were differentiation syndrome and coagulopathy. All 18 patients achieved complete morphologic remission (CR) at the end of induction but still had detectable PML-RARA by PCR. 4/18 (22%) patients received the APL2000 regimen (Adès et al. JCO 2006), and 1/18 (6%) patient received the PETHEMA regimen (Sanz et al. Blood 2003). All 5 patients were hospitalized for neutropenic fever, which required significant dose reduction and treatment delay. One patient (6%) received induction with ATRA+ATO+gemtuzumab ozogamicin followed by consolidation with ATRA+ATO (Estey et al. Blood 2006). The majority of patients (12/18, 67%) received induction as per APML4 protocol (Table 2). The APL0406 regimen was administered as consolidation in 11/18 (61%) patients; and all patients except one received consolidation at local CIC. Common side effects were headache and nausea, leading to dose reduction in five patients. One patient continued with APML4 consolidation. Molecular CR was confirmed in all patients at the end of consolidation treatment. 13/18 (72%) patients did not receive maintenance therapy after consolidation. All patients were subsequently followed by RT-PCR for PML/RARA for molecular relapse. At the last follow up, all 18 patients remained to be in molecular CR with no relapsed disease. Discussion: The benefit of ATRA+ATO for low/intermediate risk APL patients is well established, but the optimal management of high-risk APL remains a challenge, especially when consolidation is given in CIC. Our series of patients provides real world evidence that ATRA+ATO consolidation as per APL0406 regimen is effective in high-risk APL patients after induction with idarubicin+ATRA+ATO as per the APML4 regimen. Although the numbers are limited, all patients remain in molecular remission as measured from the end of consolidation, similar to the excellent outcomes of low/intermediate risk APL patients. Consolidation was well tolerated, with minimal treatment delays, suggesting that a standard consolidation regimen for low-, intermediate-, and high-risk APL patients can be successfully administered in the community (most centers typically treat 1-2 patients per year with APL). The majority of patients did not receive maintenance therapy but remained in remission, providing further evidence that maintenance therapy is unnecessary with sufficient ATRA+ATO consolidation, and simplifying therapy (Shah et al. Blood 2018). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric Acute Myeloid Leukemia with Co-Occurring BCR::ABL and CBFA2T3::GLIS2 Dual Fusion with Deep Response to FOLR1-Targeting Antibody Drug Conjugate Stro-002 and Tyrosine Kinase Inhibitor

Introduction: Pediatric acute myeloid leukemia (AML) is characterized by significant cytogenetic and molecular heterogeneity. AML with CBFA2T3::GLIS2 fusion (CBF::GLIS) involving infants and very young children is a rare but uniformly refractory disease with high mortality. Laboratory data demonstrates that this fusion is sufficient for leukemic transformation without recurrent secondary variants. These studies have also linked CBF::GLIS fusion to induction of folate receptor alpha (FOLR1) expression in transformed cells (Meshinchi et al, 2020). Incidence of Ph+ de novo (BCR::ABL minor breakpoint, p190 hybrid) disease ranges from 0.5-3% of all AML cases across the age spectrum (Soupir et al, 2017). We, for the first-time, report dual existence of both fusions in a child with AML and discuss novel interventions including combination of a BCR::ABL tyrosine kinase inhibitor (TKI) and antifolate therapy consisting of methotrexate (MTX) and FLOR1-specific antibody-drug conjugate (ADC) STRO-002 (Sutro Biopharma) based on available data regarding its in-vitro preclinical activity in CBF::GLIS disease (Meshinchi et al, ASH abstract, 2021). Case: A 2-year-old girl of Vietnamese descent received a bone marrow examination for bicytopenia and macrocytosis. Flow cytometry confirmed the poor prognostic RAM immunophenotype AML (CD45 dim, HLA-DR negative, CD38 dim, CD56 bright) and FOLR1 expression. Marrow aspirate smears showed numerous hypercellular spicules and blasts with scant cytoplasm and some blebbing but no granules or Auer rods. Marrow exhibited 100% cellularity. CNS was not involved. Review of diagnostic genomic profile mid induction showed a BCR::ABL minor breakpoint fusion as well as CBF::GLIS fusion. The child began AML induction on COG AAML1831 trial randomized to the experimental arm with CPX-351 and gemtuzumab ozogamicin (GO). Due to lack of response to AML therapy