Morphologic CR Research Articles

Disease Monitoring in Acute Myeloid Leukemia with CLEC2A qRT-PCR

CLEC2A, C-Type Lectin Domain Family 2 Member A, is a newly discovered AML-restricted antigen. Detailed characteristics, clinical correlations, and therapeutic potential of CLEC2A in AML is presented by Kirkey et. Al. (ASH 2023). CLEC2A is entirely silent in normal hematopoiesis and uniquely expressed in patients with KMT2A rearrangements, providing an ideal therapeutic target in this high-risk subtype of AML. To date, CLEC2A has not been implicated in hematologic malignancies and expression of CLEC2A is entirely absent from normal tissues other than minimal expression in the skin. In addition to its utility as a therapeutic target, CLEC2A provides a unique target for disease monitoring given its lack of expression in normal hematopoietic cells. Here we present a highly sensitive quantitative molecular assay for CLEC2A, establishing its utility for disease monitoring in patients with KMT2A-r AML. In our efforts to evaluate CLEC2A expression and demonstrate the utility of using CLEC2A for disease monitoring in KMT2A-r, we developed a qRT-PCR assay using probes directed at exon 3 of the CLEC2A gene. The assay was optimized with RNA from the OCI-AML2 cells, validated to be CLEC2A-positive by flow cytometry and RNA from Kasumi-1 and K562 cells, both CLEC2A-negative by flow cytometry. We established the sensitivity of the assay through serial dilution of OCI-AML2 in a background of CLEC2A-negative Kasumi-1 or K562 cell RNA with concentrations ranging from 100% to 0.0001% OCI-AML2 RNA. The sensitivity assay demonstrated CLEC2A was detected at a level of 0.001% in both Kasumi-1 and K562. Given the association of CLEC2A with KMT2A-r AML, we evaluated the prevalence of CLEC2A as assessed by qRT-PCR. We screened diagnostic specimens from a large cohort of patients enrolled on recent COG Phase III clinical trials (ages 0-29) with and without KMT2A fusions to establish prevalence of CLEC2A expression. Using our CLEC2A qRT-PCR assay we evaluated diagnostic specimens from 122 patients with (N=76) and without (N=46) KMT2A-r, in addition to RNA from 8 normal bone marrow (NBM) samples, as controls and validated CLEC2A was entirely absent in NBM with median expression 0.13 copies per 1e 4 GUSB, figure 1A. Of evaluable samples, CLEC2A was detected in 65 of 76 (85%) KMT2A-r cases with a median expression of 317.45 copies per 1e 4 GUSB (range 0.03 - 21134.21 copies per 1e 4 GUSB). CLEC2A was detected in 21 of 46 (45%) non-KMT2A-r cases tested with a median expression of 1.37 copies per 1e 4 GUSB (range 0 - 1204.84 copies per 1e 4 GUSB). Further evaluation of KMT2A fusion partner showed that CLEC2A was detected in 100% of MLLT4 (N=6/6), 83.3% in MLLT10 (N=20/24), 72.7% of ELL (N=8/11), and 42.9% in MLLT3 (N=21/49). While CLEC2A expression was seen in both KMT2A-r and non-KMT2A-r AML patients, CLEC2A levels were significantly higher in KMT2A-r patients compared to non-KMT2A-r and NBM samples (p < 0.0001). Since CLEC2A is expressed in AML with no expression in normal hematopoietic cells, we hypothesized that monitoring CLEC2A via qRT-PCR throughout treatment (i.e., at diagnosis, at end of induction [EOI], at relapse) would provide added specificity to traditional flow cytometric disease monitoring. Using paired RNA samples from patients across multiple timepoints, we evaluated the change in CLEC2A expression throughout treatment, figure 1B. Patients with high CLEC2A expression at diagnosis (mean 1670 copies per 1e 4 GUSB, n=39) had decreased expression at EOI (mean 199 copies per 1e 4 GUSB, n=45) indicating treatment response, however high CLEC2A expression was seen at time of relapse (mean 1849 copies per 1e 4 GUSB, n=13) with CLEC2A expression preserved at relapse for all patients. qRT-PCR was able to detect CLEC2A expression in 7 patients at EOI who were in a morphologic CR with 6/7 (86%) patients being MRD-negative, further highlighting the increased sensitivity of CLEC2A qRT-PCR. Here we describe qRT-PCR monitoring of CLEC2A, a novel AML-restricted antigen highly enriched in KMT2A-r AML, provides added specificity and sensitivity to traditional flow cytometric MRD analysis. CLEC2A monitoring via qRT-PCR can be used to assess disease response throughout treatment and evaluate impending relapse as CLEC2A expression is retained at relapse. Additionally, qRT-PCR monitoring of CLEC2A can be incorporated in to pre- and post-HSCT disease evaluations similarly to qRT-PCR analysis of other known high-risk fusions such as NUP98-NSD1 fusions.

Final Results from Phase 1 Study of Briquilimab, an Anti-CD117 Monoclonal Antibody, in Combination with Low Dose Irradiation and Fludarabine Conditioning, Shows Durable Remissions in Older Adults with Acute Myeloid Leukemia in Complete Remission and Myelodysplastic Syndrome Undergoing Allogeneic Hematopoietic Cell Transplantation

Background: Allogeneic hematopoietic cell transplantation (HCT) with non-myeloablative conditioning (NMA) is a potential cure for AML and MDS in older/frail patients. NMA is associated with better tolerability but higher relapse rates compared to more intensive regimens. In a phase 1 study (NCT#04429191), briquilimab (JSP191), a first-in-class monoclonal antibody (mAb) that inhibits stem cell factor binding to CD117 (c-Kit) to deplete hematopoietic stem and progenitor cells (HSPC), in combination with standard NMA conditioning of low dose total body radiation (TBI) and fludarabine (Flu) for HCT, was evaluated in older adults with AML and MDS. Pre-clinical experiments suggest briquilimab synergizes with low dose TBI to deplete normal and malignant HSPC to facilitate donor cell engraftment. Final 1-year efficacy endpoints for all subjects enrolled in this study are presented. Methods: 32 subjects, median age 70 yrs (range 62-79), with AML in morphologic CR (CR1 or CR2+) (n=13), MDS (n=16), or AML not in CR (n =3), and HLA-matched related or unrelated donors were enrolled. Following infusion of briquilimab 0.6 mg/kg, serum levels were assessed to determine start of Flu at 30 mg/m 2/day on Transplant Day (TD)-4, -3, -2, and TBI 2-3 Gy on TD0. Peripheral blood grafts were infused on TD0 (10-14 days after JSP191). GVHD prophylaxis was tacrolimus, sirolimus, and mycophenolate mofetil. Primary endpoints were safety, tolerability, and briquilimab pharmacokinetics. Secondary endpoints included engraftment, chimerism, measurable residual disease (MRD) clearance, acute GVHD (aGVHD), chronic GVHD (cGVHD), NRM, RFS, and OS at 1 year. Results: Summary of main efficacy outcomes is presented in Fig 1. 10 of 13 AML in CR and 14 of 16 MDS subjects had MRD at pre-HCT screening assessed by cytogenetics, flow cytometry, and/or next generation sequencing. There were no infusion toxicities and no briquilimab-related SAEs. Consistent with our previous reports, briquilimab exhibited predictable clearance prior to donor cell infusion, and all subjects engrafted, with neutrophil recovery between TD+19 and TD+26. Compared to baseline marrow, briquilimab alone depleted HSPC with a mean decrease of 62.4±22.7% prior to Flu/TBI (Fig 2). The 28 evaluable subjects at TD+90 achieved median donor myeloid chimerism of 99% (range 96-100%) and median total chimerism of 95% (range 81-100%). There was one secondary graft failure without relapse, who received a 2 nd HCT. Among 13 AML in CR subjects, by 1 year, there were 3 relapses, 1 NRM (GVHD), 9 subjects were alive, and 8 were MRD negative. The 1 subject who was MRD positive at 1 year had only a detectable DNMT3A mutation at low level. 1 of 13 AML in CR subjects had TP53+ disease at screening and this subject was alive and MRD negative at 1 year post-AHCT. Among 16 MDS subjects, by 1 year, there were 5 disease progression/relapses, 1 subject withdrew consent due to persistent disease 3 months post-HCT, 1 NRM (sepsis), 8 subjects were alive, and 6 were MRD negative. 5 of 16 MDS subjects had TP53+ disease, 3 of whom had persistent disease/progressed/relapsed prior to 1 year post-HCT, and the other 2 of whom had MRD positive disease/molecular relapse at 1 year post-HCT. Median time to MRD negativity in all subjects who converted from MRD-positive disease to MRD-negative disease was TD+28. All 3 subjects with AML not in CR had disease progression before 4 months post-AHCT. There was no apparent correlation between level of HSPC depletion at the assessed timepoint (Fig 2) and relapse or survival. Of the 32 enrolled subjects, 4 had MAGIC Grade 2 aGVHD, 1 had MAGIC grade 3-4 aGVHD, 13 had mild/moderate cGVHD, and none had severe cGVHD. Conclusions: These results demonstrate that targeting CD117 with briquilimab together with TBI/Flu as a novel conditioning regimen is safe, well-tolerated, facilitates full donor myeloid chimerism, and sustained clearance of MRD in older adults with AML in CR and MDS without TP53 undergoing NMA AHCT.

