Prognostic Factor In Acute Myeloid Leukemia Research Articles

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Introduction Regulated cell death (RCD) is a process commonly dysregulated in patients (pts) with acute myeloid leukemia (AML), resulting in the survival of genetically unstable cells. Apoptosis, as the best-known form of RCD, interacts with neddylation, a process that contributes to tumorigenesis and apoptosis resistance. Aims This study was conducted to determine the expression of the selected genes involved in the neddylation pathway and to assess their clinical importance along with selected interacting proapoptotic genes. In addition, we correlated the expression levels of these genes with known prognostic factors in AML and determined their impact on the survival of AML pts. Methods The expression of selected proapoptotic genes BIK, BAX, BAK, BCL2L11, BBC3 , PMAIP1 CASP3, CASP7, and neddylation-related genes: CUL1, CUL2 , CUL4A, CUL5, CUL7, CUL9, NEDD8 was determined in duplicates in bone marrow samples of newly diagnosed AML pts. Real-Time PCR was carried out using the TaqMan chemistry and the QuantStudio7 thermal cycler (Applied Biosystems-Thermo Fisher Scientific). Gene expression was normalized using reference genes selected by the Normirazor tool. The normalization was performed by ∆Ct = Ct (reference) - Ct (mRNA of interest). This approach results in higher values for higher mRNA expression. Differential expression analysis was performed using a Welch t-test. Kaplan-Meier method with the log-rank test was used to assess overall survival (OS). Results In a prospective study, we included 40 newly diagnosed AML pts (mean age 62; range 26-87) with a median (m) follow-up of 6.7 months (95% CI: 4.5-9.1). High-risk AML was present in 52.5%, intermediate-risk in 32.5%, and low-risk in 15% of pts, according to European LeukemiaNet (ELN) 2022 risk stratification. NPM1 mutation was present in 23%, whereas FLT3 ITD or TKD mutation in 25% of pts. Intensive treatment was introduced in 43% of pts, while 25% were treated with azacitidine+venetoclax. Non-intensive treatment with azacitidine in monotherapy was administered to the remaining pts. BAK expression was detected in 70%, BIK in 98%, while expression of the other genes was found in all pts. In the high-risk group, we detected lower expression of both proapoptotic BAX (fold change - FC=0.7, p=0.01) and BIK gene (FC=0.4, p=0.01), as well as lower expression of CUL9 (FC=0.7, p=0.03). Accordingly, in pts with NPM1 mutation we revealed upregulation of BIK (FC=6.1, p=0.001), and CUL9 (FC=1.6, p=0.01). Whereas pts with FLT3 mutation had lower expression of proapoptotic BBC3 (FC=0.5, p=0.01), and neddylation-related CUL1 (FC=0.6, p=0.01). In cases of initial bone marrow blasts infiltration above median value (>49%) BCL2L11 CUL5 , NEDD8 and PMAIP1were downregulated ( p=0.001-0.02), while CUL7 ( p=0.00) and CASP3 ( p=0.04) were upregulated. Patients with initial white blood cell count >20 G/l had lower expression of BCL2L11 ( p=0.005), and CUL4A ( p=0.02), while higher expression of CASP7 ( p=0.04). The mOS in the study cohort was not reached. OS was longer in pts with higher (> -2.061, cutoff was determined with CutOff Finder) BAX expression (log-rank: p=0.03) [Figure 1]. The mOS was not reached in this group, whereas the mOS in pts with lower BAX expression was 1.3 months (95% CI: 0.6-1.3). In univariate Cox regression analysis, higher BAX expression (HR 0.31, 95%CI: 0.11-0.84, p=0.022), higher albumin level (HR 0.13, 95%CI: 0.04-0.41, p=0.0004), and intensive treatment (HR 0.18, 95%CI: 0.04-0.69, p=0.012) were factors influencing the outcome. In the multivariate model for OS, higher expression of BAX (HR 0.03, 95%CI: 0.003-0.33, p=0.004) retained its significant protective effect in the context of established prognostic factors [Table 1]. Conclusions To our knowledge, we were the first to evaluate such a complex spectrum of neddylation and apoptosis-related genes in AML. We revealed dysregulation of these genes in AML. Our data indicate that upregulation of BAX at the diagnosis is associated with longer OS. Moreover, we reported significantly lower BAX expression in ELN high-risk group. Thus, indicating BAX may be a potential prognostic factor in AML. Our preliminary results did not prove significant differences in the effect of neddylation gene expression levels on the prognosis of AML patients. As studies are emerging on the potential of neddylation-targeted therapies, we are conducting further research to verify the effect of neddylation on AML.

Integrated Analysis of KIT Exon 17 Mutations and Flow-MRD Refines Risk Stratification in Pediatric Acute Myeloid Leukemia with RUNX1:: RUNX1T1