at end of Induction (EOI) I, she was taken off protocol and started on modified ALL regimen for Induction II consisting of cytarabine, low dose weekly methotrexate and bimonthly peg-asparaginase with addition of an oral TKI, dasatinib. EOI II marrow examination showed improvement with reduction in blasts (11%) and detectable BCR::ABL but undetectable CBF::GLIS by Q-RT-PCR. Induction III included low dose weekly methotrexate, cytarabine, GO, and dasatinib. EOI III marrow examination showed complete morphologic remission (CR) with no evidence of minimal residual disease (MRD) or fusion transcripts of either clone. Due to availability of a matched sibling donor, the child underwent hematopoietic stem cell transplantation (HSCT); however, was noted to have 3% blasts on day +83 marrow examination despite previously commencing dasatinib post-transplant prophylaxis. BCR::ABL and CBF::GLIS transcripts were again detectable at low levels. Dasatinib dosage was maximized and weekly MTX and bimonthly peg-asparaginase were reintroduced, but the marrow disease burden increased significantly to 78% blasts positive only for CBF::GLIS fusion. Given availability of FOLR1 directed ADC on single-patient compassionate use basis, patient received single agent STRO-001 on a bi-monthly basis. With rising ANC, evaluation of marrow after 2nd STRO-002 dose remarkably showed CR with MRD negative remission by flow and molecular studies. This time remission was consolidated with monthly STRO-002 and donor lymphocyte infusions (DLI). Currently, patient remains in MRD-negative remission after the 3 of 4 planned STRO-002/DLI maintenance. Patient has maintained a 100% Lansky play score and tolerated the regimen with only prolonged initial cytopenia requiring supportive care as a toxicity. Plan is to continue TKI and monthly STRO-002 as maintenance therapy. Conclusion: Complete genomic sequencing has revolutionized pediatric AML categorization. It is quite conceivable that (BCR::ABL, CBF::GLIS) dual fusion disease will become a more common finding in future. Although CBF::GLIS AML is uniformly refractory to conventional therapy, targeted therapy with FOLR1-directed STRO-002 was highly effective in eliminating CBF::GLIS clone in this patient, and combination with DLI appears to be feasible. Long-term follow-up is required to assess durability of remission. Additional testing of this approach in a larger patient population is needed to determine the role of STRO-002 in this high-risk pediatric AML population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real-World Outcomes for Acute Promyelocytic Leukemia: Need to Address Induction Mortality

Treatment outcomes of acute promyelocytic leukemia (APL) show variation between clinical trials and real-world setting largely due to exclusion of patients who present with major bleeding, severe infections and those who die within the first week. Use of differentiation agent-based therapy for low/intermediate risk group (ATRA and ATO) and limiting anthracycline use to high-risk subgroup has reduced the treatment-related mortality beyond first week. However, use of steroids for treatment of differentiation syndrome and chemotherapy can still increase the risk of infections which can compromise the outcomes, particularly in a setting with high incidence of multi-drug resistant organism and invasive fungal infections. There is a need to identify the predictors of this mortality and ways to reduce it to improve the outcomes. We analyzed the data for patients with newly diagnosed APL from two large tertiary care centers in India, registered between 2013-2018to evaluate the outcomes in all risk groups (as per Sanz score), mortality within first 30-days and prognostic factors. Patients with low/intermediate risk were treated with ATRA and ATO based therapy whereas the high-risk group received chemotherapy in addition to ATRA/ATO. We recorded demographic variables, white blood cell count and platelet count at diagnosis, Sanz score, serum albumin, serum creatinine, treatment administered, achievement of morphological complete remission post induction and complete molecular remission post consolidation, relapse and death. The primary outcome was event-free survival (EFS) which counted lack of complete remission post induction, lack of complete molecular remission post consolidation, relapse and death as event. The secondary outcomes were relapse-free survival (RFS), overall survival (OS) and induction mortality (death within first 30 days) and assessment of impact of baseline