Response In Acute Myeloid Leukemia Patients Research Articles

Erythroid Predominance in Bone Marrow Biopsies of Acute Myeloid Leukemia Patients after Decitabine Treatment Correlates with Mutation Profile and Complete Remission.

Acute myeloid leukemia (AML) patients may receive hypomethylating agents such as decitabine (DAC) as part of their treatment. Not all patients respond to this therapy, and if they do, the clinical response may occur only after 3-6 courses of treatment. Hence, early biomarkers predicting response would be very useful. We retrospectively analyzed a cohort of 22 AML patients who were treated with DAC. Histology of the bone marrow biopsy, pathogenic mutations, and methylation status were related to the treatment response. In 8/22 (36%) patients, an erythroid dominant response (EDR) pattern, defined as a ratio of myeloid cells/erythroid cells <1, was observed. In the remaining 14 cases, a myeloid predominance was preserved during treatment. No difference in the hypomethylating effect of DAC treatment was observed in patients with and without EDR, as global 5-methylcytosine levels dropped similarly in both groups. Mutational analysis by NGS using a panel of commonly mutated genes in AML showed that patients with an early EDR harbored on average less mutations, with U2AF1 mutations occurring more frequently, whereas RUNX1 mutations were underrepresented compared to non-EDR cases. Interestingly, the development of an EDR correlated with complete remission (7/8 cases with an EDR vs. only 2/14 cases without an EDR). We conclude that early histological bone marrow examination for the development of an EDR may be helpful to predict response in AML patients during treatment with DAC.

A novel immunogenic cell death-related classification indicates the immune landscape and predicts clinical outcome and treatment response in acute myeloid leukemia

BackgroundImmunogenic cell death (ICD) is closely related to anti-tumor therapy and regulates the tumor microenvironment (TME). This study aims to explore the molecular characteristics of ICD in acute myeloid leukemia (AML) and to analyze the value of ICD-related biomarkers in TME indication, prognosis prediction, and treatment response evaluation in AML.MethodsSingle-sample gene set enrichment analysis was used to calculate the ICD score. LASSO regression was used to construct a prognostic risk score model. We also analyzed differences in clinical characteristics, immune landscape, immunotherapy response, and chemotherapy sensitivity between high-risk and low-risk patients.ResultsThis study identified two ICD-related subtypes and found significant heterogeneity in clinical prognosis, TME, and immune landscape between different ICD subtypes. Subsequently, a novel ICD-related prognostic risk score model was developed, which accurately predicted the prognosis of AML patients and was validated in nine AML cohorts. Moreover, there were significant correlations between risk scores and clinicopathological factors, somatic mutations, TME characteristics, immune cell infiltration, immunotherapy response, and chemosensitivity. We further validated the model gene expression in a clinically real-world cohort.ConclusionsThe novel ICD-related signatures identified and validated by us can serve as promising biomarkers for predicting clinical outcomes, chemotherapy sensitivity, and immunotherapy response in AML patients, guiding the establishment of personalized and accurate treatment strategies for AML.Graphical

Defective Necroptosis Mediates Chemotherapy Resistance in AML

Acute myeloid leukemia (AML) is a hematopoietic malignancy that can affect individuals of any age and, if left untreated, is always fatal. Standard induction chemotherapy, known as “7+3,” using drugs like cytarabine (Ara-C) and an anthracycline (daunorubicin or idarubicin), is typically given to young and fit older patients. While this treatment regimen has shown positive outcomes in patients who respond to it, relapse and refractory disease remain the primary causes of death. Prognostic classification, which combines cytogenetic and mutational status, can estimate overall and event-free survival but is not effective in predicting induction failure. Despite decades of research and treatment with 7+3 chemotherapy, the reasons behind variable treatment responses in AML patients remain unclear, especially for those who experience induction failure. Understanding these mechanisms could aid in risk-adapted treatment decisions and the identification of new therapeutic targets. Recent research has investigated gene expression and signaling patterns in leukemic cells at the time of diagnosis to categorize patients according to their response to chemotherapy. Notably, increased NF-kB signaling at diagnosis has been linked to chemotherapy resistance. NF-kB is a family of transcription factors involved in regulating genes linked to cancer cell survival, proliferation, and therapy resistance. However, the exact mechanism by which NF-kB activation contributes to chemotherapy failure and resistance in AML is yet to be determined. Using publicly available datasets, we found that AML patients undergoing induction failure exhibited elevated expression of TNFAIP3/A20, which is driven by NF-kB signaling and leads to a worse prognosis for patients undergoing 7+3 chemotherapy. In experiments with primary AML samples, it was observed that A20-High AML are resistant to Doxorubicin treatment, whereas A20-Low AML samples are sensitive to Doxorubicin. Knockdown of A20 in A20-High AML samples resulted in increased cell sensitivity to Doxorubicin, suggesting that elevated A20 expression reduces AML cells' response to the Doxorubicin both in vitro and in vivo. To understand why A20 is overexpressed in AML, we investigated its requirement on AML cell function in various models, including human AML cell lines, patient-derived samples, and mouse models of AML. Deletion or knockdown of A20 led to reduced leukemic progenitor function, cell viability, and AML development in vitro and in vivo. A20 is a dual ubiquitin-editing protein that regulates signaling pathways by acting either as a deubiquitinase to modulate NF-kB signaling or as an E3 ligase to control necroptosis. Further analyses revealed that A20 prevents necroptosis by targeting the necroptosis effector, RIPK1, for degradation through the proteasome in AML. Preventing necroptosis, either pharmacologically or genetically, restored the viability and clonogenicity of A20-deficient AML cells in vitro and in vivo. Notably, Doxorubicin, but not AraC, induced necroptosis in AML cells, which is suppressed in leukemic clones expressing A20. Examination of single-cell RNA-sequencing data from AML patients before and after Doxorubicin treatment confirmed the persistence of A20-expressing clones during treatment. These findings demonstrate that NF-kB-driven overexpression of A20 contributes to failed induction chemotherapy in AML, highlighting the therapeutic potential of targeting an alternative cell death pathway in AML.

Booster effect of a third mRNA-based COVID-19 vaccine dose in patients with myeloid malignancies.

We have reported that seroconversion rates after the second dose of mRNA-based COVID-19 vaccines for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) were 100% and 95% respectively, with no significant difference from healthy controls (HCs).However, there are very limited data for the response to a third vaccine dose in those patients. In this complementary study, we investigated the booster effect of a third mRNA-based COVID-19 vaccine dose in patients with myeloid malignancies. A total 58 patients including 20 patients with MDS and 38 patients with AML were enrolled. Anti-SARS-CoV-2S immunoassays were performed at 3, 6, and 9 months after the second vaccine dose. Seventy-five percent of the MDS patients and 37% of the AML patients were receiving active treatment at the time of the third vaccination. Both the initial and third vaccine response in AML patients were comparable to those in HCs. In MDS patients, although the initial vaccine immunogenicity was inferior to that in HCs and AML patients, the third vaccine improved the response to a level not inferior to those in HCs and AML patients. Of note, the third vaccine resulted in a significant increase of antibodies in actively treated MDS patients who had shown a response inferior to that in untreated patients after two doses of vaccination. In patients with myeloid malignancies, the third vaccine dose showed a booster effect, and disease- and therapy-related factors associated with the booster response have been identified. The third dose of an mRNA-based COVID-19 vaccine showed a booster effect in patients with myeloid malignancies. Such a good booster response has not been reported in other haematological malignancies.

Comprehensive CRISPR-enabled functional genomics profiling to identify targetable cancer genes in primary acute myeloid leukemia (AML).

3088 Background: Acute myeloid leukemia (AML) is a life-threatening hematopoietic malignancy affecting both older and younger patient populations. Despite the approval of multiple new therapeutics during the past decade, including new targeted agents, treatment outcomes for AML remain poor, with < 10% 5-year survival in patients over 60 years of age, and < 50% 5-year survival in younger patients. Also, comprehensive genomic profiling (CGP) for common molecular lesions (e.g., FLT3 mutation) incompletely stratifies the patient population, indicating that a more comprehensive approach to personalized medicine could be of significant clinical benefit. To this end we developed Fx Heme, an in vitro functional genomics platform using CRISPR/Cas9 technology in primary cancer patient samples. Previously, we have shown it to stratify clinical response in AML patients treated with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g., GCLAM). Here we profile an expanded cohort to systematically characterize the AML functional genomics landscape and identify potential new targets for AML drug development. Methods: Unsorted primary tumor cells from patient samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library which was designed to inhibit expression of genes encoding the targets of FDA-approved targeted oncology drugs, as well as internal negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated for each gene to generate gene-level statistics, enabling identification of gene dependencies. Results: Comprehensive CRISPR/Cas9 functional genomics profiling was carried out retrospectively on pre-therapy samples from 59 AML patients. There were no false positives identified among negative controls. Functional dependencies on multiple genes were observed, with approximately half of patients (n=31; 52.5%) showing at least one gene dependency. We observed on average 6.7 dependencies per positive sample, with 5 of them revealing more than 10 dependencies. In addition to the targets of drugs approved to treat AML (FLT3, 10.2%; IDH1/2, 3.4%; SMO, 3.4%), a diversity of targetable genes for new therapeutic approaches to AML were identified across multiple known oncogenic signaling pathways. The prevalence of these recurrent gene dependencies ranged from 3.3% to 23.7% in our AML cohort. Conclusions: In summary, comprehensive functional genomic profiling using Fx Heme provides an effective platform to identify therapeutic opportunities in AML based on dependencies identified for targets of drugs not currently used to treat AML. These insights are now being utilized to develop additional treatment options and more effective personalization of treatment for AML.

The Composition of Acute Myeloid Leukemia Cell Differentiation States Predicts Response to Immune Checkpoint Blockade

Background: Given the activity of allogeneic T cells in acute myeloid leukemia (AML) patients, immunotherapies stimulating antitumor T cell responses have been explored as a promising therapeutic option. However, these therapies have been associated with modest clinical activity, and further research is needed to identify response and resistance mechanisms. A spectrum of primitive and differentiated cells comprise AML in each individual patient, and this cell composition has been previously shown to predict outcomes. In particular, high proportions of AML stem and progenitor cells have been associated with worse clinical outcomes and lack of response to induction chemotherapy (Ng et al Nature 2016; van Galen et al Cell 2019; Zeng et al Nat Med 2022). The composition of AML cell differentiation states in relation to immunotherapy response or resistance has not been elucidated; herein, we explored whether this composition may be associated with response in AML patients treated with immune checkpoint blockade. Methods: Pre- and posttreatment whole bone marrow (BM) samples were collected from 31 newly diagnosed and relapsed/refractory AML patients treated with azacitidine and pembrolizumab (NCT02845297) and 18 relapsed/refractory AML patients treated with high-dose cytarabine (HiDAC) and pembrolizumab (NCT02768792; Zeidner et al Blood Cancer Discov 2022) on two separate phase II trials. Primary patient samples and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki as per research protocols approved by the Institutional Review Boards of the participating institutions. Bulk RNA sequencing was performed on all BM samples. Differential gene expression analysis was performed using the DESeq2 package, and gene set enrichment analysis (GSEA) was performed using the clusterProfiler package. Proportions of AML cell populations present in BM samples were deconvoluted using CIBERSORTx as per Zeng et al Nat Med 2022. The LSC17 score and LinClass7 score were calculated as per Ng et al Nature 2016 and Zeng et al Nat Med 2022, respectively. All analyses and graphs were generated using R (v4.2.1). Results: GSEA using gene sets from a range of primitive and differentiated AML and normal cell types as per van Galen et al Cell 2019 was performed on pre- and posttreatment samples. Patients achieving a complete response (CR) to either immunotherapy regimen were enriched at baseline for the following differentiated AML gene sets: pro monocyte (Promono)-like AML; monocyte-like AML; conventional dendritic cell (cDC)-like AML; and myeloid-like AML (Figure 1A). Patients not responding (NR) to azacitidine/pembrolizumab were enriched for the following stem and progenitor AML gene sets: hematopoietic stem cell (HSC)-like AML; progenitor (Prog)-like AML; and HSC progenitor (HSCProg)-like AML (Figure 1A). To validate these findings, BM samples were deconvoluted into inferred fractions of AML differentiation states using CIBERSORTx and single-cell RNA sequencing expression data from Zeng et al Nat Med 2022. Patients not responding to azacitidine/pembrolizumab had a higher proportion of leukemia stem and progenitor quiescent cells (LSPC Quiescent) at baseline, while patients achieving CR to either regimen had higher baseline proportions of pro monocyte-like AML cells (Figure 1B). Additionally, patients not responding to azacitidine/pembrolizumab had higher baseline LSC17 and LinClass7 scores, two gene expression scores that are reflective of leukemia stemness (Figure 1B). In the posttreatment setting for both regimens, HSC-like, Prog-like, and HSCProg-like gene sets were associated with NR; monocyte-like and myeloid-like gene sets were associated with CR (Figure 1A). Discussion: Patients achieving CR to azacitidine/pembrolizumab and HiDAC/pembrolizumab had higher proportions of differentiated AML cells, while NR patients had a higher proportion of AML stem and progenitor cells. We hypothesize that antitumor T cells may preferentially target differentiated AML cells over AML stem and progenitor cells. Further research is needed to validate these findings in a prospective and mechanistic manner and in other immunotherapy regimens. Analyses of the proportions of AML differentiation states in patients relapsing after allogeneic hematopoietic cell transplantation are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

No Evidence that CD33 rs12459419 Polymorphism Predicts Gemtuzumab Ozogamicin Response in Consolidation Treatment of Acute Myeloid Leukemia Patients: Experience of the PETHEMA Group.

Gemtuzumab ozogamicin (GO) is a conjugate of a monoclonal antibody and calicheamicin, which has been reapproved for the treatment of acute myeloid leukemia (AML). AML patients with the CD33 rs12459419 CC genotype might benefit from the addition of GO to intensive treatment in contrast to patients with CT/TT genotypes. Nevertheless, contradictory results have been reported. We sought to shed light on the prediction of GO response in AML patients with rs12459419 polymorphism who were treated with GO in the consolidation (n = 70) or reinduction (n = 20) phase. The frequency distribution of the rs12459419 polymorphism in the complete cohort of patients was 44.4% (n = 40), 50% (n = 45), and 5.6% (n = 5) for CC, CT, and TT genotypes, respectively. Regarding the patients treated with GO for consolidation, we performed a Kaplan-Meier analysis of overall survival and relapse-free survival according to the rs12459419 polymorphism (CC vs. CT/TT patients) and genetic risk using the European Leukemia Net (ELN) 2010 risk score. We also carried out a Cox regression analysis for the prediction of overall survival, with age and ELN 2010 as covariates. We found no statistical significance in the univariate or multivariate analysis. Additionally, we performed a global Kaplan-Meier analysis for the patients treated with GO for reinduction and did not find significant differences; however, our cohort was too small to draw any conclusion from this analysis. The use of GO in consolidation treatment is included in the approval of the compound; however, evidence regarding its efficacy in this setting is lacking. Rs12459419 polymorphism could help in the selection of patients who might benefit from GO. Regrettably, in our cohort, the rs12459419 polymorphism does not seem to be an adequate tool for the selection of patients who might benefit from the addition of GO in consolidation cycles.

Clinically Relevant Oxygraphic Assay to Assess Mitochondrial Energy Metabolism in Acute Myeloid Leukemia Patients.

Simple SummaryAML mitochondrial oxidative phosphorylation has recently been identified as a biological property that influences the response to antitumor therapy. In the present study, we propose a standardized protocol to measure mitochondrial metabolic organization in patient blasts (from the blood or bone marrow) using XFe24 or XFe96 Seahorse. Monitoring mitochondrial oxygen consumption of blasts could improve the prediction of drug response in AML patients, especially in clinical trials.Resistant acute myeloid leukemia (AML) exhibits mitochondrial energy metabolism changes compared to newly diagnosed AML. This phenotype is often observed by evaluating the mitochondrial oxygen consumption of blasts, but most of the oximetry protocols were established from leukemia cell lines without validation on primary leukemia cells. Moreover, the cultures and storage conditions of blasts freshly extracted from patient blood or bone marrow cause stress, which must be evaluated before determining oxidative phosphorylation (OXPHOS). Herein, we evaluated different conditions to measure the oxygen consumption of blasts using extracellular flow analyzers. We first determined the minimum number of blasts required to measure OXPHOS. Next, we compared the OXPHOS of blasts cultured for 3 h and 18 h after collection and found that to maintain metabolic organization for 18 h, cytokine supplementation is necessary. Cytokines are also needed when measuring OXPHOS in cryopreserved, thawed and recultured blasts. Next, the concentrations of respiratory chain inhibitors and uncoupler FCCP were established. We found that the FCCP concentration required to reach the maximal respiration of blasts varied depending on the patient sample analyzed. These protocols provided can be used in future clinical studies to evaluate OXPHOS as a biomarker and assess the efficacy of treatments targeting mitochondria.

Receptor Activator of NF-κB (RANK) Confers Resistance to Chemotherapy in AML and Associates with Dismal Disease Course.

Simple SummaryAcute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Despite the emergence of new therapeutic agents in recent years, curation remains challenging, and new and better treatment options are needed. In the present study, we investigated the expression, prognostic significance, and functional role of the Receptor Activator of Nuclear Factor-κB (RANK) in AML. We found that RANK is expressed on leukemic cells in a substantial proportion of AML patients and is associated with a dismal disease course. We further demonstrated that signaling via RANK induces release of factors that favor AML cell survival and confers resistance to chemotherapeutics in AML treatment. Together, our findings identify RANK as novel prognostic marker and putative candidate for therapeutic intervention in AML to enhance response to treatment. Although treatment options of acute myeloid leukemia (AML) have improved over the recent years, prognosis remains poor. Better understanding of the molecular mechanisms influencing and predicting treatment efficacy may improve disease control and outcome. Here we studied the expression, prognostic relevance and functional role of the tumor necrosis factor receptor (TNFR) family member Receptor Activator of Nuclear Factor (NF)-κB (RANK) in AML. We conducted an experimental ex vivo study using leukemic cells of 54 AML patients. Substantial surface expression of RANK was detected on primary AML cells in 35% of the analyzed patients. We further found that RANK signaling induced the release of cytokines acting as growth and survival factors for the leukemic cells and mediated resistance of AML cells to treatment with doxorubicin and cytarabine, the most commonly used cytostatic compounds in AML treatment. In line, RANK expression correlated with a dismal disease course as revealed by reduced overall survival. Together, our results show that RANK plays a yet unrecognized role in AML pathophysiology and resistance to treatment, and identify RANK as “functional” prognostic marker in AML. Therapeutic modulation of RANK holds promise to improve treatment response in AML patients.

Identification of m6A-Related lncRNAs Associated With Prognoses and Immune Responses in Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) remains the most common type of hematopoietic malignancy in adults and has an unfavorable outcome. Herein, we aimed to construct an N6-methylandenosine (m6A)-related long noncoding RNAs (lncRNAs) signature to accurately predict the prognosis of patients with AML using the data downloaded from The Cancer Genome Atlas (TCGA) database. Methods: The RNA-seq and clinical data were obtained from the TCGA AML cohort. First, Pearson correlation analysis was performed to identify the m6A-related lncRNAs. Next, univariate Cox regression analysis was used to determine the candidate lncRNAs with prognostic value. Then, feature selection was carried out by Least absolute shrinkage and selection operator (LASSO) analysis, and seven eligible m6A-related lncRNAs were included to construct the prognostic risk signature. Kaplan–Meier and receiver operating characteristic (ROC) curve analyses were performed to evaluate the predictive capacity of the risk signature both in the training and testing datasets. A nomogram was used to predict 1-year, 2-year, and 3-year overall survival (OS) of AML patients. Next, the expression levels of lncRNAs in the signature were validated in AML samples by qRT-PCR. Functional enrichment analyses were carried out to identify probable biological processes and cellular pathways. The ceRNA network was developed to explore the downstream targets and mechanisms of m6A-related lncRNAs in AML. Results: Seven m6A-related lncRNAs were identified as a prognostic signature. The low-risk group hold significantly prolonged OS. The nomogram showed excellent accuracy of the signature for predicting 1-year, 2-year and 3-year OS (AUC = 0.769, 0.820, and 0.800, respectively). Moreover, the risk scores were significantly correlated with enrichment in cancer hallmark- and malignancy-related pathways and immunotherapy response in AML patients. Conclusion: We developed and validated a novel risk signature with m6A-related lncRNAs which could predict prognosis accurately and reflect the immunotherapy response in AML patients.

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA-CCR7-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA+CCR7-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) subsets.Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes+T-betint CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

CD33/CD3-Bispecific T-Cell Engaging (BiTE®) Antibody Constructs Efficiently Target Monocytic CD14+ hla-DRlow IDO+ aml-MDSCs

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.First, we assessed the presence of circulating CD14+ cells that co-express CD33 but lack HLA-DR expression (HLA-DRlo) in patients with newly diagnosed AML. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14+HLA-DRlo cells was significantly increased in untreated AML patients as compared to healthy controls (28,98±4,19%, n=13 versus 3,28±0,75%, n=37) but did not correlate with the number of AML blasts. Purified CD14+HLA-DRlo cells suppressed in vitro T-cell proliferation in a concentration-dependent manner allowing us their denomination as MDSCs.Interestingly, we also observed that primary AML blasts and human AML cell lines (THP, OCI-AML, and HL-60) induced HLA-DRlo cells from healthy donor-derived monocytes that were T-cell suppressive and expressed the immune regulatory indoleamine-2,3-dioxygenase (IDO). Functional and phenotypic reprogramming of monocytes was cell-contact independent.Using ex vivo co-culturing models of primary AML blasts/AML cell lines (=target cells) and T-cells (=effector cells) we observed AMG 330-triggered antibody construct-dependent cell-mediated cytotoxicity/ADCC. Short-term (three to six days) treatment of AML patient-derived peripheral blood mononuclear cells (PBMCs) with 10 or 100 pM AMG 330 led to T-cell activation based on proliferation and on increased expression/production of CD25, CD69, CD137, CD154, IL-2, and IFN-g. The initial T-cell frequency ranging from 0,16 to 14,30% had no impact on their responsiveness. Moreover, we observed a bystander activation of NK-cells. The addition of AML-MDSCs into these co-cultures did not negatively impact ADCC activity. On the contrary, T-cells formed immunological synapses with AML-MDSCs and triggered their elimination (also in presence of AML blasts) overcoming their T-cell suppressive effect. MDSC levels in AML-PBMCs had no effect on AMG 330-mediated T-cell (and bystander cell) activation, which was further corroborated in experiments where the total CD14+ cell compartment (comprising AML-MDSCs) was removed prior AMG 330 application.However, we did observe an impact of MDSCs on BiTE® mediated T-cell proliferation, which has been shown as highly relevant for clinical activity of BiTE® antibody constructs (Zugmaier et al. Blood 2015). Here we found a 1,68-fold enhanced (p=0.001) T-cell expansion in AML-PBMCs treated with 100 pM AMG 330 following CD14+ cell depletion. Given the importance of T-cell proliferation for clinical activity this finding warrants further exploration.Taken together, our results suggest that anti-CD33/CD3 bispecific BiTE® antibody constructs may achieve anti-leukemic efficacy not only through direct T cell mediated cytotoxicity against AML blasts but also through circumventing immune evasion via MDSCs targeting. Although therapeutic targeting of MDSCs in patients has not yet been successfully accomplished, bystander killing of CD33+ MDSCs via anti-CD33/CD3-bispecific BiTE® antibody constructs could represent a very promising approach to increase the anti-leukemic T-cell response in AML patients and to reverse immune evasion. DisclosuresMougiakakos:AMGEN: Research Funding. Kischel:AMGEN: Employment. Michael:AMGEN: Employment. Dos Santos:Amgen Inc.: Employment. Jitschin:AMGEN: Research Funding. Mackensen:AMGEN: Research Funding.

Leukemic Stem Cell Expression Signatures Identify Novel Therapeutics for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has a relatively poor 5-year survival rate of 30% in adults, largely due to high rates of relapse, which is thought to be driven by chemoresistant leukemic stem cells (LSCs). A therapy would need to eradicate all LSCs to obtain durable responses in AML patients. Unlike typical cancerous cells, LSCs are often quiescent, allowing them to evade standard therapies and serve as a reservoir for relapse. Thus, the identification of novel anti-LSC therapies could result in the improvement of the clinical outcome of patients suffering from AML.To identify compounds predicted to impede LSC function while not affecting normal hematopoietic stem cells (HSCs), we probed datasets of drug-gene interaction with our previously defined LSC and HSC gene expression signatures generated from 16 primary AML samples and 4 pooled cord blood samples (Eppert et al. Nat Med, 2011), and an additional LSC-signature from 86 AML patients (unpublished data; Ng et al. Nature, 2016). We identified 152 molecules predicted to target LSCs without harming HSCs. Eighty-three of these molecules were screened against a primary AML sample (8227) with a known LSC-containing population (confirmed as CD34+CD38- by xenotransplantation). Viability and phenotype were assessed by flow cytometry. We identified multiple hits in two classes of drugs: 3 drugs in a subclass of steroids and 3 drugs in a specific group of ion pump inhibitors, that preferentially eliminated CD34+CD38- cells over CD15+ blast cells in 8227. Many of these candidates are already in use for other clinical settings. The steroids induced differentiation in the lower nanomolar range (IC50: 0.44-1.30nM), shown by a depletion of primitive CD34+CD38- cells and subsequent expansion of terminally differentiated CD15+ cells. The ion pump inhibitors preferentially targeted the CD34+CD38- cells (IC50 CD34+CD38-: 11.55nM, 24.69nM, 21.78nM) over the CD15+ blast cells (IC50 CD15+: 17.72nM, 32.73nM, 45.09nM). To determine the toxicity of our candidates towards normal stem and progenitor blood cells, we exposed CD34+ enriched human cord blood to the steroids and ion pump inhibitors. All candidates had minimal effects against the CD34+ cord blood cells compared to CD34+ 8227 cells. We then tested the compounds against multiple primary AML samples and observed that at low nanomolar concentrations (0.3-4.0nM) the steroids differentiated AML #9642 (M4e, NPM1-WT, FLT3-WT) but had no effect on AML #9706 (M1, NPM1-WT, FLT3-ITD). For the ion pump inhibitors, AML #184 (M4, NPM1-MT, FLT3-TKD) and AML #9642 were sensitive while AML #116 (M0, NPM1-WT, FLT3-WT) and AML #9706 were more resistant. This variability in sensitivity to both groups of candidates suggests a possible link to subtype or genotype to responsiveness. To evaluate the effect of the steroids and ion pump inhibitors on functional leukemic progenitor cells, 8227 cells were treated with the steroids, ion pump inhibitors or control (DMSO) and then assessed by colony formation unit assay. Treatment with the steroids or ion pump inhibitors resulted in a 2-fold decrease in progenitors (colony formation) compared to DMSO. Next, we will expose a panel of AML patient samples to the candidate compounds in vitro to further elucidate whether there is a link between certain clinical or genotypic features and responsiveness. We will also perform xenotransplantation of treated AML samples into immunodeficient mice to assess the effects on functional LSCs.Overall, we have shown that both the subclass of steroids and specific ion pump inhibitors identified by our bioinformatic approach have anti-AML and anti-LSC properties against particular AML samples in vitro . Future results from our study will provide valuable insight to LSC biology and can lead to new therapeutic approaches for targeting the LSCs, the root of therapy resistance and relapse in AML. DisclosuresNo relevant conflicts of interest to declare.

Blast MRD AML-1 Trial: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease in Acute Myeloid Leukemia (AML) 1- an Investigator-Initiated, CTEP-Sponsored, Randomized Phase 2 Study of the Anti-PD-1 Antibody Pembrolizumab in Combination with Conventional Intensive Chemotherapy (IC) As Frontline Therapy in Patients with Acute Myeloid Leukemia (AML)

Study Background: The presence of minimal residual disease (MRD) after IC in AML patients is often a harbinger of relapse, and therapeutic targeting of MRD has emerged as an important strategy to improve long-term outcomes. The importance of the PD-1/PD-L1 pathway in immune evasion of AML has been demonstrated in murine AML models whereby PD-L1 and PD-1 were upregulated in leukemia cells, and PD-1 blockade suppresses invivo leukemia cell proliferation and improved survival in AML bearing mice (Zhang et al., 2009; Zhou et al., 2010). Combining IC with immune checkpoint inhibition (ICPI) has resulted in a significant clinical activity in some advanced solid malignancies including non-small-cell lung cancer. A recent single-arm phase 2 study combining IC with nivolumab showed the combination was feasible and resulted in undetectable MRD in 53% of patients with Complete response (CR)/CR with incomplete count recovery (CRi) (Ravandi et al, 2019). Here we report on the design of the first randomized multi-center clinical trial of IC+ICPI in fit AML patients. Methods: The primary objective of this investigator-initiated Cancer Therapy Evaluation Program (CTEP)-sponsored multi-institutional, randomized phase II study (NCT04214249) is to assess the percentage of patients with MRD negative CR (MRD- CR) as measured by flow cytometry at the end of first cycle of consolidation therapy with IC+ pembrolizumab and compare between the two study arms (Figure 1). Secondary objectives include rates of CR/CRi, complete remission with partial recovery count (CRh) and hematologic improvement (HI) to red blood cells and platelets, MRD negativity at Day 14, MRD-negative CR at end of induction therapy and MRD negative CR after last consolidation cycle, event free survival (EFS), relapse free survival (RFS), duration of response (DOR), and overall survival (OS), and proportion of patients who develop severe toxicity. Important exploratory objectives include MRD assessment by duplex sequencing (DS) and comparing DS and multiparameter flow cytometry for MRD detection. The study will consist of an induction, consolidation, and maintenance phase of therapy. A total of 124 patients will be included (62 patients in the intervention arm and 62 patients in the control arm). Planned stratification factors include 1) age (younger than 65 vs. 65 and older), 2) cytogenetics by FISH or metaphase karyotype (presence vs. absence of core binding factor inversions and translocations), and 3) t-AML or AML arising from prior/antecedent MDS. All patients will receive cytarabine at 100 mg/m2/day continuous infusion on Days 1-7 and either idarubicin 12 mg/m2/day IV on Days 1-3 or daunorubicin 60 mg/m2/day IV on Days 1-3. on Day 8 of the induction chemotherapy, half of the patients will receive pembrolizumab IV (intervention arm), and continue Q3 weeks throughout Induction/Consolidation, and thereafter for up to 2 years from the start of maintenance. The other half will not receive pembrolizumab (control arm). After 31 efficacy-evaluable patients (patients who had a bone marrow biopsy done after the first consolidation cycle) in each of the two study arms have had their primary endpoint results available, a formal futility and toxicity analysis will be conducted. Follow up will continue up to 2 years after the last patient is randomized. Eligible patients are aged ≥18 and ≤75 years with newly diagnosed and pathologically-confirmed AML, including secondary AML arising from prior MDS and therapy-related AML. Patients with FLT3-mutated AML are excluded as well as those with prior allogeneic stem cell transplant, active infection requiring systemic therapy, and requiring use of high dose corticosteroids. Responses in AML patients will be assessed using European Leukemia Net 2017 response criteria. EFS will be calculated as the time from initial treatment to either disease relapse or death. OS will be calculated from time from initial treatment to death. Biomarker analyses will include an extensive array of immunologic and correlative studies that will evaluate PD-1 and PD-L1 expression, immune cell subset analysis, leukemia specific T-cell response, cytokine level, T-cell receptor (TCR) repertoire, as well as RNA-seq, gut microbiome characterization and metabolomics, and whole exome sequencing for tumor and germline DNA. Figure 1 Disclosures Zeidan: Epizyme: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Cardinal Health: Consultancy, Honoraria; Acceleron: Consultancy, Honoraria; Leukemia and Lymphoma Society: Other; Jazz: Consultancy, Honoraria; Celgene / BMS: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; BeyondSpring: Consultancy, Honoraria; Cardiff Oncology: Consultancy, Honoraria, Other; Takeda: Consultancy, Honoraria, Research Funding; Ionis: Consultancy, Honoraria; MedImmune/Astrazeneca: Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Aprea: Research Funding; ADC Therapeutics: Research Funding; Astex: Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Taiho: Consultancy, Honoraria; CCITLA: Other; Novartis: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria. Radich:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Jazz: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding.

Blast MRD AML-2: Blockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease (MDR) in Acute Myeloid Leukemia (AML) 2- a Randomized Phase 2 Study of the Venetoclax, Azacitidine, and Pembrolizumab Versus Venetoclax and Azacitidine As First Line Therapy in Older Patients with AML Who Are Ineligible or Who Refuse Intensive Chemotherapy

Study Background: Survival outcomes of older patients with AML are dismal with a 3-year survival of < 10%. The combination of hypomethylating agents (HMA) with venetoclax has emerged as the new standard of care for older unfit patients with AML, but median survival remains less than 15 months. Patients with undetectable MRD after this regimen seem to have the best long-term clinical outcomes. In this randomized phase 2 trial, the goal with combining pembrolizumab is to facilitate a more effective CTL mediated destruction of leukemic blasts resulting in improved rates of CR without MRD, which would hopefully increase duration of response and lower relapse rates. Here we report on the design of the first randomized multi-center clinical trial of HMA+venetoclax +/- ICPI in unfit AML patients. Methods: The primary objective of this Cancer Therapy Evaluation Program-approved multi-institutional, randomized phase II study (NCT04284787) is to assess the percentage of patients with MRD negative CR (MRD- CR) as measured by flow cytometry with chemotherapy + pembrolizumab during the first 6 cycles and compare between the two study arms (See study Schema). Secondary objectives include rates of CR / complete remission with incomplete count recovery (CRi), complete remission with partial recovery count (CRh) and Hematologic improvement (HI) to red blood cells and platelets, MRD negativity at Day 14, MRD-negative CR at end of induction therapy and MRD negative CR after last consolidation cycle, event free survival (EFS), relapse free survival (RFS), duration of response (DOR), and overall survival (OS), and proportion of patients who develop severe toxicity. Important exploratory objectives include MRD assessment by duplex sequencing (DS), and comparing DS and multiparameter flow cytometry for MRD detection. A total of 76 patients will be included (38 patients in the intervention arm and 38 patients in the control arm). Planned stratification factors include 1) cytogenetics (intermediate/unknown vs. adverse karyotype) 2) antecedent hematologic disorder defined as prior MDS, MPN, or aplastic anemia (present vs. absent); and 3) reason to not receive intensive chemotherapy (ineligibility vs refusal). All patients will receive azacitidine at 75 mg/m2 IV over 10-40 minutes or SQ x 7 days on Days 1-7 or Days 5-2 (to avoid weekend administration) and, including a ramp-up phase to 400 mg /day venetoclax on Days 1-4. Venetoclax will be administered at 400 mg/day on the remaining days of the cycle for the first cycle and on Days 1-21 or 1-28 for subsequent cycles (depending on count recovery). Half of the patients will receive pembrolizumab intravenously starting on Day 8 of the first cycle and then q 3 weeks in cycles 2-6 (intervention arm), up to 2 years of maintenance. The other half will not receive pembrolizumab (control arm). After 15 efficacy-evaluable patients in each of the two study arms have had their primary endpoint results available, a formal futility and toxicity analysis will be conducted. Follow up will continue up to 2 years after the last patient is randomized Eligible patients are aged ≥60 years with newly diagnosed and pathologically-confirmed AML, including secondary AML arising from prior MDS and therapy-related AML, who are deemed ineligible for intensive chemotherapy according to treating physician's assessment or who refuse intensive chemotherapy. Candidates with high risk MDS and secondary AML arising from myeloproliferative neoplasm or other hematologic disorders are not allowed. Additional exclusion criteria include CBF-AML, prior allogeneic stem cell transplant, receipt of prior hypomethylating therapy for antecedent MDS, active infection requiring systemic therapy, and use of corticosteroids. Responses in AML patients will be assessed using European Leukemia Net 2017 response criteria. EFS will be calculated as the time from initial treatment to either disease relapse or death. OS will be calculated from time from initial treatment to death. Biomarker analyses will include an extensive array of immunologic and correlative studies that will evaluate PD-1 and PD-L1 expression, immune cell subset analysis, leukemia specific T-cell response, cytokine level, T-cell receptor (TCR) repertoire, as well as RNA-seq, gut microbiome characterization and metabolomics, and whole exome sequencing for tumor and germline DNA. Disclosures Zeidan: Abbvie: Consultancy, Honoraria, Research Funding; MedImmune/Astrazeneca: Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Celgene / BMS: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; BeyondSpring: Consultancy, Honoraria; Cardiff Oncology: Consultancy, Honoraria, Other; Takeda: Consultancy, Honoraria, Research Funding; Ionis: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Leukemia and Lymphoma Society: Other; CCITLA: Other; Astex: Research Funding; Aprea: Research Funding; ADC Therapeutics: Research Funding; Taiho: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Cardinal Health: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria. Radich:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Jazz: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding.

Abstract 1921: The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial

Abstract Polymorphisms in DNA damage repair (DDR) genes could serve as predictors of response to gemtuzumab ozogamicin (GO), a CD33 targeting antibody conjugated to the DNA damaging agent calicheamicin approved for use in immunotherapy for acute myeloid leukemia (AML). GO is internalized upon binding to the myeloid cell surface antigen CD33, allowing calicheamicin-mediated DNA damage and subsequent apoptosis. Following concerns over safety, GO was withdrawn in 2010 but was re-approved in 2017 with the emergence of new evidence of its clinical efficacy and safety. In clinical trials, significantly improved event free survival (EFS), disease free survival (DFS), and relapse risk (RR) were observed in a subset of AML patients with the addition of GO to conventional AML chemotherapy. Combined with low overall survival (OS) and high RR with the relatively unchanged conventional AML chemotherapy, GO usage is anticipated to increase. We used samples from the Children's Oncology Group (COG) trial AAML0531 to determine the association between polymorphism in DDR genes and OS, EFS, DFS, and RR with GO treatment as the primary end points. In this trial, newly diagnosed AML patients were randomized to receive either the standard 5 course chemotherapy alone (Arm A) or with two doses of GO (3mg/m2/dose) during induction 1 and intensification 2 (Arm B). Briefly, genomic DNA samples from a total of 947 patients from Arm A and Arm B were genotyped for 131 SNPs across 43 key genes involved in calicheamicin-mediated DDR. Overall, 28 SNPs in 21 genes were significantly associated with OS, EFS, DFS, and RR. Of these, 21 SNPs in 16 genes were associated with response to GO treatment (Arm B; p ≤ 0.05) and not with conventional AML chemotherapy (Arm A; p ≥ 0.05). The top 5 SNPs occurred within DDB2, XPA, XPC, and XRCC1 – genes involved in the nucleotide excision repair (NER) DDR pathway. The intronic variation rs830083 (G>C) in DDB2 had the poorest outcome (OS Cox p = 0.01; EFS cox p = 0.004; DFS p = 0.000; RR reg p = 0.0006). Furthermore, rs830083 was also most significantly associated with standard risk patients (DFS p = 0.003), EFS (p=0.01) and RR (p = 0.0006). DDB2 has been reported to play a critical role in apoptotic processes post-DNA damage induction by regulating p53, lending further importance to the association between rs830083 and poor response to treatment. In conclusion, the identification of several variants associated with treatment outcomes in our study underscores the importance of a pathway-directed SNP-based candidate gene approach to identify personalized approaches for AML treatment. Specifically, screening DDB2 SNPs could be applied in patient selection and predicting response to GO treatment. Citation Format: Vivek M. Shastri, Lata Chauhan, Todd A. Alonzo, Yi-Cheng Wang, Richard Aplenc, Betsy A. Hirsch, Alan S. Gamis, Soheil Meshinchi, Jatinder K. Lamba. The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1921.

Dactinomycin in Acute Myeloid Leukemia with NPM1 Mutations

NPM1 mutations are frequent in acute myeloid leukemia (AML) and define a distinct entity according to the 2016 WHO classification. NPM1 mutations are generally associated with chemosensitivity and a favorable prognosis. However, outcome may vary according to co-mutational events, and still approximately 40% of patients relapse after achieving complete response. Wild-type NPM1 is mainly located in the nucleolus where it plays a key role in the regulation of ribosome biogenesis, protein synthesis and tumor suppression through TP53 activation. Mutated NPM1 loses its predominant nucleolar location and accumulates in cytoplasm contributing to leukemogenesis (Falini B, Blood 2011). Moreover, this mutational event leads to haploinsufficiency and cytoplasmic retention of wild type NPM1 creating a vulnerability to nucleolar stress. Indeed, complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug (Falini B, NEJM 2015). Here, we report our experience of off-label dactinomycin in untreated or relapsed/ refractory NPM1-mutated AML. Inclusion criteria for this retrospective study were: age ≥ 18 years-old, AML with NPM1 mutation, relapsed or refractory disease as well as treatment-naive patients unfit for intensive chemotherapy. Patients should also have completed one cycle of dactinomycin 12.5 µg/kg/day for 5 days every 28 days. From September 2015 to February 2019, 26 patients received dactinomycin. Median age was 62.5y, WBC count was > 50 giga/L in 8 patients (31%), 13 patients (50%) had FLT3-ITD mutation whereas 10 (38%) and 11 (42%) patients were classified as favorable or intermediate-I according to the ELN-2010 classification. There were 7 (27%) relapses post-allogeneic transplantation. Median number of dactinomycin cycle was 1 (1-8) and 7 patients (27%) received more than 3 cycles. Sorafenib was added in 6 patients with associated FLT3-ITD mutations whereas 2 others patients received ATRA in combination with dactinomycin. Dactinomycin was administered in different situations: front-line treatment in 4 unfit patients (16%); morphologic (n=16, 62%) or molecular relapses (n=4, 16%) following intensive chemotherapy, refractory disease (n=1, 13%) or post remission therapy in second complete response (CR) following salvage chemotherapy (n=1, 13%). Three out of 17 patients (18%) in morphologic relapse or refractory to chemotherapy reached complete remission after the first cycle of dactinomycin. The duration of response was 4 and 6 months in 2 patients whereas the third patient is still in CR 3 years after dactinomycin. One out of 4 patients in molecular relapses achieved a complete molecular remission with dactinomycin. None of the 4 patients unfit for intensive chemotherapy responded to dactinomycin as front-line therapy. The only patient treated in post-CR2 with dactinomycin achieved a complete molecular remission before allogeneic transplantation. Overall, 5 patients (19%) appeared to benefit from dactinomycin treatment. Grade 3-4 adverse events were thrombocytopenia (n=11, 42%), neutropenia (n=11, 42%), GI toxicity (n=6, 23%), mucositis (n=5, 19%), lung infection (n=5, 19%) and skin rash (n=2, 7.6%). Dactinomycin is an inexpensive and easily available drug that may induce significant responses in AML patients with NPM1 mutations with an acceptable safety profile. Prospective and controlled clinical trials are mandatory to clearly define the role of this agent in AML with NPM1 mutations. Disclosures Tavitian: Novartis: Membership on an entity's Board of Directors or advisory committees. Bertoli:Sanofi: Honoraria. Huguet:Incyte Biosciences: Honoraria; Servier: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria. Bories:Abbvie: Consultancy. Recher:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: NPM1 mutations are frequent in acute myeloid leukemia (AML) and define a distinct entity according to the 2016 WHO classification. Wild-type NPM1 is mainly located in the nucleolus where it plays a key role in the regulation of ribosome biogenesis, protein synthesis and tumor suppression through TP53 activation. Mutated NPM1 loses its predominant nucleolar location and accumulates in cytoplasm contributing to leukemogenesis (Falini B, Blood 2011). Moreover, this mutational event leads to haploinsufficiency and cytoplasmic retention of wild type NPM1 creating a vulnerability to nucleolar stress. Indeed, complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug (Falini B, NEJM 2015).

BrUOG 345: Fractionated Gemtuzumab Ozogamicin Followed By Non-Engraftment Donor Leukocyte Infusions for Relapsed/Refractory Acute Myeloid Leukemia

Background: Allogeneic stem cell transplant (allo-SCT) is a cornerstone in the treatment of Acute Myeloid Leukemia (AML) exerting much of its therapeutic efficacy through the graft-versus-leukemia effect. The administration of immunoregulatory cells via engrafted donor lymphocytes is essential to the eradication of residual malignancy and long-term survival. Many patients, however, either do not have an allo-SCT donor or cannot withstand the toxicity of allo-SCT. Previous studies have shown durable responses in AML patients following donor lymphocyte infusions in the absence of engraftment in both the frontline and relapsed setting (Dey et al, BJH 2005, Colvin et al, BBMT 2009, Ai et al, Blood 2010). In this clinical trial we propose a role for donor leukocyte infusions (DLI) in the absence of engraftment. Without the need for engraftment patients will not need to receive high dose chemotherapy or radiation and the toxicities that accompany these therapies. Instead, allogeneic donor cells are infused into patient with relapsed/refractory (R/R) AML to serve as a potent immune stimulator. Prior to DLI, patients will receive fractionated dosing of gemtuzumab ozogamicin (GO). GO is an anti-CD-33 antibody drug conjugate approved in combination with induction therapy for de novo AML and in R/R disease. Patients who demonstrate a CR or CRi to therapy will go on to have up to 2 additional GO + DLI treatments. Bone marrow and blood samples will be obtained from patients before, during, and after treatment to determine immune effector cells (both donor and patient), cytokine release profiles, and extracellular vesicle components. Study Design and Methods: Our study, BrUOG 345 [NCT03374332], evaluates the combination of fractionated GO with non-engraftment DLI in the treatment of patients with R/R AML. Adults patients 18 years of age and older with histologically confirmed R/R AML who have had recurrence or progression after at least 1 prior standard treatment are eligible. Enrollees must have no active systemic infections and have adequate lung, liver, cardiac, and renal function with an ECOG PS 0-1. Fractionated GO 6-9mg/m2 will be administered on days 1, 4, and 7 followed by infusion of 1-2x108 CD3 cells/kg from a 0-3/6 HLA mismatched related donor cell. Patients that are in CR or CRi can receive up to 2 additional treatments with GO+DLI (Figure 1). The primary objective of the phase 1 portion is to determine the maximum tolerated dose (MTD) of DLI in combination with GO while the primary objective of Phase 2 portion is to assess the response rate after one cycle of fractionated GO followed by non-engraftment DLI in patients with relapsed/refractory AML. The study will initially utilize a 3+3 design in Phase 1 to determine if 1-2x108 CD3 cells/kg can be safely administered with GO. This study will target a response rate of 57% considered to be interesting enough to warrant further study in a randomized setting. With this hypothesis in mind, the phase 2 portion of the study will use Simon's two-stage design. The null hypothesis that the true response rate (CR and CRi) is only 29% will be tested against a one-sided alternative. In the first stage, 9 subjects will be accrued. Patients treated at MTD in the Phase 1 portion of the study will be included in this cohort. If there are 3 or fewer responses in these 9 patients, the study will be stopped for futility. Otherwise, 6 additional subjects will be accrued for a total of 15. The null hypothesis will be rejected if 7 or more responses are observed in 15 patients. This design yields a type I error rate of 0.1 and power of 80% if the true response rate is 57%. A continuous assessment of toxicity will be utilized for the Phase 2 portion of this study. Sequential boundaries will be used to monitor dose-limiting toxicity rate for patients after the initial MTD is determined. Accrual will be halted if excessive numbers of dose-limiting toxicities are seen. The primary outcome of the Phase 2 portion is the CR/CRi rate following GO and non-engraftment DLI. Secondary outcomes will include survival, both progression-free (PFS) and overall (OS) until two years post treatment, and dose limiting toxicities until 16 weeks post-infusion. Additional lab correlative studies will be performed including CD33 expression before, during, and after GO infusion and T-cell activation markers, antigen presenting cell/macrophage amounts, cytokine release profiles, and extracellular vesicle measurements (Figure 1). Disclosures Olszewski: TG Therapeutics: Research Funding; Spectrum Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech: Research Funding. Reagan:Pfizer: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees.

The prognostic impact of tet oncogene family member 2 mutations in patients with acute myeloid leukemia: a systematic-review and meta-analysis

BackgroundThe impact of Tet oncogene family member 2 (TET2) mutations on the prognosis of acute myeloid leukemia (AML) is still controversial. A meta analysis is needed in order to assess the prognostic significance of TET2 mutation in AML.MethodsFive databases including PubMed, Cochrane, EMBase, China National Knowledge Internet (CNKI) and Wanfang database were retrieved to search studies that investigated the correlation between TET2 mutations and outcomes of AML patients. Pooled hazard ratios (HRs) and odds ratios (ORs) were used to assess the effects of TET2 mutations.ResultsSixteen studies were included. TET2 mutation was an unfavorable prognostic factor for overall survival (OS: HR = 1.386; P < 0.001) and event-free survival (EFS: HR = 1.594; P = 0.002) in patients with AML. For patients under 65 years of age, TET2 mutation predicted an inferior OS (HR = 1.310, P = 0.051) and EFS (HR = 1.429, P = 0.027). For patients with intermediate-risk cytogenetics (IR-AML), mutant TET2 had a significant association with adverse OS (HR = 0.474; P < 0.001). For patients with normal cytogenetics (CN-AML), mutant TET2 also conferred adverse OS (HR = 1.425; P < 0.001) and EFS (HR = 1.450, P < 0.001). Further, among patients with CN-AML, mutant TET2 was associated with inferior OS (HR = 2.034, P < 0.001) and EFS (HR = 2.140, P < 0.001) in the ELN favorable-risk subgroup and an inferior EFS (HR = 1.487; P < 0.001) in the ELN intermediate-Isubgroup. With respect to treatment outcome, TET2 mutation predicted a significantly lower rate of complete remission (CR) in cases with ELN favorable-risk cytogenetics (OR = 0.460, P = 0.011).ConclusionsTET2 mutation had adverse impacts on survival and treatment response in AML patients and will contribute to risk-stratification, prognosis prediction and therapy guidance.