Positive Acute Myeloid Leukemia Research Articles

Label-Free Identification of AML1-ETO Positive Acute Myeloid Leukemia Using Single-Cell Raman Spectroscopy.

Acute myeloid leukemia (AML) is a malignant hematological tumor disease. Chromosomal abnormality is an independent prognostic factor in AML. AML with t(8:21) (q22; q22)/AML1-ETO (AE) is an independent disease group. In this research, a new method based on Raman spectroscopy is reported for label-free single-cell identification and analysis of AE fusion genes in clinical AML patients. Raman spectroscopy reflects the intrinsic vibration information of molecules in a label-free and non-destructive manner, and the fingerprint Raman spectrum of cells characterizes intracellular molecular types and relative concentration information, so as to realize the identification and molecular metabolism analysis of different kinds of cells. We collected the Raman spectra of bone marrow cells from clinically diagnosed AML M2 patients with and without the AE fusion gene. Through comparison of the average spectra and identification analysis based on multivariate statistical methods such as principal component analysis and linear discriminant analysis, the distinction between AE positive and negative sample cells in M2 AML patients was successfully achieved, and the single-cell identification accuracy was more than 90%. At the same time, the Raman spectra of the two types of cells were analyzed by the multivariate curve resolution alternating least squares decomposition method. It was found that the presence of the AE fusion gene may lead to the metabolic changes of lipid and nucleic acid in AML cells, which was consistent with the results of genomic and metabolomic multi-omics studies. The above results indicate that single-cell Raman spectroscopy has the potential for early identification of AE-positive AML.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

Research Progress of Isocitrate Dehydrogenase Mutation-Positive Acute Myeloid Leukemia --Review

Isocitrate dehydrogenase (IDH) is an enzymes involved in a variety of metabolic and epigenetic processes. IDH can be detected in approximately 20% of patients with acute myeloid leukemia (AML), the mutated IDH enzyme acquires new oncogenic enzyme activity and converts α-ketoglutaric acid (α-KG) to the tumor metabolite 2-hydroxyglutaric acid (2-HG), which accumulates at high levels in cells and hinders the function of αKG-dependent enzymes, including epigenetic regulators, resulting in DNA hypermethylation, abnormal gene expression, cell proliferation, and abnormal differentiation, and contributes to leukemia disease progression. IDH mutations have different effects on the prognosis of patients with AML depending on the location of the mutation and other co-occurring genomic abnormalities. This paper will review the latest research progress on the IDH positive AML gene changes, prognosis, and inhibitors.

Abstract 3604: NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts

Abstract Introduction: Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD+) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D CAR and membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD−). In this study, we utilize a flow cytometry-based approach to (i) evaluate the cell surface expression of NKG2D-Ls on normal cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine NKX101 cytotoxic killing of LSC and non-LSC AML blasts. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients (n=20) and healthy donors (n=10) in accordance with approved IRB protocols. AML somatic mutation status was determined using an Illumina TruSight® Myeloid Sequencing Panel. NKX101 cytotoxicity against patient-derived AML blasts was assessed using a flow cytometry-based assay that measured specific cytotoxicity and AML blast/LSC markers. NKG2D-L expression was determined using flow cytometry with antibodies specific to individual NKG2D-Ls. Results: Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on LSCs compared to non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 potently kills patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 had similar cytotoxic potency against both LSC and non-LSC AML blasts. Conclusions: In summary, we show that NKG2D-Ls are expressed on the surface of both LSC and non-LSC AML blasts and at levels above those observed in normal hematopoietic cells. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSCs in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR-NK therapy for the treatment of r/r AML. Citation Format: Kyle Hansen, Cynthia Cho, Nishi Kothari, David Shook, James Trager. NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3604.

Abstract 6712: LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia

Abstract Background: Acute myeloid leukemia (AML) is a malignant clonal disorder linked to a broad spectrum of molecular alterations. It is still an incurable cancer, the prognosis is even worse in elderly patients, where overall survival (OS) at 1 year is approximately 10-15%. Now, several CD3 bispecific immunotherapies that harness T cells against AML are at various stages of preclinical and clinical development, but most of them are hampered by the lack of specific targets to distinguish tumor cells from normal hematopoietic stem cells (HSC). Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immune checkpoint inhibitory receptor, which is highly expressed on FAB M4 and M5 AML cells with a lack of expression on normal HSC and progenitor cells. LILRB4 expression level inversely correlates with OS of patients with M4 and M5 AML. All of these indicate that LILRB4 represents a promising target for developing a T cell redirecting bispecific antibody to treat patients with AML, especially for more aggressive M4 and M5 subtype. Herein a novel bispecific antibody targeting LILRB4 and CD3, LBL-043, is developed to specifically kill LILRB4 positive tumor cells by engaging T cells. Methods: LBL-043 was constructed using our proprietary LeadsBodyTM T cell engager platform, which is a 2:1 format bispecific antibody with two VHH arms targeting LILRB4 with high affinity and one scFv arm targeting CD3 with fine-tuned low affinity. The binding affinity of LBL-043 to LILRB4 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-043 was investigated in B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 (human AML-M5a) tumor model. Results: The binding affinity of LBL-043 to LILRB4 and CD3 protein was 0.724 nM and 35.7 nM, respectively. In CD3 reporter gene assays, LBL-043 could activate the NFAT reporter signaling through LILRB4 binding dependent CD3 cross-linking with an EC50 value of 2.459 nM. LBL-043 was shown a potent TDCC activity in different LILRB4 expression level MOLM-13 cells and HL-60 cells with T cell activation and cytokine release. In B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 tumor cells, LBL-043 was shown significant anti-tumor activity. Simultaneously, LBL-043 can effectively improve the survival rate and maintain relatively stable body weight in the study compared to the vehicle group. Conclusion: LBL-043, a novel bispecific antibody targeting CD3 and LILRB4 with affinity differentiation, induces T cell killing of AML cells by simultaneously binding LILRB4 expressed on tumor cells and CD3 on T cells, redirecting T cells to efficiently killing of AML cells. It is also shown a great anti-tumor efficacy in CDX mouse models. These data support LBL-043 as a novel therapeutic bispecific antibody for the treatment of LILRB4 positive AML and CMML leukemia patients. Citation Format: Yujia Dang, Xiao Huang, Yurong Qin, Xiaoya Liu, Min Chen, Duqing Jiang, Guojin Wu, Mi Ye, Jianming Sun, Baohui Wang, Jing Guan, Tingting Li, Jordan Zhu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6712.