disease and host factors on the EFS, RFS, OS and induction mortality. A total of 238 patients were included in this analysis. The median age was 34 years (range 2-72 years) with male: female ratio of 1.5:1. At presentation the median WBC count was 6790 per cumm (range 300-237,000per cumm) and median platelet counts were 21000 per cumm (3000-266,000 per cumm). As per Sanz score - 12% were low-risk, 48% were intermediate risk and 40% were high-risk. Median fibrinogen levels were 239 (range 36-883). A total of 231 patients received therapy. A total of 204 patients were assessed for response at the end of induction and all but one patient achieved complete response (87.8% as per intention to treat). The rest were not assessable due to death during induction (19) and loss to follow up (15). The induction mortality was 8.2% (infections-6, coagulopathy-9, differentiation syndrome-2 and others-2).170/185(92%) patients achieved post consolidation complete molecular remission. A total of 19 patients relapsed during the follow up (bone marrow-14, CNS alone-2 and combined -3). The median time to relapse was 23.9 months (IQR 13.6-42.9 months). At a median follow up of 51.2 months,3-year EFS, RFS and OS were 79%,92% and 88%. There was no statistically significant difference between low, intermediate and high-risk group for any of the survival outcomes. Similarly, age, gender, fibrinogen and albumin at diagnosis did not have any adverse impact. Baseline WBC of >40000/cumm and serum creatinine of >2 mg/dL at diagnosis adversely affected the overall survival with HR of 3.22 (95% CI 1.42-7.29, p-0.005) and 11.9 (95% CI-2.78-50.82)respectively. However, the number of patients with raised creatinine were very small (n=4). These two factors had adverse impact on induction mortality too (WBC>40,000 per cumm - HR -3.32, 95% CI-1.31-8.42, p-0.012 serum creatinine >2 mg/dL- HR 14.69, 95% CI-3.36-64.18, p<0.0001). The survival probabilities at 3, 4 and 5 years using 30-day landmark analysis were 96%, indicating that there are very few deaths after the first 30 days of induction. Fig 1 depicts the overall survival. This retrospective analysis highlights that ATO+ATRA and minimal anthracycline based therapy has led to similar outcomes in all risk groups and the present need to address induction mortality. High induction mortality in patients with WBC >40,000/cum indicates the potential contribution of infections due to use of steroids to treat differentiation and underlying coagulopathy or a different biology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blinatumomab As Toxicity Sparing First Line Treatment of Children and Young Persons with B-Precursor Acute Lymphoblastic Leukaemia (B-ALL)

Introduction Recent studies have demonstrated that Blinatumomab is more effective and less toxic than intensive chemotherapy in children and young persons (CYP) with relapsed B-ALL. Although Blinatumomab is standard of care in adults with persistent MRD during first line therapy based on the BLAST study, there is no published evidence in CYP of its efficacy and toxicity in this setting. We report our experience of Blinatumomab as a toxicity sparing alternative to chemotherapy in first line treatment of CYP with B-ALL. Patients and Methods We audited the outcome of patients treated according to a consensus UK guideline for first line treatment with Blinatumomab of CYP patients with B-ALL who were considered unfit for post-remission intensive chemotherapy or CR1 HSCT because of toxicity to previous chemotherapy or significant co-morbidities. Treating clinicians obtained consent for treatment with Blinatumomab as standard of care after counselling patients and carers about the risk and benefits of this approach. The guideline mandated discussion of all such patients at national tumour board meetings and data was captured through a referral proforma and follow-up questionnaire. It recommended replacing consolidation and/or interim maintenance phases with 2 cycles of blinatumomab as a single agent with a 1-2 week break between cycles. Patients who obtained an MRD response (defined as MRD <0.01%) received delayed intensification (DI) followed by maintenance, or maintenance therapy alone if they were unfit for DI. Patients who had persistent MRD >0.01% were categorised as Blinatumomab failures and received either further chemotherapy, HSCT or CART depending on performance status. Events were defined as death, relapse, or secondary cancer. Results Sixty patients were eligible for analysis, including 4 cases of Ph+ ALL and 8 