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RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer., Significance: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations., (©2023 American Association for Cancer Research.)

Background: Myelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome genes, including U2AF1 , are attractive therapeutic targets as they are prevalent in MDS and sAML, arise early in neoplastic cells, and are generally absent from normal cells, including normal hematopoietic cells. MDS and sAML are susceptible to T cell-mediated killing, and thus engineered T-cell immunotherapies hold promise for their treatment. We hypothesized that targeting spliceosome mutation-derived neoantigens with transgenic T-cell receptor (TCR) T cells would selectively eradicate malignant cells in MDS and sAML., Methods: We identified candidate neoantigen epitopes from recurrent protein-coding mutations in the spliceosome genes SRSF2 and U2AF1 using a multistep in silico process. Candidate epitopes predicted to bind human leukocyte antigen (HLA) class I, be processed and presented from the parent protein, and not to be subject to tolerance then underwent in vitro immunogenicity screening. CD8 + T cells recognizing immunogenic neoantigen epitopes were evaluated in in vitro assays to assess functional avidity, confirm the predicted HLA restriction, the potential for recognition of similar peptides, and the ability to kill neoplastic cells in an antigen-specific manner. Neoantigen-specific TCR were sequenced, cloned into lentiviral vectors, and transduced into third-party T cells after knock-out of endogenous TCR, then tested in vitro for specificity and ability to kill neoplastic myeloid cells presenting the neoantigen. The efficacy of neoantigen-specific T cells was evaluated in vivo in a murine cell line-derived xenograft model., Results: We identified two neoantigens created from a recurrent mutation in U2AF1 , isolated CD8 + T cells specific for the neoantigens, and demonstrated that transferring their TCR to third-party CD8 + T cells is feasible and confers specificity for the U2AF1 neoantigens. Finally, we showed that these neoantigen-specific TCR-T cells do not recognize normal hematopoietic cells but efficiently kill malignant myeloid cells bearing the specific U2AF1 mutation, including primary cells, in vitro and in vivo., Conclusions: These data serve as proof-of-concept for developing precision medicine approaches that use neoantigen-directed T-cell receptor-transduced T cells to treat MDS and sAML., Competing Interests: Competing interests: MB is a Founder and Scientific Advisory Board member of HighPassBio, and a Scientific Advisory Board member of Orca Bio, and has also received compensation from Miltenyi Biotec for presentations at conferences and corporate symposia pertaining to research unrelated to the current manuscript. MB and MAB have filed a provisional patent application number 63/274,681 covering applications of T cell immunotherapy for U2AF1-mutated malignancies., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Somatic mutations in isocitrate dehydrogenase (IDH) genes occur frequently in adult acute myeloid leukemia (AML) and less commonly in pediatric AML. The objective of this study was to describe the prevalence, mutational profile, and prognostic significance of IDH mutations in AML across age. Our cohort included 3141 patients aged between <1 month and 88 years treated on Children's Cancer Group/Children's Oncology Group (n = 1872), Southwest Oncology Group (n = 359), Eastern Cooperative Oncology Group (n = 397) trials, and in Beat AML (n = 333) and The Cancer Genome Atlas (n = 180) genomic characterization cohorts. We retrospectively analyzed patients in 4 age groups (age range, n): pediatric (0-17, 1744), adolescent/young adult (18-39, 444), intermediate-age (40-59, 640), older (≥60, 309). IDH mutations (IDHmut) were identified in 9.2% of the total cohort (n = 288; IDH1 [n = 123, 42.7%]; IDH2 [n = 165, 57.3%]) and were strongly correlated with increased age: 3.4% pediatric vs 21% older, P < .001. Outcomes were similar in IDHmut and IDH-wildtype (IDHWT) AML (event-free survival [EFS]: 35.6% vs 40.0%, P = .368; overall survival [OS]: 50.3% vs 55.4%, P = .196). IDH mutations frequently occurred with NPM1 (47.2%), DNMT3A (29.3%), and FLT3-internal tandem duplication (ITD) (22.4%) mutations. Patients with IDHmut AML with NPM1 mutation (IDHmut/NPM1mut) had significantly improved survival compared with the poor outcomes experienced by patients without (IDHmut/NPM1WT) (EFS: 55.1% vs 17.0%, P < .001; OS: 66.5% vs 35.2%, P < .001). DNTM3A or FLT3-ITD mutations in otherwise favorable IDHmut/NPM1mut AML led to inferior outcomes. Age group analysis demonstrated that IDH mutations did not abrogate the favorable prognostic impact of NPM1mut in patients aged <60 years; older patients had poor outcomes regardless of NPM1 status. These trials were registered at www.clinicaltrials.gov as #NCT00070174, #NCT00372593, #NCT01371981, #NCT00049517, and #NCT00085709., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Purpose: Optimized strategies for risk classification are essential to tailor therapy for patients with biologically distinctive disease. Risk classification in pediatric acute myeloid leukemia (pAML) relies on detection of translocations and gene mutations. Long noncoding RNA (lncRNA) transcripts have been shown to associate with and mediate malignant phenotypes in acute myeloid leukemia (AML) but have not been comprehensively evaluated in pAML., Methods: To identify lncRNA transcripts associated with outcomes, we evaluated the annotated lncRNA landscape by transcript sequencing of 1,298 pediatric and 96 adult AML specimens. Upregulated lncRNAs identified in the pAML training set were used to establish a regularized Cox regression model of event-free survival (EFS), yielding a 37 lncRNA signature (lncScore). Discretized lncScores were correlated with initial and postinduction treatment outcomes using Cox proportional hazards models in validation sets. Predictive model performance was compared with standard stratification methods by concordance analysis., Results: Training set cases with positive lncScores had 5-year EFS and overall survival rates of 26.7% and 42.7%, respectively, compared with 56.9% and 76.3% with negative lncScores (hazard ratio, 2.48 and 3.16; P < .001). Pediatric validation cohorts and an adult AML group yielded comparable results in magnitude and significance. lncScore remained independently prognostic in multivariable models, including key factors used in preinduction and postinduction risk stratification. Subgroup analysis suggested that lncScores provide additional outcome information in heterogeneous subgroups currently classified as indeterminate risk. Concordance analysis showed that lncScore adds to overall classification accuracy with at least comparable predictive performance to current stratification methods that rely on multiple assays., Conclusion: Inclusion of the lncScore enhances predictive power of traditional cytogenetic and mutation-defined stratification in pAML with potential, as a single assay, to replace these complex stratification schemes with comparable predictive accuracy.

