RESUMO
With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.
Assuntos
Diferenciação Celular , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Proliferação de Células , Linfócitos T/metabolismo , Linfócitos T/citologia , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Linfócitos T CD8-Positivos/imunologiaRESUMO
Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.
Assuntos
Receptores de Antígenos Quiméricos , Rabdomiossarcoma , Animais , Criança , Humanos , Camundongos , Linhagem Celular Tumoral , Imunoterapia Adotiva , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Antígenos Quiméricos/genética , Rabdomiossarcoma/tratamento farmacológicoRESUMO
BACKGROUND: Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. METHODS: We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. RESULTS: We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. CONCLUSIONS: Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , NF-kappa B/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Ácidos Borônicos/administração & dosagem , Bortezomib , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos , Camundongos SCID , Análise em Microsséries , Neuroblastoma/metabolismo , Pirazinas/administração & dosagem , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.
Assuntos
Neuroblastoma , Receptores de Antígenos Quiméricos , Antígenos de Neoplasias/genética , Antígenos B7 , Linhagem Celular Tumoral , Glipicanas/genética , Humanos , Imunoterapia Adotiva , Neuroblastoma/genética , Neuroblastoma/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute myeloid leukaemia (AML) is a difficult to treat disease and strategies, such as immunotherapy, which have the potential to eliminate residual tumour cells at first remission are required to reduce the incidence of relapse with its high associated mortality rates. T cells play an important role in tumor immunity and two signals are traditionally thought to be required to activate naive T cells; signal one through the major histocompatibility:antigen:T-cell receptor complex and signal two through costimulation. Many tumor associated antigens have been identified in AML suggesting it may be possible to target the immune system of AML patients; however AML develops due to tumour and immune editing, two systems by which AML cells can escape immune surveillance. By genetically modifying AML cells to express costimulatory molecules and/or cytokines, it has been possible to transform AML cells into antigen presenting cells and this has the potential to re-activate the immune system in patients. Here we summarize the rationale for using a whole cell vaccine approach to treat AML, and discuss current progress in the field of whole cell vaccine development against AML.
Assuntos
Vacinas Anticâncer/química , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Ligante 4-1BB/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/metabolismo , Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Citocinas/metabolismo , Terapia Genética/métodos , Humanos , Sistema Imunitário , CamundongosRESUMO
The activation of T cells plays a central role in antitumor immunity. In order to activate naïve T cells, two key signals are required. Signal one is provided through the T-cell receptor (TCR) while signal two is that of costimulation. The CD28:B7 molecules are one of the best-studied costimulatory pathways, thought to be the main mechanism through which primary T-cell stimulation occurs. However, a number of molecules have been identified which serve to amplify and diversify the T-cell response, following initial T-cell activation. These include the more recently described 4-1BB:4-1BB ligand (4-1BBL) molecules. 4-1BB:4-1BBL are a member of the TNFR:TNF ligand family, which are expressed on T cells and antigen-presenting cells (APCs), respectively. Therapies utilizing the 4-1BB:4-1BBL signaling pathway have been shown to have antitumor effects in a number of model systems. In this paper, we focus on the 4-1BB:4-1BBL costimulatory molecules. In particular, we will describe the structure and function of the 4-1BB molecule, its receptor and how 4-1BB:4-1BBL costimulation has and may be used for the immunotherapy of cancer.
Assuntos
Neoplasias/terapia , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ligante 4-1BB , Sequência de Aminoácidos , Animais , Antígenos CD , Antígeno B7-1/metabolismo , Antígeno B7-1/uso terapêutico , Antígenos CD28/metabolismo , Vacinas Anticâncer/metabolismo , Vacinas Anticâncer/uso terapêutico , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasias/imunologia , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.
Assuntos
Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Rabdomiossarcoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Humanos , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Fator de Transcrição STAT3/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.
Assuntos
Neuroblastoma/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Feminino , Fator de Transcrição GATA2/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Células NIH 3T3 , Neuroblastoma/diagnóstico , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Adulto JovemRESUMO
Whole-cell vaccines allow the induction of anti-tumor immune responses without the need to define tumor antigens. We wished to directly compare, for the first time, the capacity of B7-1, B7-2 and 4-1BB ligand (4-1BBL) costimulatory molecules to convert murine and human acute myeloid leukemia (AML) cells into whole vaccines. 32Dc-kit is a murine myeloid cell line, which develops an AML-like disease over a protracted period, emulating human AML disease development. 32Dc-kit cells were modified to express elevated levels of B7-1, B7-2 or 4-1BBL, and each led to tumor rejection, although only mice injected with 32Dc-kit/B7-2 cells were able to reject subsequent parental tumor cell challenge. T-cell deficient nude mice were able to reject the 32Dc-kit variants, but they could not reject parental cell challenge; however, we found no evidence of cytotoxic T lymphocyte or natural killer (NK) activity ex vivo suggesting that tumor cell killing was mediated by an immune response that could not be recapitulated using purified NK or T cells as lone effectors. In human allogeneic mixed lymphocyte reactions (MLRs), we found no single costimulatory molecule was more effective, suggesting that the induction of a universal anti-tumor response will require a combination of costimulatory molecules.