with Down syndrome. Compared to the B-ALL cohort in a previous trial, UKALL 2003, there was a higher proportion of NCI high risk, cytogenetic high risk, end of induction MRD positive and Down Syndrome patients (Table 1). All patients were in morphological complete remission prior to receiving Blinatumomab. Blinatumomab was administered for toxicity (47/60 (78%), including G3/4 pancreatitis, typhlitis, severe infections often requiring intensive care or surgery, disseminated fungal infections, CNS haemorrhage or liver failure) or co-morbidities (Down syndrome, Li-Fraumeni syndrome (n=2), Schimke immune-osseous dysplasia and 7q11.21-23 duplication) or in one case to minimise transfusion requirements (Jehovah's witness.) In the remaining 13 cases, blinatumomab was either administered for end of consolidation MRD of ≥0.05% (n=10) in patients unfit for intensive chemotherapy/HSCT, or positive end of induction MRD in patients with HR cytogenetics (n=3). Blinatumomab replaced consolidation and interim maintenance therapy in 32/60 (53%) cases, and interim maintenance in the remaining 28/60 (47%) cases. Blinatumomab was well tolerated with 1 G3 neurotoxicity (seizure in a Down syndrome patient who had not received prophylactic anti-convulsants) and no grade 2-4 CRS. Of the 50/60 patients with positive MRD pre-Blinatumomab (median = 0.1%, range = 0.01%-8%), 48 (96%) had a reduction in level to <0.01% (45 after cycle 1, 3 after cycle 2), and 10 patients maintained MRD negativity before and after 2 cycles of Blinatumomab. In the two patients who did not respond (3%), 1 received CART followed by transplant for persistent MRD, the other is receiving intensive chemotherapy. Of the responders, 47 (80%) received DI and maintenance and 10 maintenance alone. With a median follow up of 16 months (range 2-44 months), 2 patients have relapsed (1 at 2 years, the other at 9 months) but remain alive. The 4 patients with Ph+ ALL and the 8 with Down syndrome are alive and in remission, with a median follow up of 12.5 months (range 7-22 months). Conclusion In this large case series, we found Blinatumomab is safe and highly active with promising short-term efficacy in a higher risk cohort of CYP patients with B-ALL in first remission. Our results support its use in place of intensive chemotherapy for patients with relative chemo-intolerance. Furthermore, if the efficacy is sustained on longer follow-up, the results argue for replacing parts of post-remission intensive chemotherapy with Blinatumomab to reduce attendant morbidity and mortality. SS and AV are joint senior authors, AH and AM joint first authorsFigure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cox Proportional Hazards Deep Neural Network Identifies Peripheral Blood Complete Remission (PB-CR) to be at Least Equivalent to Morphologic CR in Predicting Outcomes of Patients Treated with Azacitidine - a Prospective Cohort Study By the AGMT

Background Myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML) are multiclonal diseases demonstrating considerable biologic, phenotypic, genetic, and clinical overlap. In recognition of this continuum, these diseases are often treated similarly. Achievement of complete remission (CR)/CR-incomplete (CRi) is deemed the major outcome associated with improved overall survival (OS), and is often used as primary endpoint in clinical AML trials, whereas red blood cell transfusion independence is often the endpoint used in MDS trials. Expert opinions questioning these endpoints are cumulating [Sekeres M, Leukemia 2019, 570; Kim N, BJH 2021, 1137; Chandhok N, Leuk Lymphoma 2022, 1], as are reports indicating that achievement of CR/CRi may not be necessary for prolonged OS in patients treated with azacitidine [Pleyer L, Annals Hematol 2014, 1825; Schuh AC, ASH 2015, P575; Pleyer L, ASH 2015, P3752; Pleyer L, ASH 2019, P3387]. All response criteria for MDS, CMML and AML require bone marrow evaluations (BME), which are only performed in ~50% of patients outside of clinical trials [Dinmohamed A, Leuk Res 2015, 177; Pleyer L, ASH 2019, P3387], indicating the real-world need for internationally accepted response endpoints that do not necessitate repeated BME. We thus introduced peripheral blood CR (PB-CR), defined as Hb ≥11.0 g/dL, PLT ≥100 and <450 G/L, ANC ≥1.0 G/L, WBC ≥1.5 and <10.0 G/L, monocytes ≤1.0 G/LPB blasts=0%, no growth factors, no transfusions). In 1441 patients treated with azacitidine, multivariate survival modelling adjusted for 17 baseline covariates, indicated that PB-CR is at least as good a predictor