Gene expression changes in normal haematopoietic cells.

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

Tumor Necrosis Factor Polymorphism Affects Transplantation Outcome in Patients with Myelodysplastic Syndrome but Not in Those with Chronic Myelogenous Leukemia, Independent of the Presence of HLA-DR15

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

Gene Expression Patterns in Myelodyplasia Underline the Role of Apoptosis and Differentiation in Disease Initiation and Progression

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. The genetic and epigenetic pathways that determine disease stage and progression are largely unknown. In the current study we used gene expression microarray methodology to examine the gene expression differences between normal hematopoietic cells and hematopoietic cells from patients with MDS at different disease stages, using both unselected and CD34+ selected cells. Significant differences between normal and MDS hematopoietic cells were observed for several genes and pathways. Several genes promoting or opposing apoptosis were dysregulated in MDS cases, most notably MCL1 and EPOR. Progression from RA to RAEB(T) was associated with increased expression of several histone genes. In addition, the RAR-RXR pathway, critical for maintaining a balance between self-renewal and differentiation of hematopoietic stem cells, was found to be deregulated in hematopoietic cells from patients with advanced MDS compared to patients with refractory anemia. These findings provide new insights into the understanding of the pathophysiology and progression of MDS, and may guide to new targets for therapy. Taken together with previous published data, the present results also underscore the considerable complexity of the regulation of gene expression in MDS

Hematopoietic Cell Transplantation as Curative Therapy for Idiopathic Myelofibrosis, Advanced Polycythemia Vera, and Essential Thrombocythemia

AbstractA total of 104 patients, aged 18 to 70 years, with a diagnosis of chronic idiopathic myelofibrosis (CIMF), polycythemia vera (PV), or essential thrombocythemia (ET) with marrow fibrosis were transplanted from allogeneic (56 related and 45 unrelated) or syngeneic (n = 3) donors. Busulfan (BU) or total body irradiation (TBI)-based myeloablative conditioning regimens were used in 95 patients, and a nonmyeloablative regimen of fludarabine plus TBI was used in 9 patients. The source of stem cells was bone marrow in 43 patients and peripheral blood in 61 patients. A total of 63 patients were alive at a follow-up of 1.3–15.2 years (median, 5.3 years), for an estimated 7-year actuarial survival rate of 61%. Eleven patients had recurrent/persistent disease, of whom 8 died. Nonrelapse mortality was 34% at 5 years. Patients conditioned with targeted BU (plasma levels 800–900 ng/mL) plus cyclophosphamide (tBUCY) had a higher probability of survival (68%) than other patients. Dupriez score, platelet count, patient age, and comorbidity score were statistically significantly associated with mortality in univariate models. In a multivariable regression model, use of tBUCY (P = .03), high platelet count at transplantation (P = .01 for PV/ET; P = .39 for other diagnoses), younger patient age (P = .04), and decreased comorbidity score (P = .03) remained statistically significant for improved survival. Our findings show that hematopoietic cell transplantation offers potentially curative treatment for patients with ICMF, PV, or ET

AML risk stratification models utilizing ELN-2017 guidelines and additional prognostic factors: a SWOG report.

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, 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 \u3c 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 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

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).</p

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).</p

Global Proteomic Profiling Identifies Novel Prognostic Factors in Undifferentiated Leukemia Blasts from Patients with NPM1 Mutations: A Previously Unreported Approach to Biomarker Discovery from the Fred Hutch and SWOG

AML blasts harbor multiple genomic, epigenomic, and transcriptomic abnormalities. Proteins are responsible for much of the functional biology derived from these transforming events, yet few studies have examined the global proteome in specimens from AML patients. To gain insight into the biology and prognostic biomarkers for AML, we examined the genome, transcriptome and proteome in highly enriched less differentiated, viable leukemic blasts (VLBs) from previously untreated patients with NPM1 mutated (NPM1+) AML. We focused on NPM1+ AML because 1) NPM1 mutations are the most common genomic abnormality used for risk stratifying AML patients; 2) clinical outcomes for NPM1+ patients vary substantially; and 3) restricting studies to VLBs from patients with NPM1+ AMLreduces the molecular diversity and confounding interactions that may impede identification of biological and clinically relevant biomarkers. Cryopreserved specimens from patients with NPM1+ AML were obtained from the SWOG Leukemia and Fred Hutch/University of Washington Hematopoietic Disease Repositories (N=178). All patients were treated with dose intensive induction and consolidation with curative intent. VLBs were isolated from specimens using fluorescence-activated cell sorting (FACS). Fragment analysis was used to identify FLT3-ITDs, while other genomic mutations were identified using targeted next generation sequencing (NGS). RNA sequencing (RNAseq) was used to quantify transcript expression, while protein expression was quantified using tandem mass tag labelling followed by liquid chromatograph with tandem mass spectrometry (TMT-LC-MS/MS). Prognostic significance of biomarkers was examined by univariate and multivariate analyses, with the later adjusting for both age and ELN2017 risk. Adequate analyte from VLBs were available for RNAseq and TMT-LC-MS/MS studies in 131 and 71 patients, respectively. Proteomic profiling identified 6712 expressed proteins. Multivariable analyses adjusting for age and ELN2017 risk identified 211, 218, and 313 proteins were significantly associated with CR, EFS, and OS, respectively, of which 116 and 174 had a HR ≥ 2 or ≤ 0.5 for EFS and OS (Fig 1A). Analyses showed an overall modest correlation between RNA and protein expression for the 6297 genes present in both RNA and protein datasets (Fig 1B, R =0.27). A total of 163 of the 174 proteomic candidates for OS had RNAseq data, but RNA expression was not significantly correlated with OS for most of these genes (Fig 1C). Similarly, the expression correlation between RNA and protein was not improved by restricting the analyses to these 163 genes (Fig 1D, R =0.28). We also compared protein expression between the NPM1+ VLBs and CD34 progenitors from healthy donors (NLCD34=5), which identified 747 proteins with significant expression differences between the two cell populations (FDR ≤ 0.1). Seventy-three of the 747 proteins displayed \u3e 3-fold increased expression in NPM1+ VLBs, some of which activate targetable pathways (HOMER-\u3eWNT/CTNNB1) and/or amenable to adoptive immunotherapies (e.g., CT45). Additional analyses identified 401 proteins with significantly increased expression in VLBs harboring a high FLT3-ITD allelic ratio (NPM1+/FLT3-ITD-H), a less favorable population of patients. In these less favorable patients, many of the same proteins remained overexpressed (e.g., CT45 with 7.85-fold increased expression, FDR=0.009), but other targetable proteins also emerged. For example, PKCD had a 3.41-fold increased expression (FDR=0.001) in the NPM1+/FLT3-ITD-H VLBs, and targetable inhibitors against this protein (e.g., Parsaclisib) are already in clinical trials for other cancers. This study examines for the first time the global proteome in a large number of VLBs from patients with AML. As with recent publications examining other cancers, the overall expression correlation between RNA and protein was modest, at best, and most of the informative prognostic proteins for OS were not significant in the transcriptome. In addition, the results show the potential to identify novel proteomic targets in subpopulations of AML patients, which may not be otherwise discovered using other means. Thus, the results demonstrate the need to include proteomic profiling in future multi-omics studies seeking to discover novel prognostic biomarkers and therapeutic targets