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Altered Hematopoietic Cell Gene Expression Precedes Development of Therapy-Related Myelodysplasia and Identifies Patients at Risk

ABSTRACT: Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. Although t-MDS/AML development is associated with known genotoxic exposures, its pathogenesis is not well understood and methods to predict risk of development of t-MDS/AML in individual cancer survivors are not available. We performed microarray analysis of gene expression in samples from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and controls that did not develop t-MDS/AML after aHCT. CD34+ progenitor cells from peripheral blood stem cell (PBSC) samples obtained pre-aHCT from t-MDS/AML cases and matched controls, and bone marrow (BM) samples obtained at time of development of t-MDS/AML, were studied. Significant differences in gene expression were seen in PBSC obtained pre-aHCT from patients who subsequently developed t-MDS/AML compared to controls. Genetic alterations in pre-aHCT samples were related to mitochondrial function, protein synthesis, metabolic regulation and hematopoietic regulation. Progression to overt t-MDS/AML was associated with additional alterations in DNA repair and DNA-damage checkpoint genes. Altered gene expression in PBSC samples were validated in an independent group of patients. An optimal 63-gene PBSC classifier derived from the training set accurately distinguished patients who did or did not develop t-MDS/AML in the independent test set. These results indicate that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and can accurately identify those at risk of developing this complication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE23025 | GEO | 2011/12/12

SECONDARY ACCESSION(S): PRJNA131487

REPOSITORIES: GEO

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Project description:Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. Although t-MDS/AML development is associated with known genotoxic exposures, its pathogenesis is not well understood and methods to predict risk of development of t-MDS/AML in individual cancer survivors are not available. We performed microarray analysis of gene expression in samples from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and controls that did not develop t-MDS/AML after aHCT. CD34+ progenitor cells from peripheral blood stem cell (PBSC) samples obtained pre-aHCT from t-MDS/AML cases and matched controls, and bone marrow (BM) samples obtained at time of development of t-MDS/AML, were studied. Significant differences in gene expression were seen in PBSC obtained pre-aHCT from patients who subsequently developed t-MDS/AML compared to controls. Genetic alterations in pre-aHCT samples were related to mitochondrial function, protein synthesis, metabolic regulation and hematopoietic regulation. Progression to overt t-MDS/AML was associated with additional alterations in DNA repair and DNA-damage checkpoint genes. Altered gene expression in PBSC samples were validated in an independent group of patients. An optimal 63-gene PBSC classifier derived from the training set accurately distinguished patients who did or did not develop t-MDS/AML in the independent test set. These results indicate that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and can accurately identify those at risk of developing this complication. PBSC samples obtained pre-aHCT and BM samples at the time of development of t-MDS/AML post-HCT were studied. The training set consisted of 18 patients who developed t-MDS/AML (M-bM-^@M-^]casesM-bM-^@M-^]) after aHCT, matched with 37 controls who underwent aHCT, but did not develop t-MDS/AML. One to three controls were selected per case, matched for primary diagnosis (HL/NHL), age at aHCT (M-BM-110years), and ethnicity (Caucasians, African-Americans, Hispanics, other). The length of follow-up after aHCT for controls was longer than the time to t-MDS/AML in the corresponding case. The results of the training set were validated in an independent group of 36 patients (test set) consisting of 16 cases that developed t-MDS/AML post-aHCT and 20 matched controls. In the test set, 55 PBSC samples from 18 cases and 37 matched controls were studied. BM samples from time of development of t-MDS/AML were available for 12 cases, and from 21 matched controls obtained at a comparable time from aHCT. For validation, 36 PBSC samples from 16 cases and 20 matched controls were studied. All samples had been cryopreserved as mononuclear cells. After thawing, samples were labeled with anti-CD34-APC and anti-CD45-FITC and CD34+CD45dim cells were selected using flow cytometry. Total RNA was extracted using the RNeasy kit. RNA from 1000 cells was amplified and labeled using GeneChipM-BM-. Two-Cycle Target Labeling and Control Reagents from Affymetrix. 15 M-BM-5g of cRNA each was hybridized to Affymetrix HG U133 plus 2.0 Arrays. Microarray data were analyzed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). Data for PBSC and BM samples were normalized separately using robust multiarray averages with consideration of GC content (GCRMA). Probesets with low expression or variability were filtered. Expression of genes represented by multiple probesets was set as the median of the probesets. Using conditional logistic model (CLM) to retain matching between cases and controls, we analyzed the magnitude of association [expressed as odds ratio (OR)] between t-MDS/AML and i) gene expression levels in PBSC at the pre-aHCT time point; ii) gene expression levels in BM at time of t-MDS/AML; and iii) change of expression of individual genes from PBSC to time of t-MDS/AML. False discovery rate (FDR) was applied to adjust for multiple testing. Gene set enrichment analysis (GSEA) was performed on ranked lists of genes differentially expressed between cases and controls. Where multiple significant gene sets were related to each other, analysis was performed to identify a subset of common enriched genes. Average gene expression was calculated for each set and heatmaps plotted to show the contrasts between cases and controls. Gene Ontology (GO) and pathway analysis was performed using DAVID 2008 and Ingenuity IPA 7.5 respectively, retaining genes with z-scores M-bM-^IM-%1.8 or M-bM-^IM-$-1.8, and M-bM-^IM-%1.5-fold change in OR between cases and controls. The association between gene expression in the PBSC product and subsequent development of t-MDS/AML identified in the training set was validated in an independent test set of 36 PBSC sample procured from patients who developed t-MDS/AML after aHCT (16 cases) or did not (20 controls). Pre-processing, normalization and filtering procedures for the test set were identical to the training set. Differential expression between cases and controls was analyzed using CLM. GSEA analysis was performed on the ranked list of differentially expressed genes. Prediction analysis of microarray (PAM) was used to derive a prognostic gene signature from the training set to classify patients as case or control. PAM uses the M-bM-^@M-^\nearest shrunken centroidM-bM-^@M-^] approach and 10-fold cross-validation to select a parsimonious gene expression signature that can classify samples with minimal misclassification. PAM was applied to genes common to both datasets. Based on the misclassification error in cross-validation, a 63-gene signature was selected for prediction using the test data.