Treatment of Molecular Relapses in Pediatric AML Saves Toxicities Prior to Hematopoietic Stem Cell Transplantation (HSCT) - Results of AMoRe2017 Trial

Introduction The long-term survival for children with AML has increased to 70 % - but relapsed AML patients still have a poor prognosis (about 30 %). Recent data suggest that in AML residual leukemic cells below the 5 % limit required for a morphologic CR can adversely affect outcome and that increasing levels of Minimal Residual Disease (MRD) are almost always followed by clinical relapse. Early MRD detection provides an opportunity to implement pre-emptive therapies as a bridge-to-transplant approach, to potentially prevent hematological relapse and increase the probability of successful HSCT. A promising approach is the use of Azacitidine (Aza) for treatment of molecular relapse which we evaluated within the clinical trial “AMoRe2017”. Aza has been shown to reverse epigenetic changes in malignant cells, restoring the normal function of genes critical for differentiation and cell cycle control. The trials' primary objective was the evaluation of the effect of Aza treatment in AML subjects at molecular relapse after CR1 regarding molecular response prior to further treatment (reinduction/HSCT). Patients and Methods AMoRe2017 enrolled children with AML (N=20), >3 months < 21 yrs., of which n=1 dropped out due to laboratory measurement error. The median age was 10.9 yrs. (range 1.7-18.9 yrs.). N=10 (53 %) had a CBFB-AML, n=5 (27 %) KMT2A-rearrangement, n=1 (5 %) NUP98::NSD1, n=1 (5 %) FLT3-ITD, n=1(5 %) PICALM::MLLT10 and n=1 (5 %) NPM1. N=8 (42 %) were classified to subgroup M2, n=4 (21 %) to M5, n=3 (16 %) into M1 and n=4 (21 %) to M4/M4 Eo. At baseline, the white blood cell count averaged 3.8x10 9/l (range 0.1-6.6x10 9/l), without leukemic blasts in PB or BM. Each patient had at least one quantifiable genetic marker (rt-PCR), achieved CR with molecular remission, following initial therapy, and subsequently had a molecular relapse (defined as an increase of the MRD level by at least 1 log, less than 5 % BM myeloblasts and absence of proven extramedullary disease). Patients were treated for up to 6 cycles with 75 mg/m 2 i.v. Aza on days 1 to 7 in a 28-day cycle. Molecular response was categorized as molecular stabilization (< 1 log decrease or increase from baseline), molecular progression (≥ 1 log increase from baseline without hematological relapse), molecular improvement (≥ 1 log decrease from baseline), and hematological relapse (at least 5 % blasts in PB and/or BM and/or proven histological extramedullary relapse). Results During Aza treatment, 10 patients (53 %) had an open relapse, which occurred in n=9 after 1 st or 2 nd cycle. These patients were monitored for KMT2A::SEPTIN6 (n=1), KMT2A::MLLT3 (n=2), PICALM::MLLT10 (n=1), FLT3-ITD (n=1), NUP98::NSD1 (n=1), CBFB::MYH11 (n=1) and RUNX1::RUNX1T1 (n=3). 9 patients (47 %; RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1, KMT2A::ELL n=2) showed a molecular response to Aza and proceeded to HSCT without receiving intensive re-induction treatment. 16 out of the 19 patients are alive (overall survival 84 %), 2 died from HSCT-associated side effects (VOD, infection), and 1 from disease progression. Considering genetics, 7 out of 11 patients with favorable genetics (core-binding leukemia n=10 or NPM1 n=1) responded to the Aza therapy ( RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1). By contrast, in cases with KMT2A-rearrangements, NUP98::NSD1 or FLT3-ITD, 2 out of 8 patients showed improvement or stabilization. In total 152 adverse events (AEs) associated to Aza were reported, the majority classified as grade 1 or 2 (n= 114; 75 %). Mainly affected was the hematopoietic system (40 %), the gastrointestinal tract (15 %), kidney and liver (14 %). Cardiotoxicity was not reported. Discussion The use of Aza as pre-emptive “bridge-to-transplant” approach treating the molecular relapse showed efficacy in patients with RUNX1::RUNX1T1, CBFB::MYH11, NPM1, and KMT2A::ELL, who could be transplanted directly. This pilot provided proof of principle that molecular relapses could be treated, and MRD levels be reversed, at least in subgroups. In addition, avoiding intensive re-induction therapy prior to HSCT is feasible. Conversely, the relatively slow-acting mechanism of epigenetic therapy seems to be ineffective in highly proliferative subgroups of AML with KMT2A-rearrangements or FLT3-ITD. Further studies are mandatory to define treatment options for molecular relapses of non-favorable pediatric AML and to confirm potential improvement of outcome post-HSCT.

Enhanced anti-tumor activity of CD5 CAR T cells manufactured with tyrosine kinase inhibitors in patients with relapsed/refractory T-ALL.