Background: Studies have not systematically compared the ability to verify performance of prognostic transcripts in paired bulk mononuclear cells versus viable CD34-expressing leukemic blasts from patients with acute myeloid leukemia. We hypothesized that examining the homogenous leukemic blasts will yield different biological information and may improve prognostic performance of expression biomarkers., Methods: To assess the impact of cellular heterogeneity on expression biomarkers in acute myeloid leukemia, we systematically examined paired mononuclear cells and viable CD34-expressing leukemic blasts from SWOG diagnostic specimens. After enrichment, patients were assigned into discovery and validation cohorts based on availability of extracted RNA. Analyses of RNA sequencing data examined how enrichment impacted differentially expressed genes associated with pre-analytic variables, patient characteristics, and clinical outcomes., Results: Blast enrichment yielded significantly different expression profiles and biological pathways associated with clinical characteristics (e.g., cytogenetics). Although numerous differentially expressed genes were associated with clinical outcomes, most lost their prognostic significance in the mononuclear cells and blasts after adjusting for age and ELN risk, with only 11 genes remaining significant for overall survival in both cell populations (CEP70, COMMD7, DNMT3B, ECE1, LNX2, NEGR1, PIK3C2B, SEMA4D, SMAD2, TAF8, ZNF444). To examine the impact of enrichment on biomarker verification, these 11 candidate biomarkers were examined by quantitative RT/PCR in the validation cohort. After adjusting for ELN risk and age, expression of 4 genes (CEP70, DNMT3B, ECE1, and PIK3CB) remained significantly associated with overall survival in the blasts, while none met statistical significance in mononuclear cells., Conclusions: This study provides insights into biological information gained/lost by examining viable CD34-expressing leukemic blasts versus mononuclear cells from the same patient and shows an improved verification rate for expression biomarkers in blasts., (© 2023. The Author(s).)

SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1 126-134 (T TCR-C4 ). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1 126-134 . An analysis of a second patient treated with T TCR-C4 demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT1 37-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (T TCR37-45 ) killed the first patients' relapsed AML resistant to WT1 126-134 targeting, as well as other primary AML, in vitro. T TCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common β-chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo., (© 2022 by The American Society of Hematology.)

The NCCN Guidelines for Older Adult Oncology address specific issues related to the management of cancer in older adults, including screening and comprehensive geriatric assessment (CGA), assessing the risks and benefits of treatment, preventing or decreasing complications from therapy, and managing patients deemed to be at high risk for treatment-related toxicity. CGA is a multidisciplinary, in-depth evaluation that assesses the objective health of the older adult while evaluating multiple domains, which may affect cancer prognosis and treatment choices. These NCCN Guidelines Insights focus on recent updates to the NCCN Guidelines providing specific practical framework for the use of CGA when evaluating older adults with cancer.

