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2.  Hematopoietic Stem Cell Transplantation for Hematologic Malignancies in Older Adults: Geriatric Principles in the Transplant Clinic 
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.
PMCID: PMC4010196  PMID: 24453296
3.  Decreased IRF8 Expression in Aging Hematopoietic Progenitor/Stem Cells 
To determine how aging impacts gene expression in hematopoietic stem cells (HSCs), human CD34+ cells from bone marrow (BMCD34+) and mobilized stem cell products (PBCD34+38-) were examined using microarray-based expression profiling. The age-associated expression changes in CD34+ cells were then compared to age-associated expression changes in murine HSCs. Interferon regulatory factor 8 (IRF8) was the only gene with age-associated expression changes in all analyses, decreasing its expression in human CD34+ cells and murine HSCs. Microarray-based expression profiling found that IRF8 expression also decreased with aging in human T-cells, suggesting that the effects of aging on IRF8 expression may extend to more differentiated populations of hematopoietic cells. Quantitative-RT/PCR studies confirmed that IRF8 mRNA expression decreased with aging in additional samples of BMCD34+, PBCD34+38-, and T-cells, and IRF8 protein expression was found to decrease with aging and to correlate with mRNA levels in PBCD34+ cells. The results suggest that IRF8 may be a novel biomarker of aging for hematopoietic cells. Given that inactivation of IRF8 causes CML-like syndromes in mice and decreased IRF8 expression occurs in human hematopoietic malignancies, it will be critical to determine if decreased IRF8 expression plays a role in the increased incidence of hematopoietic malignancies in older adults.
PMCID: PMC2640437  PMID: 18596738
IRF8; hematopoietic stem cells; microarray; aging
4.  MicroRNA-150 Expression Induces Myeloid Differentiation of Human Acute Leukemia Cells and Normal Hematopoietic Progenitors 
PLoS ONE  2013;8(9):e75815.
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.
PMCID: PMC3782459  PMID: 24086639
5.  The Prognostic Significance of IRF8 Transcripts in Adult Patients with Acute Myeloid Leukemia 
PLoS ONE  2013;8(8):e70812.
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.
PMCID: PMC3743845  PMID: 23967110
6.  Proteomic classification of acute leukemias by alignment-based quantitation of LC-MS/MS data sets 
Journal of proteome research  2012;11(10):5005-5010.
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.
PMCID: PMC3564549  PMID: 22900933
7.  Blood-Based Detection of Radiation Exposure in Humans Based on Novel Phospho-Smc1 ELISA 
Radiation Research  2010;175(3):266-281.
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-Smc1Ser-957 and phospho-Smc1Ser-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-Smc1Ser-957 and phospho-Smc1Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to 131I. 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.
PMCID: PMC3123689  PMID: 21388270
8.  Identification of Radiation-Induced Expression Changes in Nonimmortalized Human T Cells 
Radiation Research  2010;175(2):172-184.
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.
PMCID: PMC3136539  PMID: 21268710
9.  TNF Polymorphism Affects Transplant Outcome in Patients with MDS but not with CML, Independent of the Presence of HLA-DR15 
Both presence of HLA-DR15 and tumor necrosis factor-α (TNF) levels have been reported to affect outcome after hematopoietic cell transplantation (HCT). Patients with myelodysplastic syndromes (MDS) show a high prevalence of HLA-DR15 and express high levels of TNF in the bone marrow. The present analysis involving 7,950 patients showed an HLA-DR15 frequency of 31% in patients with MDS, compared to only 23% in patients with chronic myeloid leukemia (CML). HLA-DR15 was more prevalent in Caucasian than in non-Caucasian patients (p=0.01). Numbers of non-Caucasian subgroups were too small for 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, non-relapse mortality (NRM), or survival. However, there was a significant correlation of DR15 and TNF polymorphisms at position -308 among patients with MDS, and the TNF -308 AG genotype conferred an increased risk of NRM compared to GG (hazard ratio [HR) 1.49, p=.02), even after adjusting for DR15. Conversely, the TNF -863 AA genotype correlated with decreased overall mortality and NRM compared to the CC genotype (HR 0.36, p=.04, and HR 0.13, p=.04, respectively), even after adjusting for DR15. There was no significant association between TNF -308 or -863 polymorphisms and transplant outcomes in CML patients. These results suggest that TNF polymorphisms, rather than DR15 affected transplant outcome in a disease-dependent manner.
PMCID: PMC2975858  PMID: 20541027
MDS; TNF polymorphism; HLA-DR15; hematopoietic cell transplantation
10.  Comparison of Two Methods for Detecting Alternative Splice Variants Using GeneChip® Exon Arrays 
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.
PMCID: PMC3614835  PMID: 23675234
alternative splicing; exon; gene expression analysis
11.  Molecular alterations of the IDH1 gene in AML: a Children’s Oncology Group and Southwest Oncology Group study 
Recent whole-genome sequencing efforts led to the identification of IDH1R132 mutations in 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 12/274 adult patients (4.4%, 95% CI 2.3-7.5%). IDH1R132 mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1R132 mutations trended towards higher median diagnostic WBC counts (59.2 × 109/L vs. 29.1 × 109/L, 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 germline polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.
PMCID: PMC2945692  PMID: 20376086
Acute Myeloid Leukemia; IDH1; isocitrate dehydrogenase; pediatric AML; NPM; FLT3

Results 1-11 (11)