7002 Background: An effective CAR T cell platform for the therapy of T-ALL requires minimizing CAR-driven fratricide of normal T cells. CD5 is a pan-T-cell surface protein present in ~85% of T-ALL. We developed a 2nd generation CD5 CAR that produces minimal T cell fratricide without additional engineering. Tonic CAR signaling accelerated terminal differentiation of CD5 CAR T cells potentially reducing their potency. In this study, we evaluated whether blocking tonic signaling with tyrosine kinase inhibitors (TKI) ibrutinib and dasatinib improves anti-leukemic activity of CD5 CAR T cells in patients with r/r T-ALL (NCT03081910). Methods: In the initial cohort of patients (CH1, n = 8), we generated CD5 CAR T cells from autologous PBMCs followed by expansion in the presence of IL-7 and IL-15 without TKI. In the subsequent cohort (CH2, n = 7) we manufactured cells from autologous (Arm A; n = 4) or HSC donor-derived (Arm B; n = 3) PBMCs in the presence of TKI. A total of 14 pts were treated (9 adult and 5 pediatric; age 11-70 years [median 36 yrs]); one pediatric pt was treated on both cohorts. Results: In CH1, all pts received autologous CAR T cells: 1 pt on DL1 (1x107 CAR T cells/m2), 5 on DL2 (5x107 CAR T cells/m2), and 2 on DL3 (1x108 CAR T cells/m2). In CH2, we treated 4 pts on Arm A (one on DL2; 3 on DL3) and 3 pts on Arm B (all on DL1). The overall expansion of CAR T cells was comparable between cohorts, but we observed more sustained persistence of CD5 CAR T cells in pts on CH2. In CH1, one pt experienced a morphologic CR. Of 7 pts treated on CH2, we observed 4 MRD-negative CRs (2 on Arm A and 2 on Arm B). The CAR T cells produced minimal early toxicity with most grade ≥3 AEs related to cytopenias, febrile neutropenia and transient abnormal laboratory values. CRS occurred in 3 pts on CH1 and 4 on CH2 (Arm A: n = 1; Arm B: n = 3); all ≤ grade 2. No pts developed ICANS. Two pts in CH2 (one on Arm A and Arm B) developed EBV viremia and post-transplant/immunodeficient lymphoproliferative disease (LPD) at wks 5 and 7 post-infusion, respectively. Both were treated with intensive multiagent chemotherapy, and one pt also received EBV specific T cells (EBVSTs) with significant reduction in viral load. Unfortunately, both passed away from complications of treatment for PTLD (unrelated to EBVSTs) with no evidence of residual T-ALL. No other significant infections occurred. Conclusions: These results demonstrate CD5 CAR T cells can induce clinical responses in heavily treated patients with r/r T-ALL. Manufacturing CD5 CAR T cells with TKI improved their potency and anti-tumor activity but also possibly suppressed CD5+ virus-specific T-cells thus leading to 2 cases of EBV associated LPD. We have subsequently modified our clinical protocol to administer prophylactic rituximab to EBV seropositive pts and identify a partially matched EBVST line prior to CAR T cell infusion as a strategy to prevent additional cases of LPD in future subjects. Clinical trial information: NCT03081910 .

A Retrospective Analysis of Autologous Stem Cell Transplantation Outcomes in Adult Philadelphia Chromosome Positive-Acute Lymphoblastic Leukemia

Abstract Introduction Philadelphia chromosome positivity (Ph + ) is a poor prognostic feature in adult acute lymphoblastic leukemia (ALL). Allogenic hematopoietic stem cell transplantation in first complete remission (CR1) is recommended. There is limited literature on the role of consolidation autologous stem cell transplantation (ASCT). This study was undertaken to assess the potential of consolidation ASCT in CR1 in adults with Ph + ALL. Objectives The aim of this study was to analyze the safety and efficacy of ASCT in CR1 in adults with Ph + ALL. Materials and Methods Adult patients diagnosed with Ph + ALL who underwent ASCT in CR1 after modified ALL-BFM95 protocol from 2015 to 2017 were included. Patients who achieved major molecular response or better were considered for ASCT with cyclophosphamide-total body irradiation regimen and peripheral blood stem cells infused on day 0. Toxicities as per Common Terminology Criteria for Adverse Event v4.0, disease-free survival (DFS), and overall survival (OS) were assessed. Inclusion criteria: Following patients were included—patients aged 18 years and above diagnosed with Ph + ALL; patients receiving BFM-95 induction chemotherapy protocol; patients who achieved CR after induction therapy; nonavailability of human leukocyte antigen match from a matched sibling donor or matched unrelated donor. Exclusion criteria: Patients not willing or unfit for ASCT and patients planned for allogenic hematopoietic stem cell transplantation were excluded. Results Six adult patients with Ph + ALL underwent ASCT in CR1 (median age: 23 [range: 19–36] years, five patients were males [83%]). Imatinib was started at a median of 11 days from the start of induction IA (range: 10–21). Five patients achieved morphological CR after induction 1A and, one patient at the end of induction 1B. The median time to ASCT (from diagnosis) was 8 months (range: 6.4–13). All the six patients had disease relapse and died due to progressive ALL. The median DFS and OS were 19.2 months and 23.3 months, respectively. Conclusion Consolidation ASCT yielded poor outcomes in this study. There was a significant delay from diagnosis to ASCT, which might have impacted the results.

Poor prognostic implications of myelodysplasia-related mutations in both older and younger patients with de novo AML

A set of myelodysplasia-related (MDS-R) gene mutations are incorporated into the 2022 European LeukemiaNet risk classification as adverse genetic factors for acute myeloid leukemia (AML) based on their poor prognostic impact on older patients. The impact of these mutations on younger patients (age < 60 years) remains elusive. In the study of 1213 patients with de novo non-M3 AML, we identified MDS-R mutations in 32.7% of the total cohort, 44.9% of older patients and 23.4% of younger patients. The patients with MDS-R mutations had a significantly lower complete remission rate in both younger and older age groups. With a median follow-up of 9.2 years, the MDS-R group experienced shorter overall survival (P = 0.034 for older and 0.035 for younger patients) and event-free survival (P = 0.004 for older and 0.042 for younger patients). Furthermore, patients with MDS-R mutations more frequently harbored measurable residual disease that was detectable using next generation sequencing at morphological CR than those without MDS-R mutations. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) might ameliorate the negative impact of MDS-R mutations. In summary, AML patients with MDS-R mutations have significantly poorer outcomes regardless of age. More intensive treatment, such as allo-HSCT and/or novel therapies, is warranted for AML patients with MDS-R mutations.

Molecular Response Analysis By High Throughput Sequencing in Higher Risk Myelodysplastic Syndrome (HR-MDS) Treated Intensively with CPX-351