INTRODUCTION: Pediatric acute myeloid leukemia (AML) with RUNX1:: RUNX1T1 is considered a favorable risk group, with a high probability of event-free survival (EFS) in recent clinical trials. However, 20-30% of patients still experience relapse within three years and some patients have poor outcome. Several prognostic factors, including secondary genetic abnormalities and treatment response, have been previously reported. Flow cytometry-based measurable residual disease (flow-MRD) has been suggested as a poor prognostic factor in AML with RUNX1:: RUNX1T1. Additionally, KIT mutations, particularly mutations of exon 17, have been associated with poor EFS in both adult and pediatric patients. However, the combined impact of KIT exon 17 mutations and flow-MRD on prognosis has not been thoroughly investigated. This study aims to evaluate the clinical and genetic features of pediatric patients with RUNX1:: RUNX1T1-positive AML enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-12 trial and determine if integrated analysis of flow-MRD and KIT exon 17 mutations can improve risk stratification. METHOD: A total of 101 pediatric AML patients with RUNX1:: RUNX1T1 were enrolled in the study. The patients were treated in the JPLSG AML-12 trial, and their clinical and genetic characteristics were analyzed. Targeted capture sequencing was performed on bone marrow samples obtained at diagnosis. The custom gene panel was designed for mutation profiling of pediatric AML, including 504 genes. Flow-MRD was centrally monitored at the end of induction 1 (EOI1) and the end of induction 2 but was not used to guide subsequent therapies. Leukemia-associated immunophenotypes were identified in diagnostic specimens and marker combinations that allowed detection of leukemia with a sensitivity of at least 0.1% were applied to subsequent samples. RESULTS: Patients with KIT exon 17 mutations had a higher white blood cell count at diagnosis and a significantly higher rate of MRD positivity (≥0.1%) at EOI1 compared to patients without KIT exon 17 mutations. Immunophenotypic analysis showed no significant differences in CD117 or CD33 expression between patients with and without KIT exon 17 mutations; whereas CD19 expression was observed less frequently and, although not significantly, CD56 expression was observed more frequently in the patients with KIT exon 17 mutations. The genetic landscape analysis revealed that KIT mutations were the most common and tended to coexist with ARID1B mutations, while NRAS mutations were mutually exclusive. The 5-year EFS and overall survival (OS) for the entire cohort were 67.3% and 82.7%, respectively. Negative MRD at EOI1 was associated with significantly superior 5-year EFS and OS (71.6% and 85.9%) compared to positive MRD (12.5% and 37.5%)(p<0.01). Patients with KIT exon 17 mutations had significantly inferior 5-year EFS and OS (46.9% and 62.2%) compared to those without KIT exon 17 mutations (76.8% and 92.4%) (p<0.01). Furthermore, we evaluated a prognostic impact of MRD within the KIT exon 17 mutated and non-mutated cases. As for the non-mutated cases, the 5-year OS in the patients with negative MRD was 92.2%. Among the KIT-mutated cases, the 5-year OS in the patients with negative and positive MRD was 68.0% and 33.3%, respectively. CONCLUSION: Patients with both non-mutated KIT exon 17 and negative MRD have the best prognosis, while positive MRD and KIT exon 17 mutations are associated with poorer outcomes. These findings indicated that integrated analysis of flow-MRD and KIT exon 17 status enables optimal risk assignment strategies in pediatric AML with RUNX1:: RUNX1T1.

Fluorescence In Situ Hybridization (FISH) for Detection of c-MYC Amplifications in AML Patients: A Potential Marker for Risk Stratification

Abstract Background c-MYC is a transcription factor (located on 8q24) which regulates genes involved in proliferation, cell growth, differentiation, and apoptosis. The c-MYC amplification is related to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Gene amplification (amp) is one of the basic mechanisms connected with over-expression of oncogenes. c-MYC is routinely analyzed in mature lymphoid neoplasms especially in Burkitt’s Lymphoma while not assessed for acute myeloid leukemia (AML).Despite the fact the c- MYC oncogene is commonly over-expressed in AML it has remained an elusive target. Routine assessment for c- MYC in AML may pave the way for targeting the c-MYC in treatment of AML patients. Several experiments are ongoing investigating both direct and indirect c-MYC targeting therapies and further establishing cMYC as an oncogene of central importance to AML pathogenesis and treatment. Aim of the Work In this study we aimed to define the prognostic value of c-MYC amplification in adult AML patients, and to relate it to other prognostic factors in AML. Patients and Methods The study was a randomized control study, conducted at the Clinical Pathology Department, Ain-Shams University Hospitals during the period from January 2019 till June 2020. It was carried on 50 adult AML patients (30 newly diagnosed and 20 on Therapy) and 10 healthy age-matched volunteers as control. All subjects underwent full history taking, thorough clinical examination, laboratory investigations including; CBC, Examination of Leishman’s stained peripheral blood films, Bone marrow aspiration and examination of Leishman’s stained bone marrow smears, Cytochemical studies using Myeloperoxidase stain, Immunophenotyping of bone marrow or peripheral blood samples and Fluorescence in situ hybridization analysis (FISH). Results we had 9 patients with c-MYC amplification (6 in the newly diagnosed group and 3 in the on therapy group). So the percentage of c-MYC amplification in our patients was 9/50 (18%). To further elucidate the effect of c-MYC amplification on our patients’ clinical, laboratory and prognostic factors, an analytical comparison was done between negative and positive patients for cMYC amplification as regard-studied parameters in each group of patients. Both group of patients showed a statistical significant association between c-MYC amplification and outcome where the majority of patients with positive c-MYC amplification achieved incomplete remission. In addition, c-MYC amplification showed a statically significant association with Trisomy 8 in both patients’ groups and with 17p deletion and hepatosplenomegaly in on therapy group. Univariate logistic regression analysis for the association between c-MYC amplification and outcome showed statistically significant association between c-MYC amplification and incomplete remission in newly diagnosed AML patients with P-value = 0.013 and odds ratio (95% CI) of 14.0. Conclusion Our study has shown that c-MYC amplification is associated with adverse prognosis, poor treatment outcome and it might be involved in the process of AML progression. Targeting cMYC may be of value in the development of new-targeted AML therapeutics.