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

Prognostic impact of cooccurring mutations in FLT3-ITD pediatric acute myeloid leukemia

AbstractWe sought to define the cooccurring mutational profile of FLT3-ITD–positive (ITDpos) acute myeloid leukemia (AML) in pediatric and young adult patients and to define the prognostic impact of cooperating mutations. We identified 464 patients with FLT3-ITD mutations treated on Children’s Oncology Group trials with available sequencing and outcome data. Overall survival, event-free survival (EFS), and relapse risk were determined according to the presence of cooccurring risk stratifying mutations. Among the cohort, 79% of patients had cooccurring alterations across 239 different genes that were altered through mutations or fusions. Evaluation of the prognostic impact of the cooccurring mutations demonstrated that patients with ITDpos AML experienced significantly different outcomes according to the cooccurring mutational profile. Patients with ITDpos AML harboring a cooccurring favorable-risk mutation of NPM1, CEBPA, t(8;21), or inv(16) experienced a 5-year EFS of 64%, which was significantly superior to of 22.2% for patients with ITDpos AML and poor-risk mutations of WT1, UBTF, or NUP98::NSD1 as well to 40.9% for those who lacked either favorable-risk or poor-risk mutation (ITDpos intermediate; P < .001 for both). Multivariable analysis demonstrated that cooccurring mutations had significant prognostic impact, whereas allelic ratio had no impact. Therapy intensification, specifically consolidation transplant in remission, resulted in significant improvements in survival for ITDpos AML. However, patients with ITDpos/NUP98::NSD1 continued to have poor outcomes with intensified therapy, including sorafenib. Cooccurring mutational profile in ITDpos AML has significant prognostic impacts and is critical to determining risk stratification and therapeutic allocation. These clinical trials were registered at www.clinicaltrials.gov as NCT00002798, NCT00070174, NCT00372593, and NCT01371981.

Clinical observation on 16 cases of DEK-NUP214 fusion gene positive acute myeloid leukemia treated with allogeneic hematopoietic stem cell transplantation

Clinical observation on 16 cases of DEK-NUP214 fusion gene positive acute myeloid leukemia treated with allogeneic hematopoietic stem cell transplantation

Dasatinib induced nephrotic range proteinuria in pediatric patient with acute lymphoblastic leukemia with hyper-eosinophilia

Tyrosine kinase inhibitors (TKIs) have improved outcomes for Philadelphia chromosome positive acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). There has been reports of dasatinib induced proteinuria or nephrotic syndrome. We present a case of a 14-year-old male with high-risk B- ALL who developed nephrotic syndrome due to dasatinib. Proteinuria and nephrotic syndrome should be considered as related adverse effects from dasatinib. This is the first reported case of dasatinib induced nephrotic range proteinuria in a patient with high-risk B-ALL with hyper-eosinophilia.

Identifying Targeted Therapies for CBFA2T3- GLIS2 Acute Myeloid Leukemia

CBFA2T3- GLIS2 fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. Children with this subtype of AML frequently experience upfront chemoresistance with a high cumulative incidence of relapse (42-83%) and a dismal overall survival (~20%) even with intensive therapy. New treatment approaches are urgently needed. By interrogating the Broad Institute's Cancer Dependency Map (DepMap), a data set composed of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, we discovered that the CBFA2T3- GLIS2 AML model M-07e was highly dependent on the kinase JAK2. Indeed, M-07e was one of the most JAK2 dependent models in the data set. Using the same genome-scale library, we next performed CRISPR-Cas9 screens in WSU-AML and CMS, two additional AML cell lines harboring this fusion, and revealed JAK2 as a strong dependency shared by the 3 cell lines. Using a doxycycline-inducible knockout (KO) system, we validated JAK2 KO by western blot in three AML cell lines and cells from a patient-derived xenograft (PDX) harboring the fusion. Using two different sgRNAs targeting JAK2 (sgJAK2) and a non-targeting (sgNT) control, we validated JAK2 dependency in all 4 AML models in competitive growth assays. We also observed impaired proliferation and induced apoptosis upon JAK2 KO. To evaluate a role for JAK2 dependency in vivo, we intravenously injected NSGS mice with WSU-AML either transduced with doxycycline-inducible sgJAK2 or sgNT. After confirmation of engraftment by bone marrow evaluation, doxycycline chow was initiated. After 2 weeks of doxycycline treatment, we observed a significantly decreased tumor burden in mice with JAK2 KO AML compared to sgNT as measured by a decrement in human CD45% cells: peripheral blood (2.8% versus 12.9%), bone marrow (37.4% versus 56.3%,) and spleen (19% versus 79.5%). We next assessed chemical inhibitors of JAK2. Both the type I JAK2 inhibitor ruxolitinib and the type II JAK2 inhibitor CHZ868 exhibited strong activity in vitro in CBFA2T3- GLIS2 cell lines and PDX cells, many resistant to cytotoxic chemotherapy. Because ruxolitinib is FDA-approved for other indications, we evaluated this drug in a cell line and PDX model of CBFA2T3- GLIS2 AML in vivo. As previously reported in myeloproliferative neoplasms, JAK2 target inhibition with ruxolitinib treatment was incomplete, and accordingly, in vivo response was modest with leukemia ultimately progressing. Evaluation of CHZ868 in vivo is ongoing. Because single agent targeted therapy is typically insufficient for durable response in the treatment of most cancers, we utilized CRISPR-Cas9 anchor screening to identify candidate resistance mechanisms and potential synergistic combinations with JAK2 inhibitors in CMS and WSU-AML cells. We compared the changes in abundance of sgRNAs in the cells either treated with ruxolitinib (IC 50) or vehicle for 14 days. The correlation of hits between the two cell lines was strong, and we found that sgRNAs targeting multiple negative regulators of the MAPK pathway were significantly enriched in the ruxolitinib-treated arm. In parallel, we generated resistant CMS sublines by chronic exposure to ruxolitinib. Ruxolitinib-resistant CMS cells were found to have a pathogenic NRAS missense mutation and an associated increase in levels of phosphorylated MEK. Accordingly, they were responsive to the MEK inhibitor trametinib. Both approaches converge on activation of the MAPK pathway as a resistance mechanism to JAK2 inhibitors in CBFA2T3- GLIS2 AML. We next hypothesized that targeting the MAPK pathway could overcome resistance to JAK2 inhibitors in this disease context. Indeed, treatment with ruxolitinib and the MEK inhibitor trametinib exhibited a synergistic effect when used in combination in cell lines and PDX cells harboring the fusion in vitro with in vivo testing as a next step. In summary, we validated JAK2 as a strong dependency in CBFA2T3- GLIS2 fusion positive pediatric AML. We demonstrated that JAK2 KO or small molecule inhibitors impaired cell viability and induced apoptosis in vitro, and JAK2 KO was highly efficacious in decreasing leukemia burden in vivo. Activation of the MAPK pathway renders resistance to JAK2 inhibitors and the combination of a JAK2 with MEK inhibitor is highly synergistic.