Background: The recently updated European LeukemiaNet risk stratification guidelines combine cytogenetic abnormalities and genetic mutations to provide the means to triage patients with acute myeloid leukemia for optimal therapies. Despite the identification of many prognostic factors, relatively few have made their way into clinical practice., Methods: In order to assess and improve the performance of the European LeukemiaNet guidelines, we developed novel prognostic models using the biomarkers from the guidelines, age, performance status and select transcript biomarkers. The models were developed separately for mononuclear cells and viable leukemic blasts from previously untreated acute myeloid leukemia patients (discovery cohort, N  = 185) who received intensive chemotherapy. Models were validated in an independent set of similarly treated patients (validation cohort, N  = 166)., Results: Models using European LeukemiaNet guidelines were significantly associated with clinical outcomes and, therefore, utilized as a baseline for comparisons. Models incorporating age and expression of select transcripts with biomarkers from European LeukemiaNet guidelines demonstrated higher area under the curve and C-statistics but did not show a substantial improvement in performance in the validation cohort. Subset analyses demonstrated that models using only the European LeukemiaNet guidelines were a better fit for younger patients (age < 55) than for older patients. Models integrating age and European LeukemiaNet guidelines visually showed more separation between risk groups in older patients. Models excluding results for ASXL1 , CEBPA , RUNX1 and TP53 , demonstrated that these mutations provide a limited overall contribution to risk stratification across the entire population, given the low frequency of mutations and confounding risk factors., Conclusions: While European LeukemiaNet guidelines remain a critical tool for triaging patients with acute myeloid leukemia, the findings illustrate the need for additional prognostic factors, including age, to improve risk stratification., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Introduction: Current prognostic models for acute myeloid leukemia (AML) are inconsistent at predicting clinical outcomes for individual patients. Variability in the quality of specimens utilized for biomarker discovery and validation may contribute to this prognostic inconsistency., Methods: We evaluated the impact of sample heterogeneity on prognostic biomarkers and methods to mitigate any adverse effects of this heterogeneity in 240 cryopreserved bone marrow and peripheral blood specimens from AML patients enrolled on SWOG (Southwest Oncology Group) trials., Results: Cryopreserved samples displayed a broad range in viability (37% with viabilities ≤60%) and nonleukemic cell contamination (13% with lymphocyte percentages >20%). Specimen viability was impacted by transport time, AML immunophenotype, and, potentially, patients' age. The viability and cellular heterogeneity in unsorted samples significantly altered biomarker results. Enriching for viable AML blasts improved the RNA quality from specimens with poor viability and refined results for both DNA and RNA biomarkers. For example, FLT3-ITD allelic ratio, which is currently utilized to risk-stratify AML patients, was on average 1.49-fold higher in the viable AML blasts than in the unsorted specimens., Conclusion: To our knowledge, this is the first study to provide evidence that using cryopreserved specimens can introduce uncontrollable variables that may impact biomarker results and enrichment for viable AML blasts may mitigate this impact.

DNA methyltransferase inhibitors sensitize leukemia cells to chemotherapeutics. We therefore conducted a phase 1/2 study of mitoxantrone, etoposide and cytarabine following 'priming' with 5-10 days of decitabine (dec/MEC) in 52 adults (median age 55 (range: 19-72) years) with relapsed/refractory acute myeloid leukemia (AML) or other high-grade myeloid neoplasms. During dose escalation in cohorts of 6-12 patients, all dose levels were well tolerated. As response rates appeared similar with 7 and 10 days of decitabine, a 7-day course was defined as the recommended phase 2 dose (RP2D). Among 46 patients treated at/above the RP2D, 10 (22%) achieved a complete remission (CR), 8 without measurable residual disease; five additional patients achieved CR with incomplete platelet recovery, for an overall response rate of 33%. Seven patients (15%) died within 28 days of treatment initiation. Infection/neutropenic fever, nausea and mucositis were the most common adverse events. While the CR rate compared favorably to a matched historic control population (observed/expected CR ratio=1.77), CR rate and survival were similar to two contemporary salvage regimens used at our institution (G-CLAC (granulocyte colony-stimulating factor (G-CSF); clofarabine; cytarabine) and G-CLAM (G-CSF; cladribine; cytarabine; mitoxantrone)). Thus, while meeting the prespecified efficacy goal, we found no evidence that dec/MEC is substantially better than other cytarabine-based regimens currently used for relapsed/refractory AML.

Cancer is the leading cause of death in older adults aged 60 to 79 years. Older patients with good performance status are able to tolerate commonly used treatment modalities as well as younger patients, particularly when adequate supportive care is provided. For older patients who are able to tolerate curative treatment, options include surgery, radiation therapy (RT), chemotherapy, and targeted therapies. RT can be highly effective and well tolerated in carefully selected patients, and advanced age alone should not preclude the use of RT in older patients with cancer. Judicious application of advanced RT techniques that facilitate normal tissue sparing and reduce RT doses to organs at risk are important for all patients, and may help to assuage concerns about the risks of RT in older adults. These NCCN Guidelines Insights focus on the recent updates to the 2016 NCCN Guidelines for Older Adult Oncology specific to the use of RT in the management of older adults with cancer., (Copyright © 2016 by the National Comprehensive Cancer Network.)