Spermatogenesis associated serine rich 2 like plays a prognostic factor and therapeutic target in acute myeloid leukemia by regulating the JAK2/STAT3/STAT5 axis

BackgroundSpermatogenesis associated serine rich 2 like (SPATS2L) was highly expressed in homoharringtonine (HHT) resistant acute myeloid leukemia (AML) cell lines. However, its role is little known in AML. The present study aimed to investigate the function of SPATS2L in AML pathogenesis and elucidate the underlying molecular mechanisms.MethodsOverall survival (OS), event-free survival (EFS), relapse-free survival (RFS) were used to evaluate the prognostic impact of SPATS2L for AML from TCGA database and ourcohort. ShRNA was used to knockdown the expression of SPATS2L. Apoptosis was assessed by flow cytometry. The changes of proteins were assessed by Western blot(WB). A xenotransplantation mice model was used to evaluate in vivo growth and survival. RNA sequencing was performed to elucidate the molecular mechanisms underlying the role of SPATS2L in AML.ResultsSPATS2L expression increased with increasing resistance indexes(RI) in HHT-resistant cell lines we had constructed. Higher SPATS2L expression was observed in intermediate/high-risk patients than in favorable patients. Meanwhile, decreased SPATS2L expression was observed in AML patients achieving complete remission (CR). Multivariate analysis showed high SPATS2L expression was an independent poor predictor of OS, EFS, RFS in AML. SPATS2L knock down (KD) suppressed cell growth, induced apoptosis, and suppressed key proteins of JAK/STAT pathway, such as JAK2, STAT3, STAT5 in AML cells. Inhibiting SPATS2L expression markedly enhanced the pro-apoptotic effects of traditional chemotherapeutics (Ara-c, IDA, and HHT).ConclusionsHigh expression of SPATS2L is a poor prognostic factor in AML, and targeting SPATS2L may be a promising therapeutic strategy for AML patients.

FLOT1 knockdown inhibits growth of AML cells through triggering apoptosis and pyroptosis.

Acute myeloid leukemia (AML) is a group of hematological malignancies characterized by clonal proliferation of immature myeloid cells. Lipid rafts are highly organized membrane subdomains enriched in cholesterol, sphingolipids, and gangliosides and play roles in regulating apoptosis through subcellular redistribution. Flotillin1 (FLOT1) is a component and also a marker of lipid rafts and had been reported to be involved in the progression of cancers and played important roles in cell death. However, the role of FLOT1 in AML remains to be explored. In this study, we found that increased expression of FLOT1 was correlated with poor clinical outcome in AML patients. Knockdown of FLOT1 in AML cells not only promoted cell death in vitro but also inhibited malignant cells engraftment in vivo. Mechanically, FLOT1 knockdown triggered apoptosis and pyroptosis. FLOT1 overexpression promoted AML cell growth and apoptosis resistance. Our findings indicate that FLOT1 is a prognostic factor of AML and may be a potential target for AML treatment.

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SYNERGISTIC EFFECTS OF PIP4K2 INHIBITOR AND VENETOCLAX ON APOPTOSIS INDUCTION IN LEUKEMIA CELLS

Venetoclax, a selective inhibitor of BCL2, has been introduced in clinical practice for the treatment of patients with acute myeloid leukemia (AML). PIP4K2s comprise a family of lipid kinases responsible to produce phosphoinositides (PtdIns4,5P2) with structural and signal transduction functions. The combined expression of PIP4K2A and PIP4K2C has been identified as an independent prognostic factor in AML. Recently, a selective pharmacological inhibitor of PIP4K2s was developed (THZ-P1-2). The present study aimed to characterize the antineoplastic potential of THZ-P1-2 in combination with venetoclax in a cellular model of AML. OCI-AML3 leukemia cell line that is resistant to venetoclax was used. Cell viability was evaluated by MTT assay and apoptosis by annexin V/PI and flow cytometry. OCI-AML3 were treated with graded doses of THZ-P1-2 (0, 1.6, 3.2, 6.4, 12.5, and 25 μM) and/or venetoclax (0, 3.2, 6.4, 12.5, 25, and 50 μM) for 48 hours. The data obtained from at least three independent experiments were analyzed by linear regression for the determination of IC50, and statistical analysis was performed by ANOVA and Bonferroni post-test using GraphPad Prism software. A p-value < 0.05 was considered significant. The combination of THZ-P1-2 and venetoclax synergistically reduced the viability of OCI-AML3 cells. The IC50 values for venetoclax, venetoclax plus 6.4 μM THZ-P1-2, and venetoclax plus 12.5 μM THZ-P1-2 were 7.3, 4.4, and 1.2 μM, respectively. Apoptosis assays corroborate these findings, the levels of apoptotic OCI-AML3 cells treated with vehicle, 6.4 μM venetoclax, 6.4 μM THZ-P1-P2, 12.5 μM THZ-P1-P2, 6.4 μM venetoclax plus 6.4 μM THZ-P1-2, and 6.4 μM venetoclax plus 12.5 μM THZ-P1- 2 were 3.8 ± 0.7%, 33.3 ± 4.8%, 18.5 ± 2.8%, 64.3 ± 4.3%, 78.3 ± 1.2% and 97.4 ± 0.3%, respectively (all p < 0.05). The pharmacological inhibition of PIP4K2 potentiates venetoclax-induced apoptosis in acute myeloid leukemia cells. These findings represent new opportunities for overcoming venetoclax resistance and improving therapeutic interventions in AML. Supported by FAPESP, CNPq, and CAPES.