of OS as CR [Pleyer L, ASH 2019, P3387]. Aims To compare the predictive value of CR or CR/CRi versus PB-CR. Methods Data from 1441 patients receiving 13971 cycles of azacitidine included in the Austrian Azacitidine Registry NCT01595295 was used for expanded analyses. The endpoints OS, time to next treatment (TTNT) and time on treatment (TOT) were defined as described previously [Pleyer L, Lancet Haematology 2021, e135]. Only data from patients who had a BME during treatment, and for whom all 17 baseline variables remaining in the final Cox model [Pleyer L, ASH 2019, P3387] were available, were analyzed (n=624). DeepSurv, a Cox proportional hazards model parametrized by a deep neural network [Katzman J, BMC Med Res Meth 2018, 24], was used to assess the predictive value of CR, CR/CRi and PB-CR. The network architecture is depicted in Fig 1. To assess whether CR or CR/CRi has greater predictive value than PB-CR, model performance was evaluated on randomly corrupted (i.e. destroyed) data. Either CR, CR/CRi or PB-CR responses were randomly permutated 1000 times, thus removing the information content from the respective variable and allowing investigation of the resulting drop in model performance using the concordance index (C-index). The average validation C-index for each response type of the corrupted datasets was generated with 5-fold cross-validation (Fig 2), stratified by sex, age </≥75 years and diagnosis. Validation C-index pairs for each of the 1000 permutations were compared by Wilcoxon signed-rank test. Bonferroni-correction for multile testing was performed. Results Random permutations of the PB-CR response resulted in signficantly higher performance loss than random permutations of the response types CR or CR/CRi, indicating that PB-CR has significantly more information content than either CR or CR/CRi. The C-indices with "intact PB-CR and permutated CR (or CR/CRi) were significantly higher than for "intact CR (or CR/CRi) and permutated PB-CR", with p<0·0001 for the endpoints TOT, TTNT and OS (Fig 3). Results remained consistent irrespective of the choice of inner network architectures. Conclusions In the largest cohort of azacitidine treated patients reported to date, survival modelling with a neural Cox regression revealed a higher information content for PB-CR as compared to either CR or CR/CRi for all time-to-event endpoints analyzed. Presence of PB-CR can thus identify patients with adequate response to non-intensive therapies without repeated BM assessments. Incorporation of PB-CR as secondary endpoint in clinical trials would be ideal to confirm these results. We aim to analyse whether these findings can be recapitulated in MDS, CMML and AML patients receiving other treatments and welcome collaborations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Impact of Additional Intensive L-Asparaginase Therapy during Consolidation Phase for High-Risk Acute Lymphoblastic Leukemia: Results of a Randomized Controlled Trial in the AIEOP-BFM ALL 2009 Protocol

Introduction Treatment of childhood Acute Lymphoblastic Leukemia (ALL) with unfavorable features remains a major challenge. An earlier intervention, aiming at lowering Minimal Residual Disease (MRD) levels, might reduce the risk of relapse and the indication to allogeneic hematopoietic stem cell transplantation (HSCT) in 1st complete remission (CR). The AIEOP-BFM ALL 2009 protocol included a randomized study for patients with high-risk (HR) features, to investigate if an intensive and continuous exposure to pegylated L-Asparaginase (Peg-L-ASP), given on top of BFM consolidation phase IB, could decrease the levels of MRD and to influence long term outcome. Patients and methods From June 1, 2010 to February 28, 2017, 6136 patients with Ph’ negative ALL, aged between 1-17 years, were enrolled in the AIEOP-BFM ALL 2009 Study (Eudract number 2007-004270-43). Participating study centers were in Australia, Austria, Czech Republic, Germany, Israel, Italy and Switzerland.1097 were classified as HR at the end of induction phase IA (day 33), on the basis of evidence ofKMT2A-AFF1 rearrangement, hypodiploidy (<45 chromosomes, or DNA-index <0.8), prednisone poor response (PPR), poor bone marrow (BM) response at day 15 (i.e. ≥10% blasts by MFC), failure to achieve morphological CR. Treatment consisted of a 7-day prephase with prednisone and 1 intrathecal dose of methotrexate (ITMTX), an induction protocol IA with prednisone or dexamethasone, vincristine, daunomycin, Peg-L-ASP (2 doses), ITMTX and, only in PPR T-ALL cyclophosphamide. Patients