Context and objective Intensive chemotherapy (IC) remains an important therapeutic option in HR-MDS, but in-depth evaluation of response to IC remains ill studied. We previously reported results of CPX-351 in 31 untreated higher risk MDS patients (pts) (GFM-CPX-MDS study, NCT04273802, ASH 2021 Abst #243). Responses were associated with a significant decrease of CD34/CD117 progenitors and restoration of normal hematopoiesis patterns in bone marrow (BM) samples in multiparameter flow cytometry. In this ancillary study, the aim was to evaluate molecular responses by high-throughput sequencing (HTS) centrally. This involved description of the pts molecular landscape and an assessment of variant allele frequency (VAF) variations after induction treatment with CPX-351. Methods The mutational landscape was explored in BM nuclear cells by capture HTS of 42 genes (SureSelect XTHS2 Agilent) with unique molecular identifiers (UMI), unique dual index (UDI) and Illumina paired-end sequencing (Miseq and Nextseq). Somatic mutations (SM) were considered significant for VAFs >2%. Molecular response was evaluated by comparing baseline and post-induction. Molecular clearance was defined according to (i) clearance (<2%) of all SM excluding the "pre-leukemic” SM of DNMTA, TET2, ASXL1 (DTA) (ii) clearance of all non-DTA SM < HTS detection threshold (iii) normalized VAF decrease of the major non-DTA baseline SM (nmVAF). Results Median age of the 31 pts included was 62 years (range 31-69). There were 26 MDS-EB2 and 5 CMML-2. IPSS was int-2 in 26 pts and high in 5. Cytogenetic IPSS-R was very good (6.5%), good (71%, including normal in 22 pts), int (9.5%), poor (6.5%) or very poor (6.5%). After induction with CPX-351 (3 days cycle and a possible 2-day second induction cycle for non-responders), 16 pts (52%) reached CR, 4 (13%) CRi, 7 (22%) MLFS while 4 (13%) were in stable disease according to ELN 2017 criteria (which appeared more adequate than MDS IWG 2006, as reported at ASH 2021, Abst #243). Median follow-up was 511 days (179-710), 29/31 patients (94%) received allogeneic hematopoietic stem cell transplantation (Allo-HSCT) after, no, 1, 2, 3 or 4 CPX-351 consolidation cycles (that included only 1 day of CPX-351) in 10, 16, 7, 3 and 1 patients respectively. So far, 4 relapses have occurred, including 3 in transplanted pts. At inclusion, all pts had at least one mutation (median 3, [1-14]) with a mean VAF of 30% (2-89), implicating at least one DTA (71%; 22/31) or one non-DTA genes (93%; 29/31) (fig). Very high or high risk was seen in 19/31 (61%) pts according to the new IPSSm classification. Post induction molecular response was evaluated at a median of 53 days from CPX-351 onset (28-112) in 26 of the 27 who reached at least MLFS, and had a post-induction sample. Molecular clearance was observed (i) at the 2% threshold of non-DTA genes in 17/26 (65%) pts (ii) at the HTS detection threshold (0.1% i.e. minimum considered 3X with median coverage > 3000X) in 11/26 pts (42%) (iii) by nmVAF decrease in 25/26 pts (96%) with a median of -62% (-2 to -100%) (fig) while the remaining pt had an increase of SRSF2 (+67%) and RUNX1 (+46%) SMs but disappearance of three STAG2 SMs and was still in CR after allogeneic SCT. Of note, DTA SMs remained detectable in all pts with a median nmVAF modulation of -86% (+83% to -99.6%, fig) from baseline to post-induction. Of the 4 non-responders to CPX-351, 2 had high risk anomalies (inv(3) and EZH2 / RUNX1 SMs). The 3 relapses observed after alloHSCT, at 7, 8 and 15 months had nmVAFs decrease from baseline to post-induction of -95%, -98% and -50% respectively. Among them, one had a very complex karyotype and a homozygous TP53 mutation, the two others had a high mutational load and EZH2 mutations and one of the latest was transplanted in refractory state. Clonal evolution was observed in 5/26 pts with significant emergence of new mutations involving PPM1D, ASXL1 or JAK2 and in one pt the emergence of EZH2, RUNX1 and TET2 SMs. Four of these pts remained in morphological CR while one progressed to AML. Conclusion In 25/26 CPX-351 responders, a significant decrease of nmVAF was observed, confirming the interest of CPX-351 treatment in higher risk MDS, especially as a bridge to transplant. Follow up is ongoing and few relapses were seen to evaluate the predictive role of molecular analysis. An update will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Functional Implications of MYC Variants As a New Class of Pathogenic Variants in AML

MYC, a well-known oncogene, is implicated in a majority of cancers, but there is little data on its role in myeloid malignancies. We recently described a novel internal tandem duplication of MYC gene (MYC-ITD) in a subset of patients with AML. As the prevalence of MYC aberrations have not been well documented in AML, we interrogated the genome, transcriptome and associated clinical and laboratory data from the largest cohort of pediatric AML patients to define the prevalence, frequency of co-occurring cytomolecular alterations and outcomes associated with MYC variants. Mutation Profiling: Diagnostic specimens from 1,806 patients with AML (age 0-29 years) enrolled on COG Phase III trials were retrospectively interrogated using contemporary NGS techniques or a PCR based "Tandem Repeat" assay for MYC-ITD. Conventional karyotyping and mutational analysis, including NGS, were used to determine additional cytogenetic and molecular abnormalities. Of the 1,806 patients studied, MYC aberrations were identified in 58 patients (3.2%) with 50% (N=29) harboring MYC-ITD, 46.5% (N=27) with a SNV or indel (MYCSNV/indel) and 2 patients (3.5%) with dual MYC-ITD and SNV. For MYC-ITD, tandem duplications occurred in exon 3 (Max binding domain) with ITD lengths ranging from 252-588 bp. The majority of MYCSNV/indel (77%) were identified at the P72-P78 hotspot with 33.3% (N=9) harboring the P74>L mutation and 18.5% (N=5) having the T73>N mutation. Functional Studies: Given the novelty of the ITD variant, we initially characterized the functional properties of MYC-ITD in vitro. Similar to wild-type (MYCWT), MYC-ITD is localized to the nucleus and requires Max to bind to specific DNA sequences (E-boxes). However, in contrast to MYCWT, MYC-ITD fails to recognize sequences containing a single E-box in vitro, but exhibits higher affinity toward sequences with two or more E-boxes. MYC-ITD is consistently more efficient in activating promoters with multiple E-boxes than MYCWT. Thus, MYC-ITD may possess different DNA-binding and protein-interacting properties, potentially leading to altered transcriptional regulation and biological output. Given the alterations in DNA binding properties, we evaluated the functional consequences of MYC-ITD in our cord blood stem cell/endothelial cell (EC) co-culture system. Normal cord blood stem cells were transduced with MYC-ITD vs. MYCWT and grown in an EC co-culture system. MYC-ITD transduced cord blood stem cells showed significantly higher proliferation compared to MYCWT or control GFP-transduced cells with proliferation advantage of 37-and 45-fold after 14 and 21 days in co-culture, respectively (p<0.001). Clinical Impact: Analysis of cooperating cytomolecular lesions revealed a striking difference between MYC-ITD and MYCSNV/indel. MYC-ITD was highly enriched in core-binding factor (CBF) AML found in 86% (N=25)[(N=18, 62%) with inv(16) and (N=7, 24%) with t(8;21)] (Figure 1A), vs. 26% in the MYCWT cohort and had a paucity of somatic mutations. Conversely, MYCSNV/indel was enriched in NUP98-NSD1 fusions (N=7, 27%), FLT3-ITD (N=10, 38%) and NPM1 mutations (N=9, 33%) (Figure 1A), which was significantly higher than the WT cohort. Patients with MYC-ITD had a morphologic CR rate after course 1 of 92.3% vs. 80% (p=0.249) in patients with MYCSNV/indel with a corresponding relapse rate of 24% and 52% (p=0.046). Lower CR and higher relapse reflects the higher proportion of patients with high-risk lesions (FLT3-ITD, NUP98-NSD1 and WT1) in MYCSNV/indel and favorable CBF in MYCITD. Those with MYC-ITD and MYCSNV/indel had a 5-year event free survival (EFS) of 70% vs. 38.5%, respectively (p=0.018) with a corresponding overall survival (OS) of 76% and 47% (p=0.023) similar to patients with favorable or high risk disease (Figure 1B). Conclusion: Here we define MYC aberrations as a new class of pathogenic variants in childhood AML. We demonstrate functional and clinical characterization of a novel MYC-ITD and contrast this novel variant to that of established pathogenic MYCSNV/indel. We show the high prevalence of co-occurring core-binding factor fusions with MYC-ITD, and high frequency of co-occurring NUP98-NSD1, FLT3-ITD and NPM1 mutations in MYCSNV/indel. Patients with MYC-ITD have superior clinical outcomes while patients with MYCSNV/indel have clinical outcomes similar to high-risk patients, likely due to the associations with the disease associated fusions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

KMT2A Partial Tandem Duplications (KMT2A-PTD) Is a Rare, but Recurrent Genomic Event in Childhood AML and Associated with High Rate of Co-Occurring FLT3 Mutations