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The t(8;21) and Inv(16) translocations disrupt the normal function of core binding factors alpha (CBFA) and beta (CBFB), respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML) patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq) to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21) (N = 17), Inv(16) (N = 14), and normal karyotype (NK, N = 33) were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21) and 474 genes in Inv(16) were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10(-30)) are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs) differentially expressed across subtypes, with 337 t(8;21)-specific and 407 Inv(16)-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5 x 10(-51) and p = 1.8 x 10(-54) for the two subsets). In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six patients. Clustering of differentially expressed genes indicated that the homeobox (HOX) gene family, including two transcription factors (MEIS1 and NKX2-3) were down-regulated in CBF compared to NK samples. This finding supports existing data that the dysregulation of HOX genes play a central role in biology CBF-AML hematopoiesis. These data provide comprehensive transcriptome profiling of CBF-AML and delineate genes and pathways that are differentially expressed, providing insights into the shared biology as well as differences in the two CBF subsets.

Purpose: Younger patients with acute myeloid leukemia (AML) harboring NPM1 mutations without FLT3-internal tandem duplications (ITDs; NPM1-positive/FLT3-ITD-negative genotype) are classified as better risk; however, it remains uncertain whether this favorable classification can be applied to older patients with AML with this genotype. Therefore, we examined the impact of age on the prognostic significance of NPM1-positive/FLT3-ITD-negative status in older patients with AML., Patients and Methods: Patients with AML age ≥ 55 years treated with intensive chemotherapy as part of Southwest Oncology Group (SWOG) and UK National Cancer Research Institute/Medical Research Council (NCRI/MRC) trials were evaluated. A comprehensive analysis first examined 156 patients treated in SWOG trials. Validation analyses then examined 1,258 patients treated in MRC/NCRI trials. Univariable and multivariable analyses were used to determine the impact of age on the prognostic significance of NPM1 mutations, FLT3-ITDs, and the NPM1-positive/FLT3-ITD-negative genotype., Results: Patients with AML age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype treated in SWOG trials had a significantly improved 2-year overall survival (OS) as compared with those without this genotype (70% v 32%; P < .001). Moreover, patients age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype had a significantly improved 2-year OS as compared with those age > 65 years with this genotype (70% v 27%; P < .001); any potential survival benefit of this genotype in patients age > 65 years was marginal (27% v 16%; P = .33). In multivariable analysis, NPM1-positive/FLT3-ITD-negative genotype remained independently associated with an improved OS in patients age 55 to 65 years (P = .002) but not in those age > 65 years (P = .82). These results were confirmed in validation analyses examining the NCRI/MRC patients., Conclusion: NPM1-positive/FLT3-ITD-negative genotype remains a relatively favorable prognostic factor for patients with AML age 55 to 65 years but not in those age > 65 years., (© 2015 by American Society of Clinical Oncology.)

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

Substantial effort is currently devoted to identifying cancer-associated alterations using genomics. Here, we show that standard blood collection procedures rapidly change the transcriptional and posttranscriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways; up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms; and RNA surveillance inhibition. Affected genes include common mutational targets and thousands of other genes participating in processes such as chromatin modification, RNA splicing, T- and B-cell activation, and NF-κB signaling. The majority of published leukemic transcriptomes exhibit signals of this incubation-induced dysregulation, explaining up to 40% of differences in gene expression and alternative splicing between leukemias and reference normal transcriptomes. The effects of sample processing are particularly evident in pan-cancer analyses. We provide biomarkers that detect prolonged incubation of individual samples and show that keeping blood on ice markedly reduces changes to the transcriptome. In addition to highlighting the potentially confounding effects of technical artifacts in cancer genomics data, our study emphasizes the need to survey the diversity of normal as well as neoplastic cells when characterizing tumors.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Patients with high FLT3 internal tandem duplication allelic ratios (FLT3/ITD-ARs) have a poor prognosis. Single-nucleotide polymorphism/comparative genomic hybridization, single-cell PCR and colony-forming assays were used to evaluate genotypic evolution of high FLT3/ITD-ARs in 85 acute myeloid leukemia (AML) patients. Microarrays were used to examine molecular pathways disrupted in leukemic blasts with high FLT3/ITD-ARs. Copy-neutral loss of heterozygosity (CN-LOH) was identified at the FLT3 locus in diagnostic samples with high FLT3/ITD-ARs (N=11), but not in samples with low FLT3/ITD-ARs (N=24), FLT3-activating loop mutations (N=11) or wild-type FLT3 (N=39). Single-cell assays showed that homozygous FLT3/ITD genotype was present in subsets of leukemic blasts at diagnosis but became the dominant clone at relapse. Less differentiated CD34(+)/CD33(-) progenitor colonies were heterozygous for FLT3/ITD, whereas more differentiated CD34(+)/CD33(+) progenitor colonies were homozygous for FLT3/ITD. Expression profiling revealed that samples harboring high FLT3/ITD-ARs aberrantly expressed genes within the recombination/DNA repair pathway. Thus, the development of CN-LOH at the FLT3 locus, which results in high FLT3/ITD-ARs, likely represents a late genomic event that occurs after the acquisition of the FLT3/ITD. Although the etiology underlying the development of CN-LOH remains to be clarified, the disruption in recombination/DNA repair pathway, which is present before the development of LOH, may have a role.