eligible were randomized to receive or not 4 weekly doses of Peg-L-ASP (2500 IU/sqm x 4) on top of consolidation phase IB, which included 6-MP, cyclophosphamide, cytosine arabinoside and ITMTX. Thereafter, patients received 3 blocks of non-cross-resistant drugs, 3 reinduction phases with or without cranial radiotherapy (12 Gys), and maintenance therapy till 24 months from diagnosis. The primary endpoint was PCR-MRD reduction to less than 5x10-4at the end of phase IB; secondary endpoint was Event-Free Survival (EFS). Results Overall, 1097 patients were at HR at the end of induction phase IA and 809 (79.2%) were randomized to receive (404) or not (405) 4 doses of Peg-L-Asp during consolidation phase IB. The primary endpoint could be assessed in 732 patients (90.5%). By ITT analysis, the proportion of patients with PCR MRD ≥ 5x10-4 at the end of consolidation phase IB in the experimental arm (EA) vs the control arm (CA) was 13.9% vs 17.0% with no significant difference (p-value=0.25).There were more toxic deaths during consolidation phase IB among patients randomized to Peg-L-Asp (n=14, 12 of them due to infections), vs controls (n=1): 7 of them, all in the EA, however, occurred before starting the randomized treatment. Deaths in CR after phase IB were 24 vs 24 in the EA vs CA (of whom 10 vs 12 were after HSCT in CR1, performed in 87 and 90 patients respectively). In the EA and CA the overall 5-year EFS by ITT was 70.4% (2.3) and 75.0% (2.2) (p-value=0.18), the 5-year cumulative incidence of death in CR was 9.5% (1.5) vs 5.7% (1.2) (p-value=0.08) and that of relapse was 17.7% (1.9) vs 17.2% (1.9), respectively (p-value=0.94). Non HSCT related deaths were due to infections in 23/28 in the EA, and in 10/13 in CA patients. The 5-year overall survival probability was 81.5% (2.0) and 84.0% (1.9) respectively (p-value=0.25). Discussion In this randomized study, results (ITT analysis) show a very modest, not significant decrease in the percentage of patients with high MRD in the EA, and no significant difference in EFS between the two arms. The trial results suggest that the additional administration of intensive Peg-L-Asp in phase IB is associated with higher, although not statistically significant, mortality in CR and very similar relapse incidence. However, some fatal events in phase IB occurred very early after randomization, before the start of the planned treatment with Peg-L-Asp. Importantly, in both analyses, by ITT and by treatment given, there is no evidence of benefit on reduction of relapses by the additional therapy with Peg-L-ASP in phase IB. These findings indicate that an early intensification of chemotherapy with Peg-L-Asp in patients defined as HR does not improve the outcome of children with HR ALL treated according to AIEOP-BFM regimen, thus suggesting that further improvements with conventional chemotherapy might be difficult in the context of intensive treatment protocols. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML: Preliminary Results from a Phase 1 Trial

Background The prognosis of pediatric patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML) remains dismal. Evaluation of novel therapies such as chimeric antigen receptor (CAR) T cells is therefore urgently needed. CD123 is overexpressed on AML blasts and leukemic stem cells (LSC), making it an attractive target for CAR T-cell directed therapy. Methods and Results We designed a 'bridge to allogeneic hematopoietic cell transplant (HCT)’ Phase 1 study to evaluate the safety and feasibility of treating pediatric patients with r/r AML with CD123-CAR T-cell on four dose levels (DL): DL1: 3x105/kg, DL2: 1x106/kg, DL3: 3x106/kg, DL4: 1x107/kg (NCT04318678). CD123-CAR T-cells were generated from CD4/CD8-selected autologous leukapheresis products using a lentiviral vector, which encoded a CD123-CAR with CD28.z signaling domain and a CD20 safety switch. Patients received lymphodepleting chemotherapy with Fludarabine and Cyclophosphamide followed by a single CAR T cell infusion. To date we have enrolled 12 patients. The median age of treated patients was 17 years (range 12-21years). Except for the first patient who had primary refractory disease, all other patients had relapsed following HCT (n=1-4 previous HCTs). Despite patients being heavily pre-treated, we successfully manufactured products on all patients. The infused products had a predominantly CD4+ effector memory immunophenotype, with a median CD123-CAR+/CD20+ expression of 60.2% (range 46.1-79.4%, N=6). All infused CAR T cell products