KMT2A partial tandem duplication (KMT2A-PTD), characterized by a large internal duplication spanning 6-8 exons, has been documented in adult AML with a prevalence of 3-10% and is associated with poor outcomes. Despite the high prevalence of KTM2A fusions and known associated outcomes, prevalence and clinical implications of KMT2A-PTD has not been well characterized in childhood AML. We interrogated the transcriptome and associated clinical and genomic data from pediatric AML patients treated on COG AAML1031 to define the prevalence of KMT2A-PTD, frequency of co-occurring genetic mutations and outcomes associated with this variant.Ribo-depleted RNA seq data from 1,294 patients with AML (age 0-29 years) enrolled on COG AAML1031 with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS), disease-free survival (DFS) and relapse risk (RR).Of the 1,294 patients studied, KMT2A-PTD was identified in 20 patients (1.5%). Evaluation of karyotype demonstrated striking paucity of karyotypic alterations with 75% with normal karyotype; 3 cases (15%) of trisomy 11 were identified. In contrast to KMT2A-rearranged (KMT2A-r) AML, which has the highest prevalence in young patients (median age 3.3 years), the median age with KMT2A-PTD was 13.4 years (p<0.0001). Further analysis demonstrated a high rate of co-occurrence of FLT3 mutations; 15 patients (75%) had FLT3-ITD (N=13; 65%) and/or FLT3 activating mutations (N=5; 25%). The rate of co-occurring FLT3-ITD was significantly higher in the KMT2A-PTD cohort compared to patients without KMT2A-PTD (65% vs. 19%, p<0.001). Prevalence of KMT2A-PTD in patients with FLT3-ITD was 5.7%. Additional co-occurring genetic mutations were identified in WT1 (20%) and the RAS pathway. There was a striking enrichment of mutations that are commonly associated with AML in older adults with 8/20 patients (40%) harboring mutations in IDH1/IDH2, U2AF1, and TP53, far in excess of what is seen in other pediatric AML patients (Figure 1A).We evaluated the significance of KMT2A-PTD on clinical outcome. Patients with KMT2A-PTD had a poor response to induction therapy with a morphologic CR rate of 45% and rate of residual disease (MRD) positivity by flow cytometry after initial course of therapy was 40%. Additionally, high rates of relapse were noted for patients with KMT2A-PTD with a RR of 53%. EFS for patients with and without KMT2A-PTD was 39% vs. 46%, respectively (p=0.54) with a corresponding OS of 58% and 64% (p=0.61). Outcome analysis for the KMT2A-PTD cohort demonstrated dual KMT2A-PTD/FLT3-ITD patients had an EFS of 46% compared to 29% for KMT2A-PTD/non-ITD patients (p=0.62, Figure 1B). We inquired whether hematopoietic stem cell transplant (HSCT) may modify outcomes in patients with KMT2A-PTD. Of the 20 patients with KMT2A-PTD, 7 (35%) proceeded to HSCT in CR1, with the indication in 6 of 7 patients due to FLT3-ITD high allelic ratio (HAR, ≥0.4). Stem cell transplant recipients had a DFS of 83% vs. 0% for those who continued on protocol chemotherapy (p=0.002, Figure 1C) with corresponding OS of 100% vs. 43%, respectively (p=0.05).In this large cohort of childhood AML patients treated on AAML1031, we identified a small subset of patients with KMT2A-PTD and show the high prevalence of co-occurring FLT3 mutations. Although KMT2A-PTD patients with and without FLT3-ITD had similar outcomes, the cohort who received HSCT in CR1 (most with FLT3-ITD) experienced improved outcomes compared to the rest of the cohort, suggesting that intensification of therapy may benefit this group of patients overall. Further research into KMT2A-PTD in pediatric AML will guide future risk-adapted therapy and enhance understanding of biologic implications of this lesion, including whether altered KMT2A may serve as a therapeutic target as it may for KMT2A-r AML. [Display omitted] DisclosuresPollard: Syndax: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees.

A Comparison Study of Anti-CLL1 CART Cells Equipped with Different Co-Stimulatory Domains in the Treatment of Children with Refractory/Relapsed Acute Myeloid Leukemia

Background: It's well established that conventional chemotherapy doesn't benefit children with refractory/relapsed acute myeloid leukemia (R/R-AML). Though multiple attempts, i.e., novel target agents, and intensive chemotherapy, have been translated into clinics, few have been proved to be successful. Chimeric antigen receptor (CAR) T cell therapy is increasingly becoming a curative candidate for refractory hematological malignancies. Recently, anti-CLL1 CAR T-cells have been reported to effectively treat children with R/R-AML by several groups even though with a small sample size. However, the impact of co-stimulatory domain (CD28/CD27 versus 4-1BB) on the efficacy of CAR T-cells could not be neglected, which is not investigated so far. Thus, we here compare the impact of different costimulatory molecules on the safety and efficacy of anti-CLL1 CAR T-cells in treating children with R/R-AML.Methods: Seven R/R-AML children treated with anti-CLL1-based CAR T-cells were enrolled into this preliminary comparison study. The baseline characteristics, time of CAR T-cells generation, in vivo persistence of CAR T-cells, cytokine release profiling, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), secondary hemophagocytic lymphohistiocytosis (sHLH) or macrophage activation syndrome (MAS), and other adverse events, the length of hospitalization, remission status, and the follow-up data were collected for analysis.Results: Among these seven patients, four received CD28/CD27 based CAR T-cells, and three received 4-1BB based CAR T-cells therapy. The CD28/CD27 group consisted of two boys and two girls, with the median age of 7.5 (range; 5 to 11), while the 4-1BB group consisted of two boys and one girl, with the median age of 12 (range; 8 to 12). Generation of CD28/CD27 equipped CAR T-cells (median, 14 days) were faster than that of 4-1BB ones (median, 21 days). All patients experienced grade 1 to 3 CRS and were well managed with one patient experiencing grade 1-2 ICANS but without any sHLH or MAS. The maximum durations were 156 days for CD28/CD27, ranging from 0 to 156 days and 274 days for 4-1BB, ranging from 16 to 274 days) respect. For hospitalization, CD28/CD27 group experienced subtle longer hospital stays (median, 23 days; range, 14 to 30 days) than 4-1BB group (median, 21 days; range, 18 to 54 days). The overall response rates were 67 % and 75 % in 4-1BB group and CD28/CD27 group respectively. Three of four patients in CD28/CD27 group achieved morphological remission with MRD negativity, while two of three patients in 4-1BB group achieved morphological CR, with one as being MRD negativity. Of all the seven cases, only one patient proceeded to hematopoietic stem cell transplant (HSCT) and died of GVHD seven months after HSCT, however, another four patients who achieved CR could not received HSCT due to poor economic status. Of the remaining six patients, two non-respondent died of disease progression, one patient relapsed and subsequently died, and the other three remained alive and being followed up.Conclusion: Though the enrollment was too small for us to draw a conclusion, we have noticed a similar efficacy/safety index between 4-1BB and CD28/CD27 equipped anti-CLL1-based CAR T-cells in treating children with R/R-AML. In sum, the relatively high efficacy and low grade and manageable side effects were also validated in this study. DisclosuresNo relevant conflicts of interest to declare.

Development of Prognostic Prediction Model for Decision of Secondary Allogeneic Stem Cell Transplantation for Relapsed Acute Myeloid Leukemia after First Stem Cell Transplantation