Tegaserod maleate exhibits antileukemic activity by targeting TRPM8

To identify therapeutic targets in acute myeloid leukemia (AML), we conducted growth inhibition screens of 2040 small molecules from a library of FDA-approved drugs using a panel of 12 AML cell lines. Tegaserod maleate, a 5-hydroxytryptamine 4 receptor partial agonist, elicits strong anti-AML effects in vitro and in vivo by targeting transient receptor potential melastatin subtype 8 (TRPM8), which plays critical roles in several important processes. However, the role of TRPM8 remains incompletely described in AML, whose treatment is based mostly on antimitotic chemotherapy. Here, we report an unexpected role of TRPM8 in leukemogenesis. Strikingly, TRPM8 knockout inhibits AML cell survival/proliferation by promoting apoptosis. Mechanistically, TRPM8 exerts its oncogenic effect by regulating the ERK–CREB/c-Fos signaling axis. Hyperactivation of ERK signaling can be reversed by TRPM8 inhibition. Importantly, TRPM8 is overexpressed in AML patients, indicating that it is a new prognostic factor in AML. Collectively, our work demonstrates the anti-AML effects of tegaserod maleate via targeting TRPM8 and indicates that TRPM8 is a regulator of leukemogenesis with therapeutic potential in AML.

Analysis of m6A-related signatures associated with the tumor immune microenvironment and predict survival in acute myeloid leukemia.

BackgroundMost previous studies have focused on the intrinsic carcinogenic pathways of tumors; however, little is known about the potential role of N6-methyladenosine (m6A) methylation in the tumor immune microenvironment (TIME). To better diagnose and treat acute myeloid leukemia (AML), we sought to examine the correlation between m6A regulatory factors and immune infiltration in cases of AML. At the same time, a prognostic model was constructed to predict the survival of AML.MethodsWe extracted data from The Cancer Genome Atlas (TCGA) database, including ribonucleic acid sequencing (RNA-seq) transcriptome data and data on the corresponding clinical characteristics of AML patients. We identified two m6A modification patterns with distinct clinical outcomes and found a significant relationship between them. Simultaneous discovery of distinct m6A clusters associated with the tumor immune microenvironment [immune cell types and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm] are closely related. Next, we implemented Lasso (Least Absolute Shrinkage and Selection Operator) Cox regression to build a predictive model in the 2-m6A regulator TCGA dataset to further explore m6A prognostic features in AML, and perform correlation validation.ResultsWe identified 2 molecular subtypes (Clusters 1 and 2) by the consistent clustering of significant m6A regulators in AML. Cluster 2 was associated with a higher immune score and obvious immune cell infiltration, and thus patients in Cluster 2 had a poorer prognosis than those in Cluster 1 (P<0.05). Additionally, the 2 m6A-related signatures representing the independent prognostic factors in AML were screened to construct a prognostic risk-score model. We found that patients with low-risk scores had higher immune scores than those with high-risk scores (P<0.05).ConclusionsOur research confirmed that m6A methylation plays an important role in AML. Further provide new directions for the prognosis and treatment of AML.

Long non-coding RNA LOC644135 is a potential prognostic indicator in cytogenetically normal acute myeloid leukemia

ABSTRACT Objectives Acute myeloid leukemia (AML) is a hematological malignancy with highly clinical heterogeneity resulting in poor outcomes. We aim to identify novel prognostic lncRNA in AML expecting to provide new clues for therapy in AML. Methods Three cohorts were enrolled in this study. Differentially expressed lncRNAs between TCGA-AML cohort and GTEx cohort was identified by DESeq2. The relationship between expression level of LOC644135 and prognosis in AML was analyzed by multiple methods. Results Pan-cancer analysis indicated that LOC644135 was most highly expressed in AML across 33 types of cancer. Patients with high expression of LOC644135 had poor overall prognosis in both TCGA-AML cohort and the TARGET-AML cohort. Especially, high expression of LOC644135 indicated inferior overall survival and event-free survival in CN-AML patients in the TCGA-AML cohort. Besides, CN-AML patients had higher expression of LOC644135 than normal samples. Multivariable analysis suggested that LOC644135 was an independent prognostic factor in AML. GSEA analysis showed that LOC644135 was associated with some immune-related pathways. Besides, high expression of LOC644135 was associated with less infiltration of CD8+ T cell. Conclusion Our findings indicated that LOC644135 was an independent prognostic factor in AML and provided a new idea in the development of therapy in AML.

P506: ACHIEVEMENT OF MEASURABLE RESIDUAL DISEASE CLEARANCE IS A STRONGER PREDICTOR OF PATIENT OUTCOME THAN TREATMENT INTENSITY IN NEWLY DIAGNOSED PATIENTS WITH ACUTE MYELOID LEUKEMIA