Project description:Full Title: Multilineage Dysplasia (MLD) in AML correlates with MDS-related cytogenetic abnormalities and a prior history of MDS or MDS/MPN but has no independent prognostic relevance: A comparison of 408 cases classified as “AML not otherwise specified” or “AML with myelodysplasia-related changes” The WHO classification of acute myeloid leukemia (AML) is hierarchically structured and integrates genetic information, data on patients’ history, and multilineage dysplasia (MLD). The category “AML with MDS-related changes” (AML-MRC) is separated from AML not otherwise specified (AML-NOS) by the presence of either MLD, MDS-related cytogenetics or MDS history. To clarify whether MLD alone is clinically relevant, we analyzed 408 adult patients categorized as AML-MRC or AML-NOS. EFS (Median 13.8 months vs. 16.0 months) and 3-year-OS (45.8% vs. 53.9%) did not differ significantly between patients with MLD and without. However, MLD correlated with pre-existing MDS (p<0.001) and MDS-related cytogenetics (p=0.035). Patients with MLD as sole AML-MRC criterion (AML-MLD-sole; n=90) had less frequently FLT3-ITD (p=0.032), and a lower median age than AML-NOS (n=232), but EFS, OS, and WBC did not differ significantly. Contrarily, patients with AML-NOS plus AML-MLD-sole (n=323) versus patients with MDS history or MDS-related cytogenetics (n=85) had better EFS (16.9 vs. 10.7 months; p=0.005) and 3-year-OS (55.8% vs. 32.5%; p=0.001). Gene expression profiles were measured for a subset of 96 patients. Analysis of the expression data showed distinct clusters for AML-MLD-sole and AML-NOS versus AML with MDS-related cytogenetics or MDS history. Thus, MLD demonstrated no independent clinical impact, while cytogenetics and MDS history were of prognostic relevance. This data suggest modifications in a revised WHO proposal. All 96 bone marrow samples were obtained from untreated patients at the time of diagnosis. Cells used for microarray analysis were collected from the purified fraction of mononuclear cells after Ficoll density centrifugation.

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Altered Hematopoietic Cell Gene Expression Precedes Development of Therapy-Related Myelodysplasia and Identifies Patients at Risk

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