BackgroundStem cell transplantation (SCT) represents the effective therapeutic option for acute myeloid leukemia (AML). However, recurring relapses remains the major cause of treatment failure. Although limited therapeutic options are available after a relapse of AML following the first autologous/allogeneic SCT (SCT1), the second SCT from the allogeneic donor (allo-SCT2) could achieve durable remission in some selective patients. As a response, this study aimed to identify factors that affect survival outcomes and develop a prognostic model using identified factors for patients who are candidate for allo-SCT2.MethodA retrospective cohort of consecutive 78 adult AML patients who received allo-SCT2 following any cycles of intensive chemotherapy (IC) for the disease relapse after SCT1 was presented. Twenty-five patients underwent relapse after autologous SCT1, whereas fifty-three patients faced relapsed after allogeneic SCT1.ResultThe median age of the patients was 41 years old (19-67). At the time of allo-SCT2, 18 patients were refractory despite administration of IC, but 60 achieved response of complete remission (i.e. CR, n=27)/ CR with incomplete (CRi, n=33); after 1 cycle of IC, 48 achieved response; after 2 cycles, 11; and after 3 cycles, only 1. The median follow-up of survivors (n=22) was 40.7 months (1.8-169.2). Four years from allo-SCT2, the overall survival (OS) rate, leukemic-free survival rate, and the cumulative incidences of relapse and non-relapse mortality were 28.7%, 28.7%, 24.6%, and 43.6%, respectively. In multivariate analysis, the poor cytogenetic risk at diagnosis (p <0.001), the circulating blast ≥ 20% at relapse (p=0.007), the duration from first transplant to relapse < 9 months (p=0.003), the failure to achieve morphologic CR at allo-SCT2 (p=0.001) were potential factors associating poor OS. These factors taken into account, the Catholic AML Second transplantation (CAST) model was developed; each intermediate and poor cytogenetic risk at diagnosis [0.5 and 1 point], peripheral blast≥20% at relapse [1 point], duration from first transplant to relapse <9 months [1 point], and failure to achieve morphological CR at allo-SCT2 [1 point] added up to present a total score from 0 to 4. The CAST model identified three subgroups with 4-year OS of 100.0% in low (score 0, n=4), 46.5% in intermediate (score 0.5-1.5, n=38), and 2.8% in high risk (score≥2, n=36) groups (p <0.001).ConclusionThe CAST model offers a relevant tool to predict survival for patients receiving allo-SCT2, a fact that may be useful to making an appropriate decision in terms of selecting a candidate for allo-SCT2 in relapsed AML after SCT1. [Display omitted] DisclosuresKim:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Allogeneic Hematopoietic Cell Transplantation for Relapsed and Refractory Philadelphia Negative B Cell ALL in the Era of Novel Salvage Therapies

Introduction of novel salvage therapies and expansion of the donor pool within the past decade have allowed more patients with relapsed/refractory (r/r) B cell acute lymphoblastic leukemia (B-ALL) to receive allogeneic hematopoietic cell transplantation (alloHCT). The impact of each salvage therapy on transplant outcomes have not been compared. Our primary objective was to determine post-HCT relapse-free survival (RFS) in adult patients with r/r Philadelphia-chromosome negative (Phneg) B-ALL. We retrospectively studied alloHCT outcomes in 108 adult patients with r/r Phneg B-ALL transplanted in morphological remission achieved by salvage therapy. Salvage therapies were chemotherapy-based combination (n=45, 42%), blinatumomab (n=43, 40%), inotuzumab (n=14, 13%), or CAR T cells (n=6, 6%). The 2-year RFS and overall survival (OS) were 44% and 50%, respectively. In multivariable analysis, conditioning with reduced-intensity or non-myeloablative regimens (hazard ratio [HR]=2.23, 95% confidence interval [CI], 1.31-3.80; P=.003), having received ≥3 lines of therapies prior to transplant (HR=2.66, 95% CI, 1.56-4.54; P < .001), and inotuzumab (HR=2.42, 95% CI, 1.14-5.12; Wald P value=.021) were independently associated with lower RFS. Blinatumomab (HR=1.10, 95% CI, 0.62-1.96) had comparable RFS to chemotherapy. Incidence of hepatic sinusoidal syndrome was highest with inotuzumab (P < .001); however, 30-day mortality and intensive care unit admissions were not different per salvage therapy. The alloHCT in r/r Phneg B-ALL after remission induction with blinatumomab or chemotherapy led to encouraging outcomes if morphologic CR was achieved. In contrast, pretransplantation inotuzumab therapy was associated with inferior RFS. Larger studies are warranted to confirm our observations. Early transplantation after relapse and the utilization of myeloablative conditioning, when feasible, were key factors associated with improved outcomes after alloHCT in these patients.

AZA + Glutaminase Inhibition with Telaglenastat (CB-839) for Advanced MDS: An Updated Interim Analysis

AZA + Glutaminase Inhibition with Telaglenastat (CB-839) for Advanced MDS: An Updated Interim Analysis

Phase II Study of Blinatumomab and Concurrent Oral Tyrosine Kinase Inhibitor Therapy As Consolidation and Maintenance Therapy for Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Following Chemotherapy-Sparing Induction

Background: Combining oral ABL-targeted tyrosine kinase inhibitors (TKIs) with (w/) multi-agent chemotherapy has improved the long-term disease-free survival of adults w/ Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). However, Ph+ ALL occurs more commonly in older adults, and toxicities of multi-agent chemotherapy, including sequelae of prolonged myelosuppression, are amplified in this population. Others have previously reported rates of morphologic complete response (mCR) approaching 100% among adults w/ Ph+ ALL treated w/ corticosteroids (CS) and dasatinib (DAS) alone as induction therapy, but w/ low rates of minimal residual disease (MRD) negativity by flow cytometry (FACS) and BCR-ABL1 PCR (complete molecular response, CMR) and high rates of relapse in the absence of further consolidation. The bispecific T-cell engager blinatumomab (BLIN) has considerable efficacy in clearing MRD in patients (pts) w/ Ph- B-cell ALL. We have previously reported our institutional experience combining ABL TKIs w/ BLIN in pts w/ Ph+ ALL and MRD, including encouraging safety data and high rates of MRD eradication (King/Geyer et al., Leuk Res, 2019). The ongoing D-ALBA study (GIMEMA LAL2116) is also investigating BLIN + DAS consolidation following 12 weeks of induction (prednisone + DAS followed by DAS), w/out protocol-specified maintenance, and has demonstrated preliminary evidence of efficacy (Chiaretti et al., ASH Meeting, 2019). As such, we designed a phase II study of BLIN as part of a chemotherapy sparing strategy in pts w/ Ph+ ALL (BLISSPHALL), introducing BLIN as early as 6 weeks into treatment for pts in morphologic CR, w/ aim of enhancing early MRD negativity and suppressing resistant clones early in disease course. Study design and methods: Our institution is leading a phase II trial of TKI + BLIN consolidation and maintenance in adults w/ newly-diagnosed Ph+ ALL, w/ potential multicenter expansion (NCT04329325). Pts are eligible if they are ≥18 years-old w/ Ph+ ALL confirmed by cytogenetic or molecular studies, ECOG performance status 0-2, w/out prior therapy for ALL beyond CS, hydroxyurea, or intrathecal chemotherapy, w/out known active extramedullary disease and/or CNS-3 disease, and w/ appropriate organ function. See Figure 1: pts will receive a CS pre-phase (days [d] -6 - 0) followed by modified GIMEMA LAL1205 induction (dexamethasone [DEX] 10 mg/m2 [max 24 mg/d], d1-24, tapered off d25-32) + DAS 140 mg/d (dose adjustments or TKI change permitted per protocol) w/ intrathecal methotrexate (IT MTX) d22, 43 and bone marrow (BM) assessments including FACS and BCR-ABL1 PCR. Pts in mCR on d43 (or optional reassessment ≤ 3 weeks later) will be eligible to proceed to consolidation w/ 3 cycles (C) BLIN 28 mcg/d IVCI, d1-28, concurrent w/ TKI, w/ 14d off BLIN between cycles and BM MRD assessment/IT MTX after each cycle. C1 BLIN + TKI is required to begin inpatient x72 hours. TKI is given continuously including between BLIN cycles. Pts in CMR after consolidation may proceed to maintenance (C4-7 BLIN + TKI, 28d off between cycles). Pts may come off study to proceed to hematopoietic cell transplant (HCT) at any point, though it is recommended such pts receive ≥2C of BLIN + TKI. The primary objective is to determine the proportion of evaluable pts achieving CMR by the end of consolidation (≤ 3C BLIN + TKI). Secondary objectives include safety/toxicity of BLIN + DAS, duration of CMR, incidence of relapse, event-free/overall survival. Exploratory objectives include safety/toxicity of non-DAS TKIs + BLIN, defining patterns/mechanisms of resistance to BLIN+TKI (including ABL kinase mutations), and outcomes among pts not undergoing HCT. The trial utilizes a Simon's minimax two-stage design; 20% CMR rate is considered not promising, a 50% CMR rate is considered promising, and probabilities of type I/II error are set at 0.10/0.10. If ≥ 3 of the first 10 pts achieve CMR we will continue enrollment to max 17 pts. If ≥ 6 pts achieve CMR, then BLIN + TKI will be considered promising for further investigation. The investigators are hopeful this study will add to the currently limited prospective data supporting TKI + BLIN consolidation/maintenance for pts w/ Ph+ ALL and efforts to develop chemotherapy-sparing and immunotherapeutic strategies for older pts w/ ALL. Disclosures Geyer: Amgen: Research Funding. King:Abbvie: Other: advisory board. Park:Novartis: Consultancy; Genentech/Roche: Research Funding; Intellia: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Takeda: Consultancy, Research Funding; Servier: Consultancy, Research Funding; AstraZeneca: Consultancy; Allogene: Consultancy; Incyte: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Autolus: Consultancy, Research Funding; Minverva: Consultancy; Amgen: Consultancy, Research Funding. OffLabel Disclosure: Blinatumomab is not approved to be given in combination with ABL tyrosine kinase inhibitors for treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Newly Diagnosed Childhood AML Patients Treated with Bortezomib Show Superior Survival If CD74 Is Expressed: A Report of 991 Patients from the Children's Oncology Group AAML1031 Protocol