In the event of a nuclear incident in a heavily populated area, the surge in demand for medical evaluation will likely overwhelm our emergency care system, compromising our ability to care for victims with life-threatening injuries or exposures. Therefore, there exists a need for a rapidly deployable biological assay for radiation exposure that can be performed in the field by individuals with little to no medical training. Saliva is an attractive biofluid for this purpose, due to the relative ease of its collection and the wide array of biomolecules it contains. To determine whether the human salivary proteome is responsive to ionizing radiation exposure, we characterized the abundances of salivary proteins in humans before and after total body irradiation. Using an assay panel targeting 90 analytes (growth factors, chemokines and cytokines), we identified proteins that were significantly radiation responsive in human saliva. The responses of three proteins (monocyte chemo-attractant protein 1, interleukin 8 and intercellular adhesion molecule 1) were confirmed using independent immunoassay platforms and then verified and further characterized in 130 saliva samples from a completely independent set of 38 patients undergoing total body irradiation. The results demonstrate the potential for detecting radiation exposure based on analysis of human saliva.

Cancer is the leading cause of death in older adults aged 60 to 79 years. The biology of certain cancers and responsiveness to therapy changes with the patient's age. Advanced age alone should not preclude the use of effective treatment that could improve quality of life or extend meaningful survival. The challenge of managing older patients with cancer is to assess whether the expected benefits of treatment are superior to the risk in a population with decreased life expectancy and decreased tolerance to stress. These guidelines provide an approach to decision-making in older cancer patients based on comprehensive geriatric assessment and also include disease specific issues related to age in the management of some cancer types in older adults.

Hematopoietic cell transplantation (HCT) provides a life-prolonging or potentially curative treatment option for patients with hematologic malignancies. Given the high transplant-related morbidity, these treatment strategies were initially restricted to younger patients, but are increasingly being used in older adults. The incidence of most hematologic malignancies increases with age; with the aging of the population, the number of potential older candidates for HCT increases. Autologous HCT (auto-HCT) in older patients may confer a slightly increased risk of specific toxicities (such as cardiac toxicities and mucositis) and have modestly lower effectiveness (in the case of lymphoma). However, auto-HCT remains a feasible, safe, and effective therapy for selected older adults with multiple myeloma and lymphoma. Similarly, allogeneic transplant (allo-HCT) is a potential therapeutic option for selected older adults, although fewer data exist on allo-HCT in older patients. Based on currently available data, age alone is not the best predictor of toxicity and outcomes; rather, the comorbidities and functional status of the older patient are likely better predictors of toxicity than chronologic age in both the autologous and allogeneic setting. A comprehensive geriatric assessment (CGA) in older adults being considered for either an auto-HCT or allo-HCT may identify additional problems or geriatric syndromes, which may not be detected during the standard pretransplant evaluation. Further research is needed to establish the utility of CGA in predicting toxicity and to evaluate the quality of survival in older adults undergoing HCT.

In acute myeloid leukemia (AML) and blast crisis (BC) chronic myeloid leukemia (CML) normal differentiation is impaired. Differentiation of immature stem/progenitor cells is critical for normal blood cell function. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Aberrant miRNA expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. We have identified miRNAs differentially expressed in AML and BC CML and identified a new role for miR-150 in myeloid differentiation. Expression of miR-150 is low or absent in BC CML and AML patient samples and cell lines. We have found that expression of miR-150 in AML cell lines, CD34+ progenitor cells from healthy individuals, and primary BC CML and AML patient samples at levels similar to miR-150 expression in normal bone marrow promotes myeloid differentiation of these cells. MYB is a direct target of miR-150, and we have identified that the observed phenotype is partially mediated by MYB. In AML cell lines, differentiation of miR-150 expressing cells occurs independently of retinoic acid receptor α (RARA) signaling. High-throughput gene expression profiling (GEP) studies of the AML cell lines HL60, PL21, and THP-1 suggest that activation of CEPBA, CEBPE, and cytokines associated with myeloid differentiation in miR-150 expressing cells as compared to control cells contributes to myeloid differentiation. These data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role for this miRNA, and that absent or low miR-150 expression contributes to blocked myeloid differentiation in acute leukemia cells.

Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis, such that disruption of IRF8 activity promotes leukemogenesis. We and others have identified aberrant expression of IRF8 transcripts, including novel splice variants, in acute myeloid leukemia (AML), but studies have not investigated the prognostic significance of these transcripts. Therefore, we developed and optimized quantitative expression assays for both, the wild type, or the reference sequence (WT-IRF8) and novel splice variants (SV-IRF8). These assays were used to quantify IRF8 transcript levels in 194 adult patients with AML, and multivariate analyses investigated the prognostic significance of these expression levels. After adjusting for known prognostic factors, expression levels of WT- or SV-IRF8 transcripts were not significantly associated with complete responses or overall survival. However, increased expression of WT-IRF8 was associated with decreased relapse-free survival (RFS) in both univariate (P = 0.010) and multivariate (P = 0.019) analyses. Similarly, increased expression of SV-IRF8 was associated with a decreased RFS (univariate, P = 0.026 and multivariate, P = 0.021). These studies show for the first time that WT-IRF8 and SV-IRF8 are independent adverse prognostic factors for patients with AML. Additional studies are planned to examine the prognostic significance of IRF8 transcripts in other populations of AML patients.

Despite immense interest in the proteome as a source of biomarkers in cancer, mass spectrometry has yet to yield a clinically useful protein biomarker for tumor classification. To explore the potential of a particular class of mass spectrometry-based quantitation approaches, label-free alignment of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) data sets, for the identification of biomarkers for acute leukemias, we asked whether a label-free alignment algorithm could distinguish known classes of leukemias on the basis of their proteomes. This approach to quantitation involves (1) computational alignment of MS1 peptide peaks across large numbers of samples; (2) measurement of the relative abundance of peptides across samples by integrating the area under the curve of the MS1 peaks; and (3) assignment of peptide IDs to those quantified peptide peaks on the basis of the corresponding MS2 spectra. We extracted proteins from blasts derived from four patients with acute myeloid leukemia (AML, acute leukemia of myeloid lineage) and five patients with acute lymphoid leukemia (ALL, acute leukemia of lymphoid lineage). Mobilized CD34+ cells purified from peripheral blood of six healthy donors and mononuclear cells (MNC) from the peripheral blood of two healthy donors were used as healthy controls. Proteins were analyzed by LC-MS/MS and quantified with a label-free alignment-based algorithm developed in our laboratory. Unsupervised hierarchical clustering of blinded samples separated the samples according to their known biological characteristics, with each sample group forming a discrete cluster. The four proteins best able to distinguish CD34+, AML, and ALL were all either known biomarkers or proteins whose biological functions are consistent with their ability to distinguish these classes. We conclude that alignment-based label-free quantitation of LC-MS/MS data sets can, at least in some cases, robustly distinguish known classes of leukemias, thus opening the possibility that large scale studies using such algorithms can lead to the identification of clinically useful biomarkers.

Clonal cytogenetic abnormalities are a major risk factor for relapse after hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS). We determined the impact of the recently established 5-group cytogenetic classification of MDS on outcome after HCT. Results were compared with the impact of the International Prognostic Scoring System (IPSS) 3 cytogenetic risk groups, and the additional effect of a monosomal karyotype was assessed. The study included data on 1007 patients, 1-75 years old (median 45 years), transplanted from related (n = 547) or unrelated (n = 460) donors. Various conditioning regimens were used, and marrow, peripheral blood, or cord blood served as stem cell source. Both IPSS and 5-group cytogenetic risk classifications were significantly associated with post-HCT relapse and mortality, but the 5-group classification discriminated more clearly among the lowest- and highest-risk patients. A monosomal karyotype tended to further increase the rates of relapse and mortality, even after considering the IPSS or 5-group classifications. In addition, the pathologic disease category correlated with both relapse and mortality. Mortality was also impacted by patient age, donor type, conditioning regimen, platelet count, and etiology of MDS. Although mortality declined significantly in recent years, novel strategies are needed to overcome the barrier of high-risk cytogenetics.

IDH1 SNP rs11554137 was recently reported in association with poor prognosis in normal karyotype adult acute myeloid leukemia (AML). We aimed to determine the prevalence, clinical associations, and prognostic significance of SNP rs11554137 in unselected pediatric and adult AML patients. Diagnostic marrow specimens from 527 AML patients treated on the pediatric trial Children's Oncology Group-AAML03P1 (N = 253) or adult SWOG trials (N = 274) were analyzed for the presence of the SNP. SNP rs11554137 was present in 11% of all patients. SNP status had no prognostic impact on survival in pediatric patients. In adult AML, overall survival for SNP-positive patients was 10% versus 18% for SNP-negative patients (P = .44). Among the 142 adults who achieved complete remission, 5-year relapse-free survival was significantly worse for SNP-positive patients (0% vs 25%, P = .0014). However, among adults with normal cytogenetics, FLT3/ITD was present in 90% of SNP-positive patients versus 59% of SNP-negative patients (P = .0053). In multivariate analysis, adjusting for the effects of age, cytogenetics, and FLT3/ITD, the independent prognostic effect of SNP positivity was not statistically significant (hazard ratio = 1.72, P = .18). The clinical profile of SNP-positive patients suggests that SNP rs11554137 may have biologic effects that bear further investigation. The clinical trials in this study are registered at http://www.clinicaltrials.gov as #NCT000707174 and #NCT00899171.