Background: Modern therapy for acute myeloid leukemia (AML) can consist of either intensive or low-intensity regimens selected based on patient (pt) age/comorbidities. Measurable residual disease (MRD) has emerged as a strong independent prognostic factor in AML. It is unknown whether pts who achieve similar levels of MRD clearance experience comparable outcomes irrespective of treatment intensity. Aims: To establish the relative prognostic contribution of treatment intensity and MRD status on overall survival (OS) and relapse-free survival (RFS) in newly diagnosed AML pts who have achieved a first response. Methods: We conducted a retrospective chart review to identify non-CBF, non-APL AML pts treated at our institution between 2010 and 2021 with intensive (IA; int/high-dose cytarabine + anthracycline-based, without venetoclax) or low-intensity (Lo + VEN; low-dose cytarabine/hypomethylating agent-based, with venetoclax) regimens who had achieved CR/CRi/MLFS and undergone MRD testing by multiparameter flow cytometry at time of first response. Differences in baseline pt characteristics were evaluated using the Chi-square or Wilcoxon-Mann-Whitney tests for categorical and continuous variables, respectively. The Kaplan-Meier method was used to estimate median OS and RFS, with pts censored at the time of stem cell transplantation (SCT). Multivariate analysis was performed using a Cox proportional hazards model. Results: We identified 635 pts meeting inclusion criteria. Baseline characteristics are shown in Table 1. Compared to the IA-treated pts (n=385), pts treated with Lo + VEN (n=250) were significantly older, more likely to be male, less likely to achieve MRD clearance, and more likely to be classified as adverse risk by ELN 2017. The SCT rate in the Lo + VEN cohort was half that of the IA cohort (25% vs 50%). Median OS was 51m, 24.6m, 15m, and 9.9m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Median RFS was 27.8m, 15.4m, 7.3m, and 5.2m in the IA MRD(-), Lo + VEN MRD(-), IA MRD(+), and Lo + VEN MRD(+) groups, respectively. Pts within the same MRD category (+ or -) had significantly higher OS and numerically higher RFS if treated with IA versus Lo + VEN. When the analysis was restricted to pts aged ≥ 60 years (n=56 in IA, n=239 in Lo + VEN; Fig. A-B), the differences within MRD categories did not reach statistical significance (median OS NR in IA MRD(-) vs 24.6m in Lo + VEN MRD(-), p=0.08; median OS 13m in IA MRD(+) vs 10.6m in Lo + VEN MRD(+), p=0.50; median RFS NR in IA MRD (-) vs 15.4m in Lo + VEN MRD(-), p=0.07; median RFS 25.1m in IA MRD(+) vs 5.3m in Lo + VEN MRD(+), p=0.24). The SCT rate was 29% (87/295) in the age ≥ 60 cohort. Given the confounding effect of unbalanced pt characteristics between the IA and Lo + VEN cohorts, we performed a multivariate analysis on the full population (n=635) taking into consideration the effects of age, treatment intensity, MRD status, and ELN category on OS and RFS (all significant by univariate analysis). In the multivariate analysis for OS, ELN adverse risk was the strongest predictor (HR 2.09, p<0.001), followed by MRD(+) status (HR 1.68, p<0.001), while treatment intensity and age were not significantly predictive. For RFS, ELN adverse risk (HR 2.31, p<0.001) and MRD(+) status (HR 1.98, p<0.001) were also the only two significant predictors. Image:Summary/Conclusion: In newly diagnosed AML patients, MRD status at time of first response and ELN risk are stronger predictors of pt outcome than intensity of therapy received.

FISH improves risk stratification in acute leukemia by identifying KMT2A abnormal copy number and rearrangements

Most cases of acute leukemia (AL) with KMT2A rearrangement (KMT2A-r) have a dismal prognosis. Detection of this aberration in Chinese adult patients relies on reverse transcription polymerase chain reaction (RT-PCR) and chromosome banding analysis (CBA). The fluorescence in situ hybridization (FISH) probe for KMT2A detects KMT2A-r and copy number variation (CNV) but is not routinely used as a detection technique. This study investigated the potential value of FISH in the treatment of AL by performing FISH along with CBA and RT-PCR in 269 de novo cases of AL. The three detection techniques were compared in identification of KMT2A-r, and the applicability of FISH for detecting KMT2A CNV was evaluated. Twenty-three samples were identified as positive for KMT2A-r (20 using FISH, 15 using RT-PCR, 16 using CBA, and eight according to all three). FISH also identified 17 KMT2A CNV, 15 with gains and two with deletions. Ten patients with acute myeloid leukemia (AML) harboring KMT2A CNV had a complex karyotype, a negative prognostic factor in AML. Adding FISH of KMT2A to routine detection leads to more accurate detection of KMT2A-r and improved identification of KMT2A CNV, which would benefit patients by improving the risk stratification in AL.

Rac GTPases in acute myeloid leukemia cells: Expression profile and biological effects of pharmacological inhibition

Acute myeloid leukemia (AML) is a highly heterogeneous hematological neoplasm with low survival rates. Thus, the investigation of new therapeutic targets is essential. The Rac subfamily of GTPase proteins has been shown to participate in the physiopathology of hematological malignancies. However, their expression and function in AML remain unclear. In this study, we evaluated Rac1, Rac2 and Rac3 gene expressions in AML and their impact on clinical outcomes. We further investigated the effects of the in vitro treatment with a Rac inhibitor (EHT-1864) on AML cell lines.Rac3 expression was increased in AML derived from myelodysplastic syndromes compared to healthy donors. Rac2 expression did not differ between AML patients and healthy donors, but de novo AML patients with higher Rac2 presented lower overall survival. Oncogenic pathway gene-sets related to AKT/mTOR were identified as associated with Rac1, Rac2 and Rac3 expressions. EHT-1864 treatment reduced the viability of OCI-AML3, KG1 and Kasumi-1 cells in a time and dose-dependent manner. In OCI-AML3 cells, treatment with EHT-1864 induced apoptosis, autophagy, and led to the accumulation of cells in the G1 phase of the cell cycle. These changes were concomitant with alterations in p53 and cyclins. Dowregulation of the PI3K/AKT/mTOR pathway was also observed. Interestingly, the combined treatment of EHT-1864 and low doses of daunorubicin enhanced OCI-AML3 cell apoptosis.In conclusion, Rac2 expression is a prognostic factor in AML and our preclinical results suggest that Rac inhibition may be an attractive mechanism to compose the antineoplastic strategy for this disease.