Introduction: CD74 is a type II transmembrane protein expressed on antigen-presenting cells and an MHC class II chaperone. It has been associated with tumor progression and metastasis in solid tumors, and its expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression associated with oncogenesis. As the expression of CD74 has been associated with response to bortezomib in multiple myeloma, we inquired whether such correlation might be seen in pediatric AML. We prospectively evaluated CD74 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome as part of Children's Oncology Group protocol AAML1031 that randomized patients younger than 30 years of age with de novo AML to standard treatment with (Arm B) or without (Arm A) bortezomib. The addition of bortezomib to standard chemotherapy increased toxicity but did not improve survival. Given the association of CD74 with B-lymphoid neoplasms and bortezomib's known efficacy in B-cell neoplasms and multiple myeloma, we hypothesized that within Arm B, the patients with CD74 expression would have a more favorable outcome. Methods: In total, 1,139 newly diagnosed pediatric patients with de novo AML were randomized to standard chemotherapy (n=561, Arm A) or standard chemotherapy with bortezomib (n=578, Arm B). All patients received the identical chemotherapy backbone with either four intensive chemotherapy courses or three courses followed by allogeneic hematopoietic stem cell transplantation for high-risk patients. To qualify for this correlative study, 991 patients satisfied 2 criteria: (1) submitting a bone marrow aspirate for ΔN at diagnosis and (2) providing consent for correlative biology studies. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD74 by ΔN. AML was considered to be CD74-positive if the MFI was more than two times above background autofluorescence and more than 40% of the leukemia was above background autofluorescence. Results: Among 991 patients, 263 were CD74-positive (26.5%) by ΔN, with similar prevalence in Arm A (27.9%) and Arm B (25.2%). Correlation of CD74 expression with clinical characteristics showed that those with CD74 expression had higher median age (p&amp;lt;0.001), lower median WBC (p&amp;lt;0.001), higher prevalence of low risk protocol status (p=0.039), lower frequency of CEBPA mutation (p=0.039), inv(16) (p=0.001), and KMT2A rearrangements (p=0.002), and were enriched for t(8;21) (p&amp;lt;0.001) and t(6;9) (p=0.014) fusions. All these features retained significance when patients were sub analyzed by respective treatment arms. CD74-positive patients had a higher morphologic CR rate (p=0.016), however, measurable residual disease by flow cytometry was not significant in the entire cohort or in the sub analysis of treatment arms (p=0.155). CD74-positive patients showed superior 5-year OS (70.6% vs 61.8%, p=0.003) and EFS survival (51.2% vs 43.1%, p=0.007) compared to those who were CD74-negative. For patients in Arm A (no bortezomib), the differences in OS (66.1% vs 61.0%, p=0.138) and EFS (48.9% vs 41.7% p=0.088) were not significant between those that were CD74-positive and those that were CD74-negative (Figure 1). However, patients in Arm B receiving bortezomib that were CD74-positive showed a significant improvement in OS (75.3% vs 62.5%, p=0.006) and EFS (53.6% vs 44.3%, p=0.028) compared to those who were CD74-negative (Figure 1). Comparison of the outcomes for CD74-positive patients with and without bortezomib exposure showed a difference in OS of 66.1% vs. 75.3% for those in Arm A vs. Arm B but did not reach significance (p=0.155). Multivariable analysis for OS yielded a hazard ratio of 0.67 (95% CI: 0.44 - 1.02) and p=0.061, approaching, but not reaching, statistical significance. Conclusions: These data demonstrate that CD74 expression is associated with more favorable disease characteristics and survival. Patients receiving bortezomib that were CD74-positive showed a superior response to therapy compared to patients who did not express CD74, by both OS and EFS, suggesting that CD74-positive childhood AML patients stand to benefit from bortezomib therapy. Bortezomib may induce a mechanistic response in CD74-positive AMLs similar to that in bortezomib-treated B-cell neoplasms and/or multiple myeloma, where bortezomib has proven to be beneficial. Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019). Pollard:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Allogeneic stem cell transplant (HSCT) for acute lymphoblastic leukaemia (ALL) using CD34 selected stem cells followed by prophylactic infusions of pathogen-specific and CD19 CAR T cells

Background & Aim Disease relapse, graft versus host disease (GVHD) and infection are responsible for the majority of deaths after HSCT. We assessed a cellular engineering approach to simultaneously reduce the incidence of these complications using CD34+ stem cell selection combined with post-transplant infusion of pathogen-specific and leukemia-specific T cells. Methods, Results & Conclusion METHODS We conducted a pilot study in which patients with ALL in remission were treated with a CD34-selected stem cell transplant followed by two planned prophylactic infusions of donor-derived T cells targeting opportunistic pathogens (CMV, EBV and Aspergillus), and leukaemia (CD19 CAR T cells). Cellular products were manufactured locally. Patients did not receive anti-thymocyte globulin or post-transplant GVHD prophylaxis. RESULTS We performed matched sibling donor transplants on two patients aged 45 and 27 years with ALL in morphological CR. Patient 1 had Ph1 positive ALL with detectable BCR-ABL at the time of transplant (0.001%). Patient 2 was Ph1 negative and had no detectable disease by flow cytometry pre-transplant. Conditioning was cyclophosphamide 120mg/kg and TBI 1200cGy. Patients received CD34 selected stem cells (total CD34+ doses 3.5 and 3.6 × 106/kg; total CD3+ cell doses 1.3 and 0.2 × 104/kg). Neutrophil engraftment (>0.5) occurred on days 11 and 12, platelet engraftment (>20) on days 8 and 13. Patients received infusions of pathogen specific T cells (day 21) and CD19 CAR T cells (days 27 and 21). CRS (grades 1 and 2) and neurotoxicity (grade 1) developed in both patients; treated with tocilizumab, patient 1 also received dexamethasone. CAR T cells proliferated in vivo despite low disease burden and persisted in blood for at least 6 weeks in both patients (Figure 1). Patient 1 received foscarnet for 9 days and a second pathogen-specific T cell infusion on day 47 post-transplant. Patient 2 developed asymptomatic reactivation of HHV6 and BK virus. At 364 and 350 days post-transplant, both patients are well, free of GVHD and remain in remission. CONCLUSION This is the first description of a strategy that combines prophylactic donor-derived CAR T cell and pathogen-specific T cell administration after HSCT in the context of CD34+ selection to minimise development of GVHD. Our results demonstrate absence of GVHD, minimal treatment for viral reactivation and remission at 12 months post-transplant suggesting that this approach has promise for improving GVHD relapse free survival following HSCT.