This phase I/II study was conducted to determine the maximum tolerated dose, toxicity, and efficacy of clofarabine in combination with high dose cytarabine and granulocyte colony-stimulating factor (G-CSF) priming (GCLAC), in the treatment of patients with relapsed or refractory acute myeloid leukaemia (AML). Dose escalation of clofarabine occurred without dose-limiting toxicity, so most patients were treated at the maximum dose, 25 mg/m(2) per day with cytarabine 2 g/m(2) per day, each for 5 d, and G-CSF 5 μg/kg, beginning the day before chemotherapy and continuing daily until neutrophil recovery. The complete remission (CR) rate among the 46 evaluable patients was 46% (95% confidence interval [CI] 31-61%) and the CR + CR but with a platelet count <100 × 10(9)/l rate was 61% (95% CI 45-75%). Multivariate analysis showed that responses to GCLAC were independent of age, cytogenetic risk category, and number of prior salvage regimens. GCLAC is highly active in relapsed and refractory AML and warrants prospective comparison to other regimens, as well as study in untreated patients., (© 2011 Blackwell Publishing Ltd.)

The Affymetrix GeneChip Exon Array can be used to detect alternative splice variants. Microarray Detection of Alternative Splicing (MIDAS) and Partek(®) Genomics Suite (Partek(®) GS) are among the most popular analytical methods used to analyze exon array data. While both methods utilize statistical significance for testing, MIDAS and Partek(®) GS could produce somewhat different results due to different underlying assumptions. Comparing MIDAS and Partek(®) GS is quite difficult due to their substantially different mathematical formulations and assumptions regarding alternative splice variants. For meaningful comparison, we have used the previously published generalized probe model (GPM) which encompasses both MIDAS and Partek(®) GS under different assumptions. We analyzed a colon cancer exon array data set using MIDAS, Partek(®) GS and GPM. MIDAS and Partek(®) GS produced quite different sets of genes that are considered to have alternative splice variants. Further, we found that GPM produced results similar to MIDAS as well as to Partek(®) GS under their respective assumptions. Within the GPM, we show how discoveries relating to alternative variants can be quite different due to different assumptions. MIDAS focuses on relative changes in expression values across different exons within genes and tends to be robust but less efficient. Partek(®) GS, however, uses absolute expression values of individual exons within genes and tends to be more efficient but more sensitive to the presence of outliers. From our observations, we conclude that MIDAS and Partek(®) GS produce complementary results, and discoveries from both analyses should be considered.

MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.

The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966). Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1(Ser-957) and phospho-Smc1(Ser-966) in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to (131)I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.

In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15-12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.

Both the presence of HLA-DR15 and tumor necrosis factor (TNF)-α levels have been reported to affect outcome after hematopoietic cell transplantation (HCT). Patients with a myelodysplastic syndrome (MDS) show a high prevalence of HLA-DR15 and express high levels of TNF-α in the bone marrow. The present analysis involving 7950 patients showed an HLA-DR15 frequency of 31% in patients with MDS, compared with only 23% in patients with chronic myelogenous leukemia (CML). HLA-DR15 was more prevalent in Caucasian patients than in non-Caucasian patients (P = .01). The numbers of patients in the non-Caucasian subgroups were too small to allow further analysis. Among Caucasian patients with MDS and CML, the presence of HLA-DR15 did not significantly affect the occurrence of graft-versus-host disease, relapse, nonrelapse mortality (NRM), or survival. However, there was a significant correlation between DR15 and TNF polymorphisms at position -308 among patients with MDS, and the TNF-308 AG genotype conferred an increased risk of NRM compared with the GG genotype (hazard ratio [HR], 1.49; P = .02), even after adjusting for DR15. Conversely, the TNF-863 AA genotype was correlated with decreased overall mortality and NRM compared with the CC genotype (HR, 0.36, P = .04 vs HR, 0.13, P = .04), even after adjusting for DR15. There was no significant association between TNF-308 or -863 polymorphisms and transplantation outcome in CML patients. These results suggest that TNF polymorphisms, but not DR15, affect transplantation outcome in a disease-dependent manner., (Copyright © 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Recent studies of WT1 mutations in acute myeloid leukemia (AML) mostly report an association with unfavorable clinical outcome. We screened 842 patients treated on 3 consecutive pediatric AML trials for WT1 zinc-finger mutations. Eighty-five mutations were detected in 70 of 842 patients (8.3%). Mutations occurred predominantly in exon 7 (n = 74) but were also found in exons 8 (n = 5) and 9 (n = 6). Normal karyotype was observed in 35.3% of WT1(mut) patients, whereas 27.5% WT1(mut) patients harbored favorable risk cytogenetics. Patients with or without mutations had similar rates of complete remission after one course of induction chemotherapy. Overall survival (OS) for patients with WT1 mutations was 41% versus 54% for those without mutations (P = .016). Corresponding event-free survival (EFS) was also significantly worse for those with WT1 mutations (28% vs 42%; P = .01). However, FLT3/ITD was present in 36% of the WT1(mut) cohort; WT1(mut) patients without FLT3/ITD had similar OS (56% vs 56%, respectively; P = .8) and EFS (35% and 44%, respectively; P = .34) to patients who were wild type for both mutations. In current risk stratification schemes incorporating cytogenetics and FLT3/ITD status, the presence of WT1 mutations has no independent prognostic significance in predicting outcome in pediatric AML. The clinical trials are registered at www.clinicaltrials.gov as #NCT00002798 and #NCT00070174.