exhibited potent antitumor activity in vitro when cultured against CD123+ targets (including autologous blasts for 2 patients). We infused 2 patients on DL1 and 3 patients on DL2. Two patients were infused on single patient protocols. All infusions were well tolerated, without any adverse infusion events. Post infusion, we observed isolated fevers that resolved within 24 hours (possible Grade 1 Cytokine Release Syndrome (CRS)). No Grade 2 ≥ CRS or neurotoxicity was seen. Multiplex cytokine analysis showed statistically significant increase of IL15 after lymphodepletion without significant increase in Th1 cytokines or IL6 after T cell infusion. While transient cytopenias were observed, patients did not develop persistent marrow aplasia. No dose limiting toxicities were noted. Disease evaluation was performed at 4-6 weeks following CAR T cell infusion. The 2 patients on DL1 showed no response. In the 3 patients on DL2, we observed: reduction in blast percentage without complete remission (CR) in 1 patient, no response in 1 patient and CR in 1 patient. The patient with CR had isolated extramedullary disease, showing complete resolution of all lesions by week 4 by PET imaging (Figure 1). She recurred 2 months following infusion, coinciding with loss of CAR T-cell detection by qPCR. She received a second infusion off study and again achieved a short-lived CR. One additional patient was infused on dose level 2 off protocol. She achieved morphological CR at day 28 with low level minimal residual disease (0.19%). Correlative studies revealed CD123-CAR T cell expansion in patients on DL2 (Figure 2), but not on DL1. However, peak expansion on DL2 was low in comparison to published reports on CAR T cells for Acute Lymphoblastic Leukemia. Detailed phenotypic analysis revealed that CD123-CAR T cell products were predominantly effector memory (CD45RO+CCR7-) with <2% naïve-like (CD45RO-CCR7+ or CD45RO-CD62L+) T cells. In addition, PD1, TIM3 and CD39 were expressed at a median of 43, 81 and 16% in CD4+ and 3, 72 and 45% in CD8+ T cells. This phenotype could be completely reversed by inhibiting CAR signaling with dasatinib during the manufacturing process, resulting in a CAR T cell product dominated by naïve-like T cells that did not express TIM3, PD1 or CD39, leading to enhanced T-cell effector function. Mechanistic analysis revealed that the observed phenotype could be explained by the transient, increased cell surface expression of CD123 on T cells upon activation during manufacturing, and not by tonic CAR signaling. Conclusions Our initial clinical experience with CD123-CAR T cells for pediatric patients with r/r AML demonstrates feasibility, safety, and evidence of anti-leukemic activity. Upcoming CD123-CAR T cell products will be manufactured in the presence of dasatinib to limit T-cell differentiation and exhaustion as we continue to explore our CD123-CAR T cell therapy approach for pediatric r/r AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Measurable residual disease by flow cytometry in acute myeloid leukemia is prognostic, independent of genomic profiling

Measurable residual disease (MRD) assessment provides a potent indicator of the efficacy of anti-leukemic therapy. It is unknown, however, whether integrating MRD with molecular profiling better identifies patients at risk of relapse. To investigate the clinical relevance of MRD in relation to a molecular-based prognostic schema, we measured MRD by flow cytometry in 189 AML patients enrolled in ECOG-ACRIN E1900 trial (NCT00049517) in morphologic complete remission (CR) (28.8 % of the original cohort) representing 44.4 % of CR patients. MRD positivity was defined as ≥ 0.1 % of leukemic bone marrow cells. Risk classification was based on standard cytogenetics, fluorescence-in-situ-hybridization, somatic gene analysis, and sparse whole genome sequencing for copy number ascertainment. At 84.6 months median follow-up of patients still alive at the time of analysis (range 47.0–120 months), multivariate analysis demonstrated that MRD status at CR (p = 0.001) and integrated molecular risk (p = 0.0004) independently predicted overall survival (OS). Among risk classes, MRD status significantly affected OS only in the favorable risk group (p = 0.002). Expression of CD25 (α-chain of the interleukin-2 receptor) by leukemic myeloblasts at diagnosis negatively affected OS independent of post-treatment MRD levels. These data suggest that integrating MRD with genetic profiling and pre-treatment CD25 expression may improve prognostication in AML.