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

A Phase Ib/II Study of the Histone Methyltransferase Inhibitor Pinometostat in Combination with Azacitidine in Patients with 11q23-Rearranged Acute Myeloid Leukemia

Clinical Activity of CC-90009, a Cereblon E3 Ligase Modulator and First-in-Class GSPT1 Degrader, As a Single Agent in Patients with Relapsed or Refractory Acute Myeloid Leukemia (R/R AML): First Results from a Phase I Dose-Finding Study

Clinical Activity of CC-90009, a Cereblon E3 Ligase Modulator and First-in-Class GSPT1 Degrader, As a Single Agent in Patients with Relapsed or Refractory Acute Myeloid Leukemia (R/R AML): First Results from a Phase I Dose-Finding Study

Adult Case Series of Extramedullary Relapsing Acute Myeloid Leukemia (AML) after Allogeneic Stem Cell Transplantation in Whom a Rapid Response and a Reasonably Long Remission Could be Achieved with Gemtuzumab Ozogamycin (GO)

Gemtuzumab Ozogamycin (GO) is a drug conjugated monoclonal antibody which targets CD33 an antigen highly expressed on the surface of AML blasts. GO is approved for the treatment of both newly-diagnosed and relapsed AML. Despite growing evidence regarding its efficacy and safety among AML patients there is limited data about the use of GO in isolated extra-medullary relapsed AML. Extramedullary AML remains as an unmet clinical need regarding the poor prognosis and lack of a standard therapeutic approach. Our cases demonstrated a rapid and long lasting response with a favorable toxicity profile, in patients who were treated with GO as a single agent after an isolated extramedullary relapsing disease. Our first case was a 40-years-old woman admitted to our outpatient clinic with pain in her joints and redness in her back and stomach. She was diagnosed with CD33-positive Acute Myeloid Leukemia according to the immunophenotyping of bone marrow blasts. Idarubicin and ARA-C (7+3) combination initiated as a frontline induction therapy and morphological remission was achieved after the first cycle of induction. After the first cycle of consolidation chemotherapy with high dose ARA-C she has relapsed with skin myeloid sarcomas and diffuse bone marrow infiltration. Soon after the relapsing disease she was put on to ida-flag salvage chemotherapy and she responded well with the disappearance of skin lesions and morphological complete remission of bone marrow. After achieving response, an allogeneic stem cell transplantation (HSCT) was applied from her sibling donor with a myeloablative conditioning. Unfortunately patient had a relapsing extramedullary disease with reappearance of skin lesions even with an ongoing acute skin gvhd (Figure) soon after allogeneic HSCT. Bone marrow biopsy revealed no increase in blast count. We have decided to initiate a novel targetted therapy to control the extramedullary disease. As her blasts infiltrating the skin were universally CD 33 positive we considered to put her on GO therapy with the approval obtained from health authority. GO was applied according to the dosage approved by FDA for relapsing disease. After the first cycle of GO she had a rapid disappearance of all skin lesions just complicated with a febrile neutropenic episode and re-activation of CMV infection which was controlled with Gancyclovir. She has retained a complete remission regarding extramedullary disease throughout the repeated courses of GO for 3 times, and she is still in remission for 4 months after the first appearance of skin lesions (Figure). Our second case was a 24-years-old boy who admitted to our outpatient clinic with a worsening fatigue and shortness of breath. He was also diagnosed with CD33-positive Acute Myeloid Leukemia according to the immunophenotyping of bone marrow blasts and was able to achieve a morphological CR after frontline induction therapy with 7+3 protocol. After two cycles of high dose ARA-C consolidation he was transplanted from a matched unrelated donor because of an intermediate cytogenetic risk profile. At the 14th month of allogeneic transplantation he had relapsed with a bone marrow blast count of 90% and harboring a monosomy on the 10th chromosome. After the first salvage chemotherapy with IDA-FLAG protocol he has achieved a morphological CR and we have decided to proceed with an alternative donor transplantation. But unfortunately soon after discharge, he has admitted to the emergency clinic with a new onset headache and nausea and vomiting. A cranial CT revealed multiple foci of solid masses with peripheral edema. We have performed a lumbar puncture and CSF fluid revealed a high number of CD33 positive blastic cells (over 100 cells per HPF). At the time of central nervous system (CNS) relapse his bone marrow was free of blastic infiltration. We decided to initiate GO treatment with the diagnosis of isolated central nervous system relapse of AML, accompanying intra-thecal chemotherapy via an Omaya Reservoir. He has received two cycles of GO which was complicated with neutropenic fever and grade 4 leukopenia and thrombocytopenia, and his CNS lesions also responded well and clinically he had no CNS related signs or symptoms. The patient received additional GO with continued response but unfortunately after a severe pneumonia he passed away at intensive care unit. Our case series documented a clinically relevant therapeutic field for GO in isolated extramedullary relapse of AML. Disclosures No relevant conflicts of interest to declare.

Impact of Minimal Residual Disease Detected By Multiparameter Flow Cytometry on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia Patients in First Complete Remission

Introduction. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be curative for acute myeloid leukemia (AML) patients. However, the disease relapse after allo-HSCT lead to poor outcomes almost in all cases. Minimal residual disease (MRD) is a strong prognostic marker of increased risk of relapse and reflects overall (OS) and relapse-free survival (RFS) in patients with AML. Immunophenotypic evaluation using multiparameter flow cytometry (MFC) is an important strategy for detecting MRD, because of its simplicity and wide availability. Aim. Determine the impact of MRD detected by MFC before allo-HSCT on clinical outcomes. Patients and methods. To assess the impact of MRD status before transplantation we include 56 AML patients who underwent allo-HSCT in National Research Center for Hematology between July 2016 and June 2019. Patient's characteristics are shown in table 1. All these patients were in first compete morphologic remission at the time of allo-HSCT. MRD was evaluated by MFC. Bone marrow samples were obtained before allo-HSCT and were analyzed by 6-color MFC (BD FACS Canto II, USA). Positive MRD was identified as a cell population deviating from the normal patterns of antigen expression on specific cell lineages at specific stages of maturation for all patients. In addition, for patients with LAIP at diagnosis, positive MRD was also defined as a cell population carrying LAIP markers at diagnosis. The probabilities of OS and RFS were estimated using the Kaplan-Meier method. Results. Among 56 AML patients before allo-HSCT (all were in CR at the time of MRD analysis). Median of follow-up was 9.3 months.11 patients (19,6%) were identified as MRD+ (0,011%-5,08%, mean 1%). And 5 of them relapsed (45,5%). Out of 45 MRD negative patients before HSCT relapse was registered in one (relapse rate 2,2%). Statistical analysis revealed significant differences between MRD- and MRD+ patients: MRD+ had much worse OS (22,9% vs. 85.5%, p=0.0047, Fig.1A) and RFS (26% vs. 97.4%, p&lt;0,0001, Fig.1B). Conclusions. Despite the presence of morphological CR before allo-HSCT, MRD persistence can lead to worse probabilities of OS and RFS, which is comparable to patients who were transplanted in relapse. Because of the association between MRD and relapse risk, it can be assumed, that testing for MRD before allo-HSCT may help to identify a subgroup of patients at high risk of relapse who might benefit from preemptive posttransplantation treatment. Disclosures No relevant conflicts of interest to declare.