Recent whole-genome sequencing efforts led to the identification of IDH1(R132) mutations in acute myeloid leukemia (AML) patients. We studied the prevalence and clinical implications of IDH1 genomic alterations in pediatric and adult AML. Diagnostic DNA from 531 AML patients treated on Children's Oncology Group trial COG-AAML03P1 (N=257), and Southwest Oncology Group trials SWOG-9031, SWOG-9333 and SWOG-9500 (N=274), were tested for IDH1 mutations. Codon R132 mutations were absent in the pediatric cohort, but were found in 12 of 274 adult patients (4.4%, 95% CI 2.3-7.5). IDH1(R132) mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1(R132) mutations trended toward higher median diagnostic white blood cell counts (59.2 x 10(9) vs 29.1 x 10(9) per liter, P=0.19) than those without mutations, but the two groups did not differ significantly in age, bone marrow blast percentage, overall survival or relapse-free survival. Eleven patients (2.1%) harbored a novel V71I sequence alteration, which was found to be a germ-line polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.

Acute myeloid leukemia (AML) is the most common form of leukemia in adults, and despite some recent progress in understanding the biology of the disease, AML remains the leading cause of leukemia-related deaths in adults and children. AML is a complex and heterogeneous disease, often involving multiple genetic defects that promote leukemic transformation and drug resistance. The cooperativity model suggests that an initial genetic event leads to maturational arrest in a myeloid progenitor cell, and subsequent genetic events induce proliferation and block apoptosis. Together, these genetic abnormalities lead to clonal expansion and frank leukemia. The purpose of this chapter is to review the biology of receptor tyrosine kinases (RTKs) in AML, exploring how RTKs are being used as novel prognostic factors and potential therapeutic targets.

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

CEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations-N-terminal truncating mutations and in-frame bZip-domain mutations-were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis.

The complexity of the healthy haematopoietic system is immense, and as such, one must understand the biology driving normal haematopoietic expression profiles when designing experiments and interpreting expression data that involve normal cells. This article seeks to present an organised approach to the use and interpretation of gene profiling in normal haematopoiesis and broadly illustrates the challenges of selecting appropriate controls for high-throughput expression studies.

Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow microenvironment confers protection from chemotherapy-induced apoptosis. One mechanism for retention of blasts within the bone marrow is adhesion via very late antigen-4 (VLA-4), the alpha(4)beta(1) integrin heterodimer that binds to its main ligands, fibronectin, and vascular cell adhesion molecule-1 (VCAM-1). To examine the relationship of functional expression of VLA-4 to prognosis in AML, we studied marrow samples from 175 adult AML patients who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group trials. The studies included flow cytometry and functional in vitro assays for ligand binding and maximal beta(1) activation. VLA-4 expression varied widely, with mean expression 60.6% for alpha(4), and was not significantly associated with response to chemotherapy, relapse-free, or overall survival (OS). However, increased binding of soluble VCAM-1 via VLA-4 was significantly associated with longer OS, corrected for age (P = .033). Estimated 5-year OS was 31% (95% confidence interval, 14%-48%) in 30 patients with soluble VCAM-1 binding greater than or equal to 40%, compared with 10% (confidence interval, 3%-17%) in 72 patients with lower binding. Adhesion and migratory properties of AML blasts thus appear to influence chemosensitivity and therefore may be therapeutic targets.

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

FLT3 internal tandem duplication (FLT3/ITD) is a common somatic mutation in acute myeloid leukemia (AML) with significant variation in the position, length, and number of duplications of the FLT3 gene. We evaluated these physical characteristics in FLT3/ITD-positive patients who were treated on CCG-2941/2961 and correlated them with clinical outcome. Fiftynine of 77 FLT3/ITD-positive patients (77%) had a single ITD, 16 (21%) had 2 ITDs, and 2 (3%) had 3 ITDs. The length of the duplicated region varied from 6 to 51 amino acids, and in all cases amino acid residues Y591-Y597 were duplicated. Structural analysis demonstrated that Y591-Y597 encodes the switch and zipper regions of the juxtamembrane domain of FLT3. In addition, 24 of 77 patients (31%) had duplication of the critical STAT5 docking sites Y589/591. Patients with longer ITDs had a worse relapse-free survival (19% vs 51%, P = .035), while the presence of more than 1 ITD was not clinically significant. Physical characteristics including the length of FLT3/ITD may influence FLT3 activation state by altering its structure and may impact response to therapy.