The FLT3 internal tandem duplication mutation at disease diagnosis is a negative prognostic factor in myelodysplastic syndrome patients

The role of the FMS-like tyrosine kinase 3 (FLT3) gene in acute myeloid leukemia (AML) has been well documented and the FLT3-internal tandem duplication (FLT3-ITD) mutation has been identified as a prognostic factor in AML. Due to its low incidence, the role of the FLT3 mutation remains unclear in myelodysplastic syndrome (MDS) patients. To investigate the impact of the FLT3-ITD status on the prognosis of MDS at diagnosis, we retrospectively analyzed 72 MDS patients admitted to Teikyo University Hospital. FLT3-ITD was examined by a reverse transcription-polymerase chain reaction using complementary DNA synthesized from mRNA extracted from bone marrow mononuclear cells at the diagnosis of MDS. Fifteen patients (20.8 %) were positive for FLT3-ITD and had significantly worse overall survival (OS) and progression-free survival (PFS) than patients who were negative (P < 0.001 and P < 0.001, respectively) in a multivariate analysis. We also investigated whether the Wilms’ tumor gene-1 (WT-1) copy number was associated with the FLT3-ITD mutational status using data available on WT-1 from 57 patients. A WT-1 transcript copy number ≥50/μg total mRNA in peripheral blood was detected in 35 patients (61.4 %). All FLT3-ITD-positive patients showed WT-1 ≥50. The FLT3-ITD-positive group showed significantly higher WT-1 transcription levels than the negative group. These results indicate that the FLT3-ITD mutation has a prognostic impact at the diagnosis of MDS and is associated with a high level of WT-1.

Expression Changes of CX3CL1 and Interleukin-6 Genes During Remission Induction Therapy in Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) syndrome is a hematologic malignancy due to the extensive clonal proliferation of leukemic precursor cells and is rapidly fatal unless treated or in response to chemotherapy. Cytogenetic findings have an important role in the prognosis and categorization of AML. This study aimed to investigate the expression changes in CX3CL1 and Interleukin-6 (IL-6) genes before and after chemotherapy as remission induction therapy in AML patients. Materials and Methods: In this study, 69 patients (36 males, 33 female) with AML were selected from tertiary medical health centers. A quantitative polymerase chain reaction was performed for mRNA expression of CX3CL1 and IL-6 genes before and after induction chemotherapy using the 2-ΔΔCT method. Results: The expression of CX3CL1 and IL-6 were significantly increased after induction chemotherapy. Also, the ΔCt mean of CX3CL1 and IL-6 mRNA was not significant between AML subtype groups. Conclusion: We showed that chemotherapy significantly increases the expression of CX3CL1 and IL-6, which can be used as a prognostic factor of AML. [GMJ.2021;10:e2288]

Tim-3 Mutation Might Play in the Pathophysiology of Acute Myeloid Leukemia

Genetic polymorphisms and expression of T-cell immunoglobulin mucin-3 (Tim-3) were associated with susceptibility and prognosis in some tumors, but the relationship between Tim-3 mutation and acute myeloid leukemia (AML) was rarely reported. This study explored the effects of Tim-3 mutations on AML. Four hundred and ninety-one de novo newly diagnosed AML patients were enrolled in this exploratory study. A genomic panel of 167 gene targets were detected by next-generation sequencing (NGS). A matched-pair analysis was designed for prognosis based on 1:2 ratio. 26 patients carried Tim-3 mutations, including 20 Tim-3 p.Y82C and other 6 mutations of p.E273K, p.E182N190del, p.Ter302E, p.A28V, p.R184W, p.G54fs. The median number of somatic mutations apart from Tim-3 was 6 and 7 in Tim-3 + and Tim-3 - groups (P=0.543), respectively. Tim-3 expression was lower, and white blood cell (WBC) at diagnose was higher (P=0.045) in Tim-3 + than Tim-3 - groups. The 2-year cumulative incidence of relapse was 50.8% and 28.7% (P=0.043), overall survival (OS) was 42.6% and 68.5% (P=0.048), and event-free survival (EFS) was 26.8% and 49.6% (P=0.028) in both groups, respectively. Multivariate analysis showed that WBC at diagnose was the risk factor (P=0.004, 0.011 and 0.000, respectively) while transplantation was the protective factor (P=0.015，0.000 and 0.000) for relapse, OS and EFS, and Tim-3 mutation was the risk factor for relapse (P=0.006) in the normal karyotype patients. In vitro, AML cell lines with Tim-3 p.Y82C overexpression revealed lower Tim-3 expression and apoptotic rate as well as lower sensitivity to cytarabine. The bioinformatic analysis also indicated Tim-3 low expression was associated with lower survival in AML patients (P=0.0009). Our findings provide new viewpoint that Tim-3 mutation results in Tim-3 lower expression and is a poor prognostic factor in AML. DisclosuresNo relevant conflicts of interest to declare.