Quantum-First: Safety By Treatment Phase and By Age in Newly Diagnosed (nd) Patients (pts) with FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD) Positive Acute Myeloid Leukemia (AML)

Background Results from the phase 3 QuANTUM-First study (NCT02668653) showed that in nd FLT3-ITD+ AML pts, the addition of the oral, highly potent, selective, type 2 FLT3 inhibitor quizartinib (Quiz) to standard chemotherapy ± allogeneic hematopoietic cell transplantation (allo-HCT), followed by Quiz monotherapy for up to 36 cycles (3 years [yrs]), decreased the relative risk of death by 22.4% vs placebo (PBO), with a generally manageable safety profile (PMID: 37116523). We describe safety by treatment phase (Induction [IND], Consolidation [CONS], Continuation [CONT]) and by age group (&amp;lt;60, 60-64, ≥65 yrs). Methods Eligible pts (18-75 yrs) with nd FLT3-ITD+ AML were randomized 1:1 to Quiz (40 mg/d) or PBO + standard IND chemotherapy. Pts in complete remission (CR) or CR with incomplete neutrophil or platelet recovery received standard CONS chemotherapy + Quiz (40 mg/d) or PBO and/or allo-HCT (Quiz/PBO not given with allo-HCT), followed by CONT of single-agent Quiz (30-60 mg/d) or PBO for up to 36 cycles. Safety was evaluated in pts treated with ≥1 dose of Quiz or PBO. Treatment-emergent adverse events (TEAEs) were coded by MedDRA v24.0, severity by NCI-CTCAE v4.03. Results In both arms, median treatment durations were 2 weeks in IND, 4 weeks in CONS, and 1.3 yrs in CONT. Rates of TEAEs and grade ≥3 TEAEs were similar in both arms in IND &amp; CONS, although grade ≥3 TEAEs were more common with Quiz vs PBO in CONT (Table 1). In IND &amp; CONS, the most common TEAEs were gastrointestinal (GI) AEs (diarrhea, nausea, vomiting), infections (pneumonia, sepsis), hypokalemia, pyrexia, febrile neutropenia, and rash. In IND &amp; CONS, myelosuppression occurred in most pts in the Quiz and PBO arms, with a median blood count recovery time &amp;lt;4 weeks from the time of onset. In CONT, most common TEAEs were upper respiratory tract infections, GI AEs, cytopenias, and ALT increase. In CONT, more pts with Quiz had myelosuppression, with trends toward earlier onset and delayed recovery with Quiz. ECG QT prolonged was more common with Quiz vs PBO in all phases, QTcF &amp;gt;500 ms was low (2.3%) and observed in IND &amp; CONS only. Ventricular arrythmias were few with Quiz (2 pts [0.8%] had cardiac arrest/ventricular fibrillation with severe hypokalemia). Infections were the most common serious TEAEs (TESAEs), with febrile neutropenia, pneumonia, and sepsis in IND &amp; CONS and pneumonia and viral infections in CONT. TEAEs leading to death were numerically higher with Quiz vs PBO in IND &amp; CONS, mainly due to infections in older pts, but numerically lower with Quiz vs PBO in CONT. In IND &amp; CONS, TEAEs leading to dose interruptions or dose reductions were similar in both arms, whereas TEAEs leading to discontinuation (disc) were higher with Quiz vs PBO. TEAEs leading to dose interruptions, dose reductions, or disc in CONT were higher with Quiz vs PBO. TEAE leading to disc of Quiz were mostly infections in IND &amp; CONS and cytopenias in CONT. Cytopenias and QT prolonged were the most common AEs leading to dose modifications and were more common with Quiz. There was no evidence of increasing toxicity with long-term Quiz therapy for up to 36 cycles in CONT, and no new AEs were observed in pts receiving &amp;gt;12 cycles. Rates of TEAEs and grade ≥3 TEAEs were similar in both arms across age groups (Table 2). TESAEs, TEAEs leading to death, and TEAEs leading to disc were more common in older pts (≥65 yrs [n=134]) vs younger pts (&amp;lt;60 and 60-64 yrs [n=399]) in both arms. Infections in the elderly were most commonly severe, serious, and fatal AEs and a main cause of more early deaths (&amp;lt;60 days of Quiz start). TEAEs leading to dose modifications were higher with Quiz vs PBO across age groups. In the Quiz arm, cytopenias, headache, rash, cough, ALT increase, hypomagnesemia, oropharyngeal pain, and back pain were more common in younger vs older pts; ECG QT prolonged was highest in the 60-64 yrs group. GI AEs and were more common in older pts with Quiz. QTcF &amp;gt;500 ms occurred mainly in pts 60-64 yrs with Quiz. Conclusions In QuANTUM-First, infections and cytopenias associated with Quiz were observed across phases. Fatal infections were more common with Quiz in IND &amp; CONS but not in CONT. Rates of prolonged QTcF &amp;gt;500 ms were low overall, occurring mostly in IND &amp; CONS, not in CONT. Pts ≥65 yrs had higher TESAEs, TEAEs leading to death (including early death), and TEAEs leading to disc (mainly infections) vs younger pts in both arms. Quiz safety profile in different treatment phases and age subgroups supports an overall positive benefit/risk.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) &amp;gt;2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM &amp;lt;1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

