This study assessed the incidence of cytomegalovirus (CMV) infection after transplantation of cord blood (CB) from unrelated donors and evaluated strategies for screening CB donors. Posttransplantation CMV infection, reported in 23% of 1221 CB recipients, was associated with patient pretransplantation CMV serology (P < .001), but not with CMV serology in CB donors or their mothers. A total of 26 988 infant CB donors were evaluated by viral culture of saliva. Subgroups were evaluated by polymerase chain reaction in CB (CB-PCR) in 2 case-control studies. In the first study, 33 of 47 saliva culture-positive CB donors were confirmed by CB-PCR. All mothers of the 33 infants with confirmed CMV infection were CMV-total antibody positive, but only 1 of 3 had CMV-IgM antibody. The second study evaluated infants born to mothers with CMV-IgM antibody. Of these, 5 of 170 saliva culture-negative infants were positive by CB-PCR. The incidence of congenital CMV infection in CB donors was low (0.12%). Maternal serology had poor predictive value for CMV infection in their infant CB donors and bore no detected relationship to CMV infection in CB recipients. Saliva culture for CMV had both false-positive and -negative results. CB-PCR was a useful alternative for detecting CMV in CB donors.

The HLA class I/II alleles and the tumor necrosis factor alpha (TNFA) locus are closely linked in the MHC complex. We have characterized the causative factor VIII mutation, HLA alleles as well as 4 polymorphisms (-827C>T, -308G>A, -238A>G, and 670A>G) in the TNFA gene in 164 patients (124 severe, 26 moderate, and 14 mild) in 78 families with hemophilia A enrolled in the Malmö International Brother Study (MIBS). Inhibitors were identified in 77.8% of patients with a single haplotype (Hap 2) and 72.7% of the patients with the TNFA -308 A/A genotype within this haplotype compared with 39.7% for TNFA -308 G/G patients and 46.9% for TNFA -308 G/A heterozygotes (OR 4.0; 95% CI, 1.4-11.5; P = .008). The association between the -308 A/A genotype and inhibitors was enhanced in subgroups of patients with severe hemophilia (OR 19.2; 95% CI 2.4-156.5; P < .001) and with inversions (n = 75; OR, 11.8; 95% CI, 1.3-105.1; P = .013). Associations were found for the HLA A26 and B44 alleles, but these were not consistent in the subgroup analysis. Our data imply that the TNFA -308G>A polymorphism within Hap 2 is a useful marker and potential modulator of the immune response to replacement therapy in patients with hemophilia.

Recently, protocols using high-dose melphalan chemotherapy and autologous peripheral blood stem cell transplantation (HDM/SCT) have been developed for the treatment of patients with immunoglobulin light chain (AL) amyloidosis. Although peritransplantation mortality is greater than for other hematologic diseases, treatment leads to durable hematologic complete responses, improvements in organ function and quality of life, and extended survival in a substantial proportion of patients. To determine whether this treatment can be applied to older patients, we have analyzed HDM/SCT treatment outcomes for 65 patients (aged 65 years or older) with AL amyloidosis compared with outcomes for 280 younger patients. For patients over age 65 years who meet the same eligibility criteria as younger patients, toxicity, hematologic remission rate, and survival were not significantly different from those observed in younger patients, indicating that older patients should not be excluded a priori from consideration for HDM/SCT treatment.

The t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation.

Previous studies demonstrated that circulating dendritic cells (DCs) in myeloma patients were functionally abnormal. However, the phenotype and function of patients' monocyte-derived DCs (MoDCs), which are commonly used for immunotherapy, were poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterparts, patient-derived, mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR and were poor at activating alloreactive T cells, presenting recall antigen, and activating autologous antigen- and myeloma-specific T cells. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and, more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield but also produced MoDCs that were as functional as their normal counterparts. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy in this disease.

There is conflicting information about the influence of body mass index (BMI) on the pharmacokinetics, toxicity, and outcome of chemotherapy. We compared pharmacokinetics, outcome, and toxicity data across 4 BMI groups (underweight, BMI < or = 10th percentile; normal; at risk of overweight, BMI > or = 85th and < 95th percentile; overweight, BMI > or = 95th percentile) in 621 children with acute lymphoblastic leukemia (ALL) treated on 4 consecutive St Jude Total Therapy studies. Chemotherapy doses were not adjusted to ideal BMI. Estimates of overall survival (86.1% +/- 3.4%, 86.0% +/- 1.7%, 85.9% +/- 4.3%, and 78.2% +/- 5.5%, respectively; P = .533), event-free survival (76.2% +/- 4.2%, 78.7% +/- 2.1%, 73.4% +/- 5.5%, and 72.7% +/- 5.9%, respectively; P = .722), and cumulative incidence of relapse (16.0% +/- 3.7%, 14.4% +/- 1.8%, 20.6% +/- 5.1%, and 16.7% +/- 5.1%, respectively; P = .862) did not differ across the 4 groups. In addition, the intracellular levels of thioguanine nucleotides and methotrexate polyglutamates did not differ between the 4 BMI groups (P = .73 and P = .74, respectively). The 4 groups also did not differ in the overall incidence of grade 3 or 4 toxicity during the induction or postinduction periods. Further, the systemic clearance of methotrexate, teniposide, etoposide, and cytarabine did not differ with BMI (P > .3). We conclude that BMI does not affect the outcome or toxicity of chemotherapy in this patient population with ALL.

Thymus-derived CD4+ CD25+ regulatory T cells suppress autoreactive CD4+ and CD8+ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4+ CD25+ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7- and L-selectin (CD62L)-coexpressing cells within expanded CD4+ CD25high T cells maintain phenotypic and functional characteristics of regulatory T cells. Further analysis revealed that these cells originate from CD45RA+ naive cells within the CD4+ CD25high T-cell compartment, as only this subpopulation homogeneously expressed CD62L, CCR7, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 (FOXP3), produced no inflammatory cytokines and maintained robust suppressive activity after expansion. In contrast, cell lines derived from CD45RA- memory-type CD4+ CD25high T cells lost expression of lymph node homing receptors CCR7 and CD62L, contained interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) as well as IL-10-secreting cells, showed only moderate suppression and, most importantly, did not maintain FOXP3 expression. Based on these unexpected findings, we suggest that isolation and expansion of CD45RA+ naive CD4+ CD25high T cells is the best strategy for adoptive regulatory T (Treg)-cell therapies.

We investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.

An activating JAK2 mutation (JAK2 V617F) is present in the chronic myeloproliferative disorders (MPDs), polycythemia vera (PV), idiopathic myelofibrosis (IMF), and essential thrombocytosis (ET). JAK2 is also a chaperone for Mpl and responsible for its cell-surface expression. We observed a reciprocal relationship between neutrophil JAK2 V617F allele percentage and platelet Mpl expression in JAK2 V617F-positive PV, IMF, and ET patients. However, severely impaired platelet Mpl expression was present in JAK2 V617F-negative MPD patients. While JAK2 V617F allele status did not necessarily correlate with the clinical MPD phenotype, the degree of impaired platelet Mpl expression did. We conclude that multiple molecular abnormalities are involved in the pathogenesis of the MPDs and that aberrant Mpl expression may be a common denominator of aberrant signaling in both the JAK2 V617F-positive and JAK2 V617F-negative MPDs.

Activating mutations of the FLT3 gene occur because of an internal tandem duplication of the juxta-membrane domain (FLT3/ITD) or point mutation of the activation loop domain (FLT3/ALM). The presence of FLT3 mutations as well as the allelic ratio of FLT3/ITD (ITD-AR, mutant-wild type ratio) may have prognostic significance. FLT3 mutation status of 630 children with de novo acute myeloid leukemia (AML) treated on CCG-2941 and -2961 was determined, and ITD-AR was calculated for patients with FLT3/ITD. Clinical characteristics and outcomes for patients with FLT3/ALM and FLT3/ITD at varying ITD-ARs was determined and compared with those without FLT3 mutations (FLT3/WT). FLT3/ITD and FLT3/ALM were detected in 77 (12%) and 42 (6.7%) of the patients. Progression-free survival (PFS) was similar in patients with FLT3/ALM and FLT3/WT (51% versus 55%, P = .862). In contrast, PFS at 4 years from study entry for patients with FLT3/ITD was inferior to that of patients with FLT3/WT (31% versus 55%, P < .001). PFS decreased with increasing FLT3/ITD-AR (P < .001), and those with ITD-AR greater than 0.4 had a significantly worse PFS than those with lower ITD-AR (16% versus 72%, P = .001) or with FLT3/WT (55%, P < .001). ITD-AR defines the prognostic significance in FLT3/ITD-positive AML, and ITD-AR greater than 0.4 is a significant and independent prognostic factor for relapse in pediatric AML.

The dynamics of human T-lymphotropic virus type-1 (HTLV-1) provirus expression in vivo are unknown. There is much evidence to suggest that HTLV-1 gene expression is restricted: this restricted gene expression may contribute to HTLV-1 persistence by limiting the ability of the HTLV-1-specific CD8(+) cell immune response to clear infected cells. In this study, we tested the hypothesis that derepression of HTLV-1 gene expression would allow an increase in CD8(+) cell-mediated lysis of HTLV-1-infected cells. Using histone deacetylase enzyme inhibitors (HDIs) to hyperacetylate histones and increase HTLV-1 gene expression, we found that HDIs doubled Tax expression in naturally infected lymphocytes after overnight culture. However, the rate of CD8(+) cell-mediated lysis of Tax-expressing cells ex vivo was halved. HDIs appeared to inhibit the CD8(+) cell-mediated lytic process itself, indicating a role for the microtubule-associated HDAC6 enzyme. These observations indicate that HDIs may reduce the efficiency of cytotoxic T-cell (CTL) surveillance of HTLV-1 in vivo. The impact of HDIs on HTLV-1 proviral load in vivo cannot be accurately predicted because of the widespread effects of these drugs on cellular processes; we therefore recommend caution in the use of HDIs in nonmalignant cases of HTLV-1 infection.

The translocation t(12;22) involves MN1 and TEL and is rarely found in acute myeloid leukemia (AML). Recently, it has been shown in a mouse model that the fusion protein MN1-TEL can promote growth of primitive hematopoietic progenitor cells (HPCs) and, in cooperation with HOXA9, induce AML. We quantified MN1 expression by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 142 adult patients with AML with normal cytogenetics treated uniformly in trial AML-SHG 01/99. AML samples were dichotomized at the median MN1 expression. High MN1 expression was significantly correlated with unmutated NPM1 (P < .001), poor response to the first course of induction treatment (P = .02), a higher relapse rate (P = .03), and shorter relapse-free (P = .002) and overall survivals (P = .03). In multivariate analysis, MN1 expression was an independent prognostic marker (P = .02) in addition to age and Eastern Cooperative Oncology Group (ECOG) performance status. Excluding patients with NPM1(mutated)/FLT3ITD(negative), high MN1 expression was associated with shorter relapse-free survival (P = .057). MN1 was highly expressed in some patients with acute lymphoblastic but not chronic lymphocytic or myeloid leukemia. MN1 was highly expressed in HPCs compared with differentiated cells and was down-regulated during in vitro differentiation of CD34(+) cells, suggesting a functional role in HPCs. In conclusion, our data suggest MN1 overexpression as a new prognostic marker in AML with normal cytogenetics.

Circulating endothelial progenitor cells (EPCs) are thought to contribute to angiogenesis following vascular injury, stimulating interest in their ability to mediate therapeutic angiogenesis. However, the number of EPCs in the blood is low, limiting endogenous repair, and a method to rapidly mobilize EPCs has not been reported. In this study, healthy donors were mobilized sequentially with the CXCR4 antagonist, AMD3100, and G-CSF. The number of EPCs and circulating angiogenic cells (CACs) in the blood and pheresis product was determined and the angiogenic capacity of each cell population assessed. Compared with baseline, treatment with AMD3100 or G-CSF increased the number of blood CACs 10.0-fold +/- 4.4-fold and 8.8-fold +/- 3.7-fold, respectively. The number of EPCs in the blood increased 10.2-fold +/- 3.3-fold and 21.8-fold +/- 5.4-fold, respectively. On a percell basis, CACs harvested from G-CSF-mobilized blood displayed increased in vivo angiogenic potential compared with AMD3100-mobilized CACs. Mobilized EPCs displayed a greater proliferative capacity than EPCs isolated from baseline blood. Both CACs and EPCs were efficiently harvested by leukapheresis. Cryopreserved CACs but not EPCs retained functional activity after thawing. These data show that AMD3100 is a potent and rapid mobilizer of angiogenic cells and demonstrate the feasibility of obtaining and storing large numbers of angiogenic cells by leukapheresis.

Tandutinib (MLN518/CT53518) is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases: FMS-like tyrosine kinase 3 (FLT3), platelet-derived growth factor receptor (PDGFR), and KIT. Because of the correlation between FLT3 internal tandem duplication (ITD) mutations and poor prognosis in acute myelogenous leukemia (AML), we conducted a phase 1 trial of tandutinib in 40 patients with either AML or high-risk myelodysplastic syndrome (MDS). Tandutinib was given orally in doses ranging from 50 mg to 700 mg twice daily The principal dose-limiting toxicity (DLT) of tandutinib was reversible generalized muscular weakness, fatigue, or both, occurring at doses of 525 mg and 700 mg twice daily. Tandutinib's pharmacokinetics were characterized by slow elimination, with achievement of steady-state plasma concentrations requiring greater than 1 week of dosing. Western blotting showed that tandutinib inhibited phosphorylation of FLT3 in circulating leukemic blasts. Eight patients had FLT3-ITD mutations; 5 of these were evaluable for assessment of tandutinib's antileukemic effect. Two of the 5 patients, treated at 525 mg and 700 mg twice daily, showed evidence of antileukemic activity, with decreases in both peripheral and bone marrow blasts. Tandutinib at the MTD (525 mg twice daily) should be evaluated more extensively in patients with AML with FLT3-ITD mutations to better define its antileukemic activity.

Peripheral T-cell lymphomas (PTCLs) are rare and have a dismal prognosis. The most frequent subtype is PTCL, unspecified. Epstein-Barr virus (EBV) has been detected in around 40% of cases, but its prognostic significance is not fully established. Lymph node samples from 110 patients with PTCL, unspecified included in LNH87 and LNH93 trials were available. EBV status was studied by EBV-encoded small RNA in situ hybridization (EBER-ISH). EBER-ISH showed positive cells in 45 (41%) of 110 patients. Pretreatment characteristics were comparable between positive and negative cases, except for male sex (80% versus 60%, respectively, P = .02). Only 50% of patients achieved complete remission with a 5-year event-free survival (EFS) and overall survival (OS) of 21% and 30%, respectively. EBER-ISH positivity was the sole factor linked with worse EFS, with a 5-year probability of 11% for positive patients. In univariate analysis, factors affecting OS were EBER-ISH positivity, high LDH level, and age older than 60 years. In multivariate analysis, EBER-ISH was associated with a worse OS in the elderly population. Time-dependent analysis showed that the negative impact of EBV was essentially seen in the first 2 years following diagnosis. These results warrant further studies regarding pathogenesis and specific treatment approaches for EBV-associated PTCL patients.

Regulatory T (Treg) cells accumulate in the lymphoid tissues of human immunodeficiency virus (HIV)-infected individuals, contributing to the inability of the immune system to control virus replication. We investigate here Treg-cell numbers and functional markers (FOXP3, CTLA-4, IDO, and TGF-beta1) in lymphoid tissues from untreated infected hosts with progressive or nonprogressive disease (HIV-infected humans and simian immunodeficiency virus [SIV]-infected macaques). We found that increased numbers of FOXP3(+) T cells as well as increased expression of Treg-cell-associated functional markers were detected only during progressive disease. Such increases were not correlated with immune activation. Of importance, a high-perforin/FOXP3 ratio was associated with nonprogressive disease, suggesting that the immune control of virus replication represents a balance between cell-mediated immune responses and Treg-cell-mediated counter regulation of such responses. Furthermore, using an in vitro model of Treg-cell-HIV interactions, we showed that exposure of Treg cells to HIV selectively promoted their survival via a CD4-gp120-dependent pathway, thus providing an underlying mechanism for the accumulation of Treg cells in infected hosts with active viral replication. Considered together, our findings imply that therapeutic manipulation of Treg-cell number and/or function could improve immune control of HIV infection.

Studies of radiation-induced acute myeloid leukemia (AML) in mice suggest that the number of target stem cells is a risk factor, and the HLX1 homeobox gene, which is important for hematopoietic development, is a candidate gene. The distribution of the C/T-3' untranslated region (UTR) polymorphism in HLX1 in patients with AML and therapy-related AML (t-AML) compared with controls was therefore determined. The presence of the variant HLX1 allele significantly increases the risk of t-AML (OR = 3.36, 95% CI, 1.65-6.84). The DNA repair gene RAD51 (135G/C-5' UTR) polymorphism also increases t-AML risk, and when combined analysis was performed on both RAD51 and HLX1 variant alleles, a synergistic 9.5-fold increase (95% CI, 2.22-40.64) in the risk of t-AML was observed. We suggest that the HLX1 polymorphism has an effect on stem cell numbers, whereas an increased DNA repair capacity (RAD51) will suppress apoptosis, a genetic interaction that may increase the number of genomes at risk during cancer therapy.

Heteroclitic peptide modifications increase immunogenicity, allowing generation of cytotoxic T lymphocytes (CTLs) against weakly immunogenic tumor-associated antigens (TAAs). A critical issue is whether T cells generated against heteroclitic peptides retain the ability to recognize and kill tumor cells expressing the original weak TAAs, and whether there is a lower threshold of binding affinity of the native peptides, below which such CTLs can still kill primary tumor cells. To examine this we used a model examining the ability of native and heteroclitic immunoglobulin (Ig)-derived peptides to generate CTLs that can kill chronic lymphocytic leukemia (CLL) cells. We demonstrate that CTLs generated against heteroclitic peptides have enhanced killing of CD40-activated B cells pulsed with either heteroclitic (P < .001) or native peptide (P = .04) and primary CLL cells (P = .01). The novel finding reported here is that the rate-limiting factor appears to be the ability to generate CTLs and that once generated, CTL lysis of primary tumor cells is independent of the binding affinity of the native peptide. These findings have implications for vaccination strategies in malignancies and are currently being further examined in vivo in murine models.

Our previous results demonstrated that B cells from a patient (pt1) with non-X-linked hyper-IgM syndrome (HIGM) possess an atypical CD23(lo) phenotype that is unaffected by CD40-mediated activation. To investigate the molecular mechanism underlying defective CD23 expression in pt1 B cells, we used lymphoblastoid cell lines that express LMP1 under the control of a tetracycline-inducible promoter (LCL(tet)). Our analysis revealed that the CD23(lo) phenotype in the pt1-LCL(tet) cells is a direct consequence of diminished CD23 transcription. We demonstrate a marked decrease in c-Rel-containing complexes that bind to the proximal CD23a/b promoters in pt1-LCL(tet) extracts, resulting from an overall lower expression of c-Rel in pt1-LCL(tet) cells. Analysis of c-Rel mRNA revealed relatively equal amounts in pt1-LCL(tet) and control LCL(tet) cells, indicating that diminished c-Rel protein expression is unrelated to decreased transcription. Finally, a critical role for c-Rel in CD23 regulation was demonstrated by effectively altering c-Rel expression that resulted in the direct modulation of CD23 surface expression. Collectively, these findings demonstrate that low levels of c-Rel are the underlying cause of aberrant CD23 expression in pt1 B cells and are likely to play a critical role in the pathophysiology of this form of HIGM.

Protein tyrosine phosphatase (PTPgamma) is a receptor-like molecule with a known role in murine hematopoiesis. We analyzed the regulation of PTPgamma expression in the human hematopoietic system, where it was detected in human peripheