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1.  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.
doi:10.1371/journal.pone.0070812
PMCID: PMC3743845  PMID: 23967110
2.  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.
doi:10.1021/pr300567r
PMCID: PMC3564549  PMID: 22900933
3.  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.
doi:10.1667/RR2402.1
PMCID: PMC3123689  PMID: 21388270
4.  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
5.  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.
doi:10.1038/leu.2008.176
PMCID: PMC2640437  PMID: 18596738
IRF8; hematopoietic stem cells; microarray; aging

Results 1-5 (5)