Efficacy and Safety of Clifutinib, a Novel, Highly Selective, Oral FLT3 Inhibitor, in Patients with FLT3-Mutated Relapsed or Refractory Acute Myeloid Leukemia：Updated Results from a Phase I Study

Background: Patients with relapsed/ refractory (R/R) FLT3-internal tandem duplication positive (FLT3-ITD+) acute myeloid leukemia (AML) have limited treatment options and poor prognosis. Clifutinib is a novel, highly selective, oral FLT-3 inhibitor with potent activity against FLT3-ITD(+) leukemic models both in vitro and in vivo. This study is a first-in-human study of clifutinib, with the purpose of evaluating the safety, tolerability, efficacy, and pharmacokinetic (PK) profile in Chinese patients with R/R FLT3-mutated AML (NCT04827069). Methods: Patients aged ≥18 years with AML who were refractory to ≥ 2 cycles of standard induction chemotherapy or relapsed after achieving remission from previous treatments were enrolled. Previous use of FLT3 inhibitors were allowed. The study included two parts, a dose-escalation part (10 to 100 mg/day cohorts) and a dose-expansion part (40 and 70 mg/day cohorts). The primary endpoints were safety and tolerability, and the secondary endpoints were efficacy [CR(complete remission)/CRh (complete remission with partial hematologic recovery) rate, composite complete remission (CRc: CR+CRh+CRi, CRi defined as complete remission with incomplete hematological recovery) rate, TTR (time to remission), duration of remission (DOR), and overall survival (OS)] and pharmacokinetics (PK). Results: As of May 2023, 75 patients (pts) were enrolled (Figure 1), with a median age of 55 years (range:18-80), and 96.0% pts were FLT3-ITD(+) or TKD(+) [80.0% with ITD(+)/TKD(-), 9.3% with ITD(+)/TKD(UK), 5.3% with ITD(+)/TKD(+), 1.3% with ITD(-)/TKD(+)], 58.7% pts had received ≥2 lines of previous treatment, and 32.0% pts exposed to FLT3 inhibitors. 3 pts experienced dose-limiting toxicities (DLT): one out of 6 pts in the 55 mg/day cohort (Grade 3 QTcF prolongation with ΔQTcF =71 ms) and 2 out of 6 pts in the 100 mg/day cohort (Grade 3 nausea and Grade 3 atrial fibrillation). Therefore, the maximum tolerated dose of clifutinib was 70 mg/day. The most common (rate ≥20%) treatment-related adverse events (TRAEs) included leukopenia (70.7%), thrombocytopenia (68%), neutropenia (64%), anemia (48%), lymphopenia (26.7%), pneumonia (26.7%), hypokalemia (26.7%), aspartate aminotransferase increased (22.7%), alkaline phosphatase increased (21.3%) and alanine aminotransferase increased (20.0%). Grade ≥3 TRAEs occurring in ≥20% pts were leukopenia, thrombocytopenia, neutropenia, anemia, lymphopenia, and pneumonia. Approximately dose-proportional PK parameters were observed across 40-100 mg/day dose range for both single and repeat dosing (the parent drug might approach saturation at 100 mg/day), and median t max ranged from 2 to 6 hours. For efficacy, responses occurred in 28 (40.6%) of 69 FLT3-ITD(+) or TKD(+) efficacy evaluable pts, including 5 (7.2%) CR, 5(7.2%) CRh, 17 (24.6%) CRi, 1 (1.4%) MLFS, and the median TTR was 32 days (range: 26-149 days). The efficacy was better in the 40 mg/day group compared to 70 mg/day and 40-100 mg/day groups, with CR/CRh rate of 21.2% (7/33) vs 8.3% (2/24) vs 14.5% (10/69), CRc rate of 48.5% (16/33) vs 33.3% (8/24) vs 39.1% (27/69), and median DOR of CR/CRh was 3.2 months (95% CI:1.9, NA) vs 0.9 months (95% CI: NA, NA ) vs 3.2 months (95% CI: 0.9, 7.4 ), the median OS was 6.5 months vs 5.4 months vs 6.3 months, respectively. Among FLT3-ITD(+)/TKD(-) subjects administered with clifutinib 40 mg/day, the CR/CRh rate was 23.1% (6/26) with 3 patients achieved CR, median DOR of CR/CRh was 3.7 months, the CRc rate was 53.8% (14/26), and median OS was 7.4 months. Compared to FLT3-ITD(+) or TKD(+) pts, 40 mg dose group exhibited better efficacy in FLT3-ITD(+)/TKD(-) pts. In addition, clifutinib exhibited more potent anti-leukaemic activity in patients received only one line of previous treatment. Among patients experienced only one line treatment of 40 mg dose group, the CR/CRh rate was 30.8% (4/13), the CRc rate was 53.8% (7/13), and the median OS was 7.9 months (Table 1) . Conclusion: This phase 1 study has demonstrated an acceptable safety profile and a significant anti-leukaemic activity of clifutinib in FLT3 mutant R/R AML, with the best response at the dose of 40 mg/day in FLT3-ITD(+)/TKD(-) AML. A confirmatory phase 3 study is currently ongoing to further evaluate the efficacy and safety of clifutinib dosed at 40 mg/day in R/R AML pts after first-line therapy with FLT3-ITD(+)/TKD(-) (NCT05586074). Keywords: Acute myeloid leukemia, FLT3 inhibitor, Clifutinib

Gemtuzumab Ozogamicin for Patients with Newly Diagnosed CD33 Positive Acute Myeloid Leukemia: Results from a French Retrospective Observational Study

Introduction: Gemtuzumab ozogamicin (GO) is an antibody-drug conjugate targeting CD33 with a calicheamicin derivative payload. GO is approved in France in combination with daunorubicin and cytarabine for treatment-naïve patients aged ≥15 years with de novo CD33-positive acute myeloid leukemia (AML). Before the European Medicines Agency granted marketing authorization in 2018, GO was available in France with an authorization for temporary use (ATU) for specific patients since 2010, and between 2018 and 2019 as part of a “post-ATU” cohort until its reimbursement. This study aimed to describe the real-world use, effectiveness, and safety of GO in treatment-naïve patients with CD33-positive AML in France, as requested by the French Transparency Commission. Methods: This retrospective, multicenter, observational study included all patients in the French ATU and post-ATU cohorts treated between 01 December 2014 and 31 October 2022. Patient and disease characteristics, treatment (dose/combination), response outcomes, and adverse events (AEs) of interest were described. Prognostic factors for event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS) were identified using Cox proportional hazard models. Results: Overall, 113 patients were included (ATU cohort, N=62; post-ATU cohort, N=51). The median age when GO was initiated was 63.0 years (range, 19-91); 54.9% (n=62) of patients were male; 81.8% (n=72/88) had an ECOG performance score 0-1, and 65.3% (n=64/98) had favorable risk according to the ELN classification 2017 ( Table). During the first induction treatment, most patients (98.1%; n=105/107) received GO in association with other agents, most commonly cytarabine and daunorubicin (60.0%; n=63/105). Most patients (78.5%, n=84/107) received 3 doses of GO during first induction. In all cases, patients received GO in second (7.1%; n=8) or subsequent (2.7%; n=3) induction courses - always in association with cytarabine and with or without daunorubicin/other agents. GO was given during the first, second, and subsequent consolidation treatment in 46.9% (n=53), 32.7% (n=37), and 2.7% (n=3) of patients, respectively, usually with cytarabine with or without daunorubicin. After a median follow-up of 44.6 months (95% CI, 33.8-69.3), 78.6% (n=88/112) of patients responded post-induction - of these, 72.3% (n=81) achieved a complete response (CR) and 6.3% (n=7) achieved a CR without platelet recovery. Minimal residual disease was evaluable in 46 patients; 52.2% (n=24) achieved a CR without minimal residual disease. Median EFS was 13.1 months (95% CI, 9.9-17.5); EFS by ELN risk group is shown in the Table. Median RFS was 17.5 months (95% CI, 12.6-35.6) and median OS was 49.8 months (95% CI, 21.8-not estimable; Figure). After a median period of 13.0 months (range, 3.0-33.0) following GO treatment, 31.5% (n=35/111) of patients received a hematopoietic stem cell transplant. Older age predicted shorter OS (hazard ratio [HR] 1.1 [95% CI, 1.0-1.1]; p&amp;lt;0.001). Having an FLT3TKD mutation (HR 4.1 [95% CI, 1.4-12.4]; p=0.013) or adverse cytogenetic classification (HR 13.0 [95% CI, 2.3-73.8]; p=0.015) predicted shorter EFS. Having ECOG-PS ≥2 (HR 6.6 [95% CI, 1.8-24.5]; p=0.005) or adverse cytogenetic classification (HR 72.7 [95% CI, 7.6-699.1]; p=0.001) predicted shorter RFS. AEs of interest were reported for 38.9% (n=44) of patients; 13.3% (n=15) were serious and 26.6% (n=30) were treatment-related (TRAE). The most common AEs were thrombocytopenia (21.2%; n=24), pyrexia (4.4%; n=5), and hepatic cytolysis (3.5%; n=4). AEs of special interest included persistent thrombocytopenia (15.9%; n=18), severe hemorrhage (5.3%; n=6), and veno-occlusive disease/sinusoidal obstruction syndrome (0.9%; n=1). Overall, 46.9% (n=53) patients died, with relapse or progressive disease accounting for 41.5% of deaths (n=22/53). Two (1.8%) patients died from TRAEs (laryngeal edema/pulmonary alveolar hemorrhage and hepatic cytolysis). Conclusions: GO was predominantly administered according to its indication. Response rates were similar to those reported in the pivotal ALFA-0701 study. Median OS was longer in this study than in ALFA-0701 (49.8 vs 27.5 months), although median RFS and EFS were reduced. No new safety signals were reported. Overall, GO appears safe and effective in real-world practice when added to induction therapy for treating patients with de novo CD33-positive AML.

Patient-Reported Outcomes in Acute Myeloid Leukemia Patients with FLT3-ITD Mutation Receiving Quizartinib Vs. Standard Chemotherapy: Results from the Quantum-First Trial

Introduction: QuANTUM-First (NCT02668653) is a multicenter, global, randomized, double-blind phase 3 clinical trial evaluating the efficacy and safety of the novel oral fms-like tyrosine kinase 3 inhibitor (FLT3i) quizartinib in combination with standard induction and consolidation chemotherapy, and as maintenance monotherapy for up to 36 cycles in patients 18-75 years old with FLT3-ITD positive acute myeloid leukemia (AML). Quizartinib demonstrated a clinically meaningful and statistically significant improvement in overall survival (OS) versus placebo. An exploratory endpoint of the trial was to assess the impact of quizartinib on patient-reported outcomes (PROs). This is the first report of the longitudinal results of the PRO measures (PROMs) collected in the QuANTUM-First trial. Methods: Patients were randomized to receive chemotherapy + quizartinib or chemotherapy + placebo. PROMs were the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQ-C30) and EuroQol EQ-5D-5L. Baseline measurement was collected on day 8 of the first induction cycle, and repeated measurements were collected on day 28 of induction cycles 1-2, day 6 and 28 of consolidation cycles 1-4, and day 1 of continuation cycles 1-34 at 3-cycle intervals. EORTC QLQ-C30 and EQ-5D-5L scores were calculated as per their respective scoring manuals and included mean (95% CI, p-value) score for each domain of the EORTC QLQ-C30 and EQ-5D-5L index score, as well as mean change from baseline score at each timepoint. A minimal clinically important difference (MCID) score ≥10 points for each domain of the EORTC QLQ-C30, was considered clinically meaningful. A mixed-effect model for repeated measures (MMRM) and time until definitive deterioration (TUDD) was used to assess the longitudinal impact of treatment on PROs. TUDD was defined as time from baseline PRO score to first deterioration of the score beyond the MCID as compared to the baseline without further improvement of more than one MCID as compared to the reference score or without any further available score. Results: A total of 509 out of 539 patients from the trial (254 quizartinib; 255 placebo) were included for PRO analysis. Patient compliance to questionnaire completion at the beginning of each phase was high and similar for both arms and most scales (Global Health Status [GHS] completion rate (%) on induction cycle 1, consolidation cycle 1, and continuation cycle 1: 99.2, 95.3, 93.4, respectively) Baseline PRO scores were comparable between arms and were lower than the general population norm EORTC QLQ-C30 and EQ-5D-5L scores for EU and US (Table 1). Per MMRM, there were clinically meaningful improvements from baseline in GHS (Figure 1) and fatigue in both arms. However, there was no significant difference between arms for the change from baseline score although placebo arm had numerically better scores specifically during the maintenance phase (treatment difference for quizartinib - placebo was -2.0 [95% CI: -4.8, 0.7], p=0.1479 for GHS and 3.0 [95% CI: -0.1, 6.1], p=0.0600 for fatigue). Similar trends towards clinically meaningful improvement in score over time for both arms and no significant differences between arms were observed in most of the functional and symptom scales of EORTC QLQ-C30 and EQ-5D-5L index scores. By TUDD analysis, quizartinib arm showed slower deterioration in several scales (specifically GHS, cognitive function, appetite loss, and constipation) but the differences did not reach statistical significance. Conclusions: QuANTUM-First is the first study to explore the impact of quizartinib on PROs. Quizartinib showed improvement in OS without any detrimental impact on quality of life and symptoms when added to standard chemotherapy followed by maintenance monotherapy in patients with newly diagnosed FLT3-ITD AML.

Engineered Natural Killer Cells Expressing Chimeric Ilt Receptors (CIR) Effectively Target HLA-G Positive AML Tumor Cells

Background Human leukocyte antigen-G (HLA-G), the immune checkpoint of pregnancy, directs immunosuppression by binding to Ig-like transcript (ILT) receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains. HLA-G is expressed in more than 50% of solid tumors and leukemias as a mechanism to evade immune attack. As a result, HLA-G is an excellent tumor-specific antigen since its expression in normal tissues is low except for cytotrophoblasts in the placenta where it protects the fetus from the mother's immune system. We present here a novel approach to target HLA-G-expressing Acute Myeloid Leukemia (AML) cells by utilizing the natural receptors of ILT2 and ILT4 coupled to positive intracellular signaling domains. Such chimeric ILT receptors, or CIRs drive activation of Natural Killer (NK) cells. Further, we have identified unique intracellular activation modules that function to robustly enhance cytotoxicity and expansion of NK cells upon engagement with tumor cells. Methods CD56 + NK cells were isolated from PBMCs, cultured with IL-15 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins (CIR.4-1BB.CD3ζ or CIR.X.Y where X and Y are candidate activation and coactivation moieties), soluble IL-15 and a ΔCD19 marker or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with GFP ffluc-expressing AML target cell lines with or without transgenic HLA-G (G1, G2 and G5) isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence in Incucyte or Celligo instruments or luciferase activity and supernatants were analyzed for IFN-γ and TNF-α release by ELISA. Transduced NK cells were labeled with nuclear-RFP for measurement of expansion in coculture. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods. Results CIR (ILT2 or ILT4.4-1BB.ζ) constructs were efficiently and stably expressed in T and NK cells (&amp;gt;80% ΔCD19 + 8- and 14-days post activation). Mock-transduced NK cells displayed innate killing activity against HLA-G negative AML cells (THP1 and KG1) that was not augmented by CIR constructs (mock = 6.16E6 ± 0.77E6 vs. CIR = 6.43E6 ± 0.37E6 in coculture against KG1 cells). In cocultures against THP1 and KG1 target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed enhanced target cell killing in 2 and 7-day assays relative to mock-transduced effector cells (mock = 1.47E6 ± 0.49E6 vs. CIR = 0.57E6 ± 0.6 in coculture against KG1-HLA-G1). Further, while mock NK cells transduced with RFP alone could kill one-third of AML cells expressing endogenous HLA-G1 and G5 (Molm13 and Kasumi1), CIR-NK cells eliminated more than 90% of these AMLs and their activivation resulted in 4-fold higher IFN-γ secretion. Notably, robust cytotoxicity of CIR-NK cells was maintained even at 28 days post activation (mock = 36.95E6 ± 21.85E6 vs. CIR = 7.38E6 ± 1.92 in coculture against Kasumi1). To further enhance the functionality of the CIR constructs, a screen of novel costimulatory molecules to replace 4-1BB and/or CD3ζ resulted in several potent candidates ultilizing MyD88 signaling that greatly improved the cytotoxicity (&amp;gt; 80% tumor elimination), proliferation (&amp;gt; 2-fold expansion) and proinflammatory cytokine production (IFN-γ and TNF-α) of NK cells in cocultures. A subset is displayed in Figure 1. Conclusions The use of natural receptors ILT2 and ILT4 to target HLA-G takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms thereby circumventing the possibility of antigen escape often observed in CAR-directed therapy. We demonstrated that engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of leukocyte activity into a target for anti-tumor control.

Phase 1/2 Study of Donor-Derived Anti-CD33 Chimeric Antigen Receptor Expressing T Cells (VCAR33) in Patients with Relapsed or Refractory Acute Myeloid Leukemia after Allogeneic Hematopoietic Cell Transplantation

Background and Significance: Although allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative therapy for patients with high-risk acute myeloid leukemia (AML), relapse occurs in 30% to 40% of alloHCT recipients and is associated with a 2-year post-relapse survival rate of less than 20%. Chimeric antigen receptor (CAR) T cells targeting tumor-associated antigens have demonstrated efficacy in relapsed/refractory (R/R) acute lymphoblastic leukemia and other hematologic malignancies, but there is limited clinical experience with CAR T cell therapies for AML. Current challenges for autologous AML CAR T cell therapy include the inability to harvest adequate numbers of functional, undifferentiated T cells as CAR T cell starting material. In addition, the potential presence of AML blasts in an autologous cell collection can interfere with T cell proliferation and function during the manufacturing process. To circumvent these impediments in patients with R/R AML after alloHCT, VCAR33 represents a CD33-directed CAR T cell product generated from lymphocytes from each patient's prior allogeneic stem cell donor. CAR T cell manufacture from HLA-matched donors will likely not only eliminate delays and failures in production due to lymphopenia but also improve T cell function by using healthy starting material. CD33 is a preferential target for AML CAR T cell therapy as it is expressed on the majority (&amp;gt;80%) of AML blasts and because prior clinical experience with gemtuzumab ozogamicin (GO; tradename: Mylotarg TM) has demonstrated the safety and efficacy of targeting CD33. Study Design and Methods: Study NCTxxxxxxxx* is a multi-center phase 1/2 study evaluating the safety and preliminary efficacy of VCAR33, donor-derived allogeneic CAR T cells targeting CD33 (Figure 1). The CAR construct used for the manufacture of VCAR33 contains a lintuzumab-derived binding domain and a CD28 co-stimulatory domain. Key inclusion criteria include adult patients with relapsed or measurable residual disease positive (MRD+) AML after undergoing alloHCT with an 8/8 HLA-matched related or unrelated donor. Patients who have undergone alloHCT with trem-cel, a CD33-deleted allogeneic stem cell product intended to facilitate the post-HCT treatment with CD33-directed therapies by reducing on-target off-tumor hematotoxicity, may also participate. Patients will be assigned to one of two study arms based on their AML disease burden: Arm A for morphologic disease with ≥ 5% blasts in the bone marrow, or Arm B for MRD+ defined as &amp;lt; 5% blasts in the bone marrow with ≥ 0.1% CD33+ leukemia cells by flow cytometry. The patient's prior stem cell donor undergoes apheresis for collection of mononuclear cells, which are used as starting material for VCAR33 manufacturing. Patients will receive lymphodepletion on days -5 to -2 with fludarabine (total 120 mg/m 2) and cyclophosphamide (total 1000 mg/m 2) followed by infusion of VCAR33 on Day 0. Each study arm will enroll and escalate independently according to a standard 3+3 trial design, starting at 1 x 10 6 CAR T cells/kg for Dose Level 1. The dose limiting toxicity monitoring period is 28 days, at which time patients will undergo evaluation of disease response. Secondary endpoints include incidence of cytokine release syndrome, graft-vs-host disease, and clinical response. Exploratory endpoints aim to correlate VCAR33 properties with clinical response by characterizing T cell phenotypes within the VCAR33 product and by examining VCAR33 expansion and persistence after infusion. Follow up on trial will continue for up to 2 years after infusion followed by long term monitoring for up to 15 years. Patients may undergo second alloHCT for consolidation of remission or for rescue of prolonged cytopenia due to on-target off-tumor effects of VCAR33. *NCT number pending. Clinical trial information submitted to CT.gov on July 19, 2023.

Characteristics of FLT3-ITD and Efficacy of Sorafenib Treatment in Pediatric AML: A Report from AML-BFM Study Group

Introduction Internal tandem duplication (ITD) of FMS-related tyrosine kinase 3 ( FLT3) gene is one of the most common somatic mutations in acute myeloid leukemia (AML). We and others have previously shown that the prognostic significance of FLT3-ITD in children and young adults relies on its characteristics e.g., co-occurrence with WT1 mutationsand NUP98::NSD1 fusions (Niktoreh et al. 2019) or higher allelic ratio (AR) (Tarlock et al. 2018) lead to worse survival outcomes. Also, recent studies suggested that treatment with FLT3 inhibitors, including sorafenib, might be effective in children with FLT3-ITD positive AML. In this report, we summarize the characteristics of FLT3-ITD and outcomes of treatment with sorafenib in patients recruited in AML-BFM consecutive trials. Methods Patients aged 0-21 years old, diagnosed with FLT3-ITD positive de novo AML between Jan. 2004 and Dec. 2021 in Germany and recruited in AML-BFM 2004 and 2012 studies or the 2012 and 2017 registries were retrospectively analyzed. All patients with FLT3-ITD with high- or intermediate risk stratification (HR or IR) were recommended to receive 100 mg/m² sorafenib taken orally twice daily on days 7 to 21 of their induction and consolidation chemotherapy blocks, the final decision however was made by the treating physicians. Patient samples were examined in AML-BFM reference laboratory in Essen, Germany using standard methods. Patients with Down syndrome and AML-M3 were excluded from the current analysis and survival rates were calculated using Kaplan-Meier method and Log-Rank test (significance threshold p&amp;lt;0.05). Results In total, 137 patients (51% male) with median age of 7.78 (1.3-19, IQR=7.27) years at diagnosis were included. Most of the patients were initially stratified in IR group (70%, N=95) followed by HR (26%, N=35). FABs M2, M4 and M1 consisted 74% of all morphologic subtypes with 26%, 25% and 23% of patients, respectively and with a median of 64,000 (500 -700,400, IQR=53,178) x10 9/L, the majority of patients showed a leukocytosis at diagnosis. The median ITD length in the 107 patients with available data was 51 (9-111, IQR=39) bps and 54% of the 91 patients with available data had an AR above 0.5. WT1 mutations and NUP98::NSD1 were detected in 25% (N=34) and 7% (N=10) of patients, respectively and 4% (N=6), harbored both mutations simultaneously. The three-year probability of overall and event-free survival (3y-OS and 3y-EFS, respectively) in all FLT3-ITD patients were 68.8% (±4.3) and 47.6% (±4.5), respectively and although harboring ITDs longer than the median length had a tendency to worsen the 3y-OS (54.4% in ITD&amp;gt;51 bps vs. 73.3% in ITDs&amp;lt;51 bps, p=0.071), it did not influence the 3-y EFS (41.7% vs. 54.2%, p=0.204). Similarly, an AR higher than 0.5 did not affect the survival outcomes (3y-OS: 67.4% vs. 60.4%, p=0.744, 3y-EFS: 46.8% vs. 47%, p=0.618). In total, 36 patients (27%) were treated with sorafenib of which, 23 (63%) received a consecutive HSCT. We observed no survival advantage between sorafenib-treated and sorafenib-naive groups of patients (3y-OS: 73.7% vs. 67.3%, p=0.408, 3y-EFS: 48.3% vs. 47.5%, p=0.929). Patients with longer ITDs did not benefit from sorafenib treatment (3y-OS: 68% vs. 54.3%, p=0.279, 3y-EFS: 48.9% vs. 38.4 %, p=0.649) but higher AR, showed a tendency of improved survival outcomes when sorafenib treatment was implicated (3y-OS: 80.8 % vs. 61.9 %, p=0.159, 3y-EFS: 67.3 % vs. 39.9 %, p=0.131). Although the number of patients were limited, we observed a possible survival advantage in double or triple FLT3-ITD, NUP98::NSD1 and WT1 mutated patients who received Sorafenib: from 28 patients with WT1 mutations only, four patients received sorafenib three of which, are currently alive and in CR but contrarily, from the 24 untreated patients, 14 (58%) are deceased (78% due to relapse). Additionally, from 10 patients with NUP98::NSD1 (w/o WT1 mutations), two who were treated with sorafenib are alive and in CR, whereas from the remaining 8 patients, 6 (75%) experienced relapse and unfortunately did not survive. Conclusions Our preliminary analysis implicates that although addition of sorafenib to the intensive chemotherapy of children and adolescence with FLT3-ITD positive AML, did not improve the survival outcomes in allpatients; specific genetic subgroups might benefit from this treatment. These results must be validated in bigger cohorts of patients with longer follow-ups.

The Diversity of FLT3-ITD and the Prognostic Impact in Adult Acute Myeloid Leukemia

Introduction FLT3 internal tandem duplication (ITD) is commonly detected in patients with acute myeloid leukemia (AML) and is known to confer poor prognosis. It has different insertion numbers, mutation burden, insertion sizes, insertion sites and insertion nucleotide sequences, showing complex diversity. However, the guideline of AML did not sufficiently consider the impact of the diversity of FLT3-ITD on treatment responses, and previous studies have not been consistent with the impact of FLT3-ITD diversity on clinical prognosis. In this study we examined the diversity of FLT3-ITD and correlated these parameters with clinical outcome in adult AML. In addition, we revealed the correlation between FLT3-ITD and drug resistance through evolutionary trajectories in primary resistant patients. Methods We included adult patients from 2016-2021 at Henan Cancer Hospital with newly diagnosed adult AML. next generation sequencing (NGS) was performed for all patients at diagnosis. The FLT3-ITD was also detected using conventional methods based on PCR, with a cut-off of 3% for positivity. The comparative analysis of NGS and PCR was done to validate the diversity of FLT3-ITD. Whole genome sequencing (WGS) data for multiple time points were used to analyze clonal evolution. Data were collected by chart review. The chi-squared or Fisher's exact test, Mann-Whitney U test, log-rank and Cox regression analyses were performed. Results Among 406 newly diagnosed adult AML patients, 24.4% (99/406) patients were detected with FLT3-ITD. The FLT3-ITD diversity was validated by NGS and PCR data from 90 FLT3-ITD positive patients. 61.1% (55/90) patients had single FLT3-ITD (sFLT3-ITD) (31M/24F, median age 46 at diagnosis, range 18-64), and 38.9% (35/90) patients carried multiple FLT3-ITDs (mFLT3-ITD) at the same time (23M/12F, median age 47 at diagnosis, range 19-64). A total of 161 FLT3-ITDs were detected, with a median mutation burden of 21.51% (0.71-93.21%) and a median insertion length of 36bp (9-189 bp). 80.20% of FLT3-ITD insertions were in the near-membrane region, and 45.54% of FLT3-ITD insertions contained exogenous nucleic acid sequences. Patients with mFLT3-ITD had a significantly lower CR rate than those with sFLT3-ITD (25.7% vs 60.0%, p=0.001), while the CR rate of patients with sFLT3-ITD was similar to patients without FLT3-ITD (60.0% vs 58.6%, p=0.521). In the setting of NPM1, the CR rate of patients with sFLT3-ITD+NPM1 mut improved significantly when compared to patients with sFLT3-ITD+NPM1 wild (76.9% vs 44.8%, p=0.015). Unfortunately, the state of NPM1 had no impact on the CR rate of patients with mFLT3-ITD. After a median follow-up of 38.9 months, the median overall survival (OS) has not been reached for patients with a single FLT3-ITD, and the median OS for patients with multiple FLT3-ITDs was only 12.4 months (95 CI, 3.8 to 20.9) ( p=0.028). Multivariate analysis confirmed its prognostic significance on CR rate (HR 2.13, 95% CI, 1.38-4.20, p=0.020) but not on OS. The mutation burden, insertion sizes, insertion sites and insertion nucleotide sequences of FLT3-ITD had no significant impact on the CR rate or OS. To explore the clonal architecture of mFLT3-ITD, single cell DNA sequencing on one patient showed that respective FLT3-ITD variants were detected in completely separated cell populations. This suggested that mFLT3-ITD independently occurred in different subclone. WGS data of persistent primary resistant patients at multiple time points were used to analyze the evolutionary trajectory under chemotherapy. We observed complete separation of evolving trajectory of different FLT3-ITD variants over the course of chemo-treatment. Importantly, FLT3-ITD subclone of diagnosis evolved into the dominant clone at resistance. Conclusion In summary, by exploring the impact of the FLT3-ITD diversity on clinical prognosis and the evolutionary trajectory of mFLT3-ITD, the FLT3-ITD number may be a biomarker to refine the risk stratification and further identify adverse populations among FLT3-ITD positive AML patients.

The study of clinical characteristics and prognosis of RUNX1-RUNX1T1 positive acute myeloid leukemia based on next-generation sequencing

The study of clinical characteristics and prognosis of RUNX1-RUNX1T1 positive acute myeloid leukemia based on next-generation sequencing