ARHGAP6 EXPRESSION IS ASSOCIATED WITH MOLECULAR RISK AND IMPACTS CLINICAL OUTCOMES IN ACUTE MYELOID LEUKEMIA

Despite the advances in the large-scale generation of genomic data from acute myeloid leukemia (AML) patients, much effort is still needed to convert these data into biological functions, which may pave avenues for the discovery of new prognostic markers and therapeutic targets. In a previous study, four cytoskeleton-related genes (ARHGAP6, EZR, MAP3K11 and PAK1) were identified as a prognostic factor in AML. In the present study, we detail the prognostic impact, as well as its functional genomic associations, of the ARHGAP6 gene in AML patients. Clinical and genomic data were obtained from the TCGA AML cohort (n = 173). The clinical impact (overall survival [OS] and disease-free survival [DFS]) for ARHGAP6 expression was investigated by the proportional hazards Cox regression model. Functional genomic analyzes were performed by gene set enrichment analysis (GSEA) and Gene Ontology (GO) from differentially expressed (DEG) genes obtained from the Galaxy tool. All p values were two sided with a significance level of 5%. ARGHAP6 expression was higher in the intermediate and high molecular risk groups in AML patients (all p < 0.01). High ARGHAP6 expression was identified as an independent risk factor for OS (HR = 1.92; p = 0.001) and DFS (HR = 1.96; p = 0.01) when analyzed with confounders, age, gender, white blood cell count, and molecular risk. In the GSEA, a total of 30 cellular and molecular events were found to be positively enriched in high ARHGAP6-expressing AML patients, which we highlighted the signaling pathways: IL6/JAK2/STAT3 (NES: 1.62; p < 0.001), NOTCH (NES: 1.60; p < 0.001), KRAS (NES: 1.59; p < 0.001), IL2/STAT5 (NES: 1.51; p < 0.001), TGF BETA (NES: 1.39; p = 0.03) and BETA CATENIN SIGNALING (NES: 1.36; p = 0.04). In GO analyses, high ARHGAP6 expression was associated with important processes for cancer development and progression such as cell signaling mediated by surface receptors, signal transduction, linkage to growth factors, and regulation of cell population proliferation (all p < 0.001). ARHGAP6 is a RhoGAP that acts converting RhoGTPases in its inactive GDP-bound state, which may regulate the interactions of signaling molecules with the actin cytoskeleton. It promotes continuous elongation of cytoplasmic processes during cell motility and simultaneous retraction of the cell body changing the cell morphology. The role of ARHGAP6 is still poorly established in cancer, but it is known to modulate pathways of RhoGTPases. In this sense, our study is a pioneer in the identification of this gene in processes related to neoplastic hematopoiesis, which will guide future investigations. ARHGAP6 gene expression was identified as an independent marker of poor prognosis in AML patients. The GSEA suggested that high ARHGAP6 expression is associated with activation of relevant processes to the malignant phenotype of AML. FAPESP, CNPq and CAPES.

Downregulation Of The Wnt Inhibitor CXXC5 Predicts a Better Prognosis In Acute Myeloid Leukemia

▪Different CXXC zinc finger proteins like MLL, DNMT1 and TET1 have been identified to play a role in leukemia development. The CXXC family member CXXC5 is located on 5q31, a region recurrently deleted in acute myeloid leukemia (AML), suggesting that inactivation of CXXC5 might be involved in leukemogenesis. CXXC5 has previously been shown to exert tumor suppressor functions in solid tumor cells and to negatively regulate Wnt signaling, a known pro-survival pathway in leukemia. Here, we investigated prognostic and biological implications of CXXC5expression in AML.CXXC5 mRNA expression was determined by real time quantitative PCR in pretreatment bone marrow samples of 186 AML patients enrolled on the Medical Research Council (MRC) AML15 and UK National Cancer Research Institute (NCRI) AML17 trials. We found downregulation of CXXC5 in AML compared to healthy controls, particularly in cases with del(5q)/-5, MLL rearrangements and t(8;21). To investigate CXXC5-associated gene expression, microarray data from 351 normal karyotype AML derived from the Microarray Innovations in LEukemia study and 529 unselected AML patients treated in Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group (HOVON/SAKK) trials were analyzed. CXXC5 was associated with expression of several genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1), indicating that alteration of CXXC5 expression is a downstream effect of different leukemic pathways. Data from the HOVON/SAKK cohort (median age 46 years (range 15-77) was further used to perform outcome analyses according to microarray-based CXXC5 expression. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P=0.007) and a better overall survival (OS: 46% vs 28%; P<0.001) and event-free survival (EFS: 36% vs 21%; P<0.001) at 5 years. This was of independent significance in multivariate analyses after adjustment for age, white blood cell count, cytogenetic risk group, FLT3-ITD status, biallelic CEBPA mutations, as well as mutations of NPM1, DNMT3A and ASXL1.DNA sequencing of CXXC5 exons 1-3 on 84 AML cases revealed no mutations, suggesting that CXXC5 is predominantly inactivated by gene regulatory mechanisms in AML. Pyrosequencing analysis showed that lower expression of CXXC5 in AML samples was correlated with hypermethylation within the promoter region. Treatment of NB4 and U937 cell lines with 5-Aza-2´-deoxycytidine led to demethylation of this region and induction of CXXC5 at the mRNA and protein level.Functional analyses confirmed Wnt inhibitor function of CXXC5 in leukemic cells. K562 cells overexpressing pEGFP-C2-CXXC5 showed impaired upregulation of active β-catenin protein, decreased upregulation of Wnt target genes Axin2 and Survivin, as well as decreased Wnt reporter activity after Wnt3a stimulation. In addition, we found evidence for involvement of CXXC5 in the DNA damage-induced p53 response in leukemia. Knockdown of CXXC5in OCI-AML3 cells harboring wildtype p53 led to attenuated p53 upregulation and cell cycle arrest after γ-irradiation.In summary, we have identified CXXC5 as a novel independent prognostic factor in AML. Our data suggest a tumor suppressor function of CXXC5 in AML and association of the gene with different leukemic pathways. These functional and prognostic implications build the framework for further investigating the role of CXXC5 in leukemia. AML patients with downregulation of CXXC5 might be candidates for evaluation of Wnt inhibition and cases with epigenetically silenced CXXC5 expression may particularly benefit from demethylating agents. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Comprehensive analysis of cytoskeleton regulatory genes identifies ezrin as a prognostic marker and molecular target in acute myeloid leukemia.

Despite great advances that have been made in the understanding of the molecular complexity of acute myeloid leukemia (AML), very little has been translated into new therapies. Here, we set out to investigate the impact of cytoskeleton regulatory genes on clinical outcomes and their potential as therapeutic targets in AML. Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) AML study and used for survival and functional genomics analyses. For pharmacological tests, AML cells were exposed to ezrin (EZR) inhibitors and submitted to several cellular and molecular assays. High EZR expression was identified as an independent marker of worse outcomes in AML patients from the TCGA cohort (p < 0.05). Functional genomics analyses suggested that EZR contributes to responses to stimuli and signal transduction pathways in leukemia cells. EZR pharmacological inhibition with NSC305787 and NSC668394 reduced viability, proliferation, autonomous clonal growth, and cell cycle progression in AML cells (p < 0.05). NSC305787 had a greater potency and efficiency than NSC668394 in leukemia models. At the molecular level, EZR inhibitors reduced EZR, S6 ribosomal protein and 4EBP1 phosphorylation, and induced PARP1 cleavage in AML cells. NSC305787, but not NSC668394, favored a gene network involving cell cycle arrest and apoptosis in Kasumi 1 AML cells. From our data we conclude that EZR expression may serve as a prognostic factor in AML. Our preclinical findings indicate that ezrin inhibitors may be employed as a putative novel class of AML targeting drugs.

Mutation Patterns Identify Adult Patients with De Novo Acute Myeloid Leukemia (AML) Aged 60 Years or Older Who Respond Favorably to Standard Chemotherapy: an Analysis of Alliance Studies

Background: Despite changes in treatment regimens, the patient's age (and the corresponding treatment intensity) remains one of the most important prognostic factors in AML. Thus far, only 5-15% of AML patients aged ≥60 years (y) are cured with chemotherapy. Identification of patients who are more (or less) likely to respond to standard cytarabine/daunorubicin-based chemotherapy might help to enable early risk stratification toward alternative treatment regimens.Aims: To provide a comprehensive molecular profile of a large cohort of adults with de novo acute myeloid leukemia (AML) aged ≥60 y, and to determine if specific molecular and/or cytogenetic features could identify patients who are more likely to respond to standard chemotherapy.Methods: We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 patients aged ≥60 y with de novo AML and available cytogenetic data similarly treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology.Results: Overall, we detected 1377 mutations, with a median of 3 mutations per patient (range: 0-8). The outcome of the whole patient cohort was poor, with complete remission (CR) rate of 55% and disease-free (DFS) and overall survival (OS) 3y-rates of 14%. In multivariable analyses (MVA), mutated NPM1 was the only factor positively associated with achieving CR (P <.001). The presence of U2AF1 mutations (P=.03), WT1 mutations (P=.01), or a cytogenetically normal karyotype (CN-AML; P=.03) were associated with lower CR rates. We also found that CR rates of NPM1 -mutated patients varied from 50 to 95% depending on specific co-mutations. Patients who in addition to a NPM1 mutation harbored mutations in cohesin complex (SMC1A, SMC3 or STAG2) or chromatin remodeling (ASXL1, BCOR, BCORL1, EZH2 or SMARCA2) genes had CR rates of 95% and 89%, respectively, and those with simultaneous FLT3 tyrosine kinase domain mutations (FLT3 -TKD) or mutations in the methylation-related (DNMT3A, IDH1 / 2 or TET2) or RAS (KRAS, NRAS, or PTPN11) genes had CR rates of ~83%, compared with only 40% in patients without these coexisting mutations (P <.006). In MVA for DFS, the presence of NPM1 (P <.001) and SF1 mutations (P=.04) associated with longer DFS, with 22% of NPM1 -mutated patients and 40% of SF1 -mutated patients being disease-free 3 y after diagnosis, compared with only 8% of patients without these mutations. The presence of FLT3 internal tandem duplication (FLT3 -ITD; P <.001), and complex karyotype (CK) (P <.001) were associated with poor DFS. In MVA for OS, NPM1 mutations associated with improved OS (P <.001). The presence of FLT3 -ITD (P <.001), BCOR mutations (P=.003), TP53 mutations (P=.003), t(9;11) (P=.03) or CK (P=.002) was associated with shorter OS. Using the variables identified in our multivariable models, refined by the consideration of the presence or absence of co-occurring mutations in NPM1 -mutated AML, we classified the patients into good, intermediate and poor risk for CR attainment, DFS and OS (Figure 1). Whereas 81% (103/127) of good-risk patients (comprising NPM1 -mutated patients harboring mutations in chromatin remodeling or cohesin complex genes, FLT3 -TKD, methylation-related genes, spliceosome genes, RAS pathway, and/or patients without FLT3 -ITD) achieved a CR,only 32% (30/95) of poor-risk patients did (comprising patients with U2AF1 mutations, WT1 mutations and/or CK). Intermediate-risk patients had a 50% (100/201) CR rate. Similarly, using NPM1 co-mutation patterns and SF1 mutation status, we identified patients with favorable DFS and OS 3-year rates of 46% and 45% (representing 8% and 10% of all patients), respectively. In contrast, patients with adverse molecular features [DFS: the presence of FLT3 -ITD, RUNX1, TP53 or U2AF1 mutations and/or typical or atypical CK; OS: the presence of BCOR mutations, FLT3 -ITD, TP53, U2AF1 or WT1 mutations, t(9;11), and/or typical CK] had DFS and OS rates of only 2% and 4%.Summary: We identified combinations of mutations whose presence is associated with favorable outcome in older AML patients treated with standard chemotherapy. Patients with intermediate and adverse molecular features should be considered for alternative treatment regimens. [Display omitted] DisclosuresStone:Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Ono: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Sumitomo: Membership on an entity's Board of Directors or advisory committees. Kolitz:Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Gilead, Magellan, and Novartis: Honoraria; Celgene, Jazz: Equity Ownership; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Novartis, and Seattle Genetics: Other: Travel Support. Powell:Rafael Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Byrd:Janssen: Research Funding; Genentech: Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S.