Assuntos
Vacinas Anticâncer , Imunidade , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/terapia , Ligante 4-1BB/imunologia , Ligante 4-1BB/uso terapêutico , Animais , Antígeno B7-1/imunologia , Antígeno B7-1/uso terapêutico , Antígeno B7-2/imunologia , Antígeno B7-2/uso terapêutico , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , CamundongosRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at < or =10 microM concentration. More interestingly, 17 compounds are active at < or =1 microM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, eleven compounds are not currently used to treat NB. Among them, nine are FDA approved compounds, and three agents are undergoing clinical trials for various malignancies. Furthermore, we identified four agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neuroblastoma/tratamento farmacológico , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Química Farmacêutica/métodos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Tempo , Triterpenos/farmacologiaRESUMO
PURPOSE: microRNAs have been shown to be involved in different human cancers. We therefore have performed expression profiles on a panel of pediatric tumors to identify cancer-specific microRNAs. We also investigated if microRNAs are coregulated with their host gene. EXPERIMENTAL DESIGN: We performed parallel microRNAs and mRNA expression profiling on 57 tumor xenografts and cell lines representing 10 different pediatric solid tumors using microarrays. For those microRNAs that map to their host mRNA, we calculated correlations between them. RESULTS: We found that the majority of cancer types clustered together based on their global microRNA expression profiles by unsupervised hierarchical clustering. Fourteen microRNAs were significantly differentially expressed between rhabdomyosarcoma and neuroblastoma, and 8 of them were validated in independent patient tumor samples. Exploration of the expression of microRNAs in relationship with their host genes showed that the expression for 43 of 68 (63%) microRNAs located inside known coding genes was significantly correlated with that of their host genes. Among these 43 microRNAs, 5 of 7 microRNAs in the OncomiR-1 cluster correlated significantly with their host gene MIRHG1 (P < 0.01). In addition, high expression of MIRHG1 was significantly associated with high stage and MYCN amplification in neuroblastoma tumors, and the expression level of MIRHG1 could predict the outcome of neuroblastoma patients independently from the current neuroblastoma risk-stratification in two independent patient cohorts. CONCLUSION: Pediatric cancers express cancer-specific microRNAs. The high expression of the OncomiR-1 host gene MIRHG1 correlates with poor outcome for patients with neuroblastoma, indicating important oncogenic functions of this microRNA cluster in neuroblastoma biology.
Assuntos
Biomarcadores Tumorais/biossíntese , MicroRNAs/biossíntese , Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Criança , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.
Assuntos
Mutação/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Rabdomiossarcoma/fisiopatologia , Animais , Ciclo Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Replicação do DNA , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/fisiopatologia , Neoplasias Pulmonares/secundário , Camundongos , Modelos Moleculares , Metástase Neoplásica , Fosforilação , Estrutura Terciária de Proteína , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/química , Rabdomiossarcoma/mortalidade , Fator de Transcrição STAT3/metabolismo , Transplante HeterólogoRESUMO
We describe the modification of tumour cells to enhance their capacity to act as antigen presenting cells with particular focus on the use of costimulatory molecules to do so. We have been involved in the genetic modification of tumour cells to prepare a whole cell vaccine for nearly a decade and we have a particular interest in acute myeloid leukaemia (AML). AML is an aggressive and difficult to treat disease, especially, for patients for whom haematopoietic stem cell (HSC) transplant is not an option. AML patients who have a suitable donor and meet HSC transplant fitness requirements, have a 5-year survival of 50%; however, for patients with no suitable donor or for who age is a factor, the prognosis is much worse. It is particularly poor prognosis patients, who are not eligible for HSC transplant, who are likely to benefit most from immunotherapy. It would be hoped that immunotherapy would be used to clear residual tumour cells in these patients in the first remission following standard chemotherapy treatments and this will extend the remission and reduce the risk of a second relapse associated with disease progression and poor mortality rates. In this symposia report, we will focus on whole cell vaccines as an immunotherapeutic option with particular reference to their use in the treatment of AML. We will aim to provide a brief overview of the latest data from our group and considerations for the use of this treatment modality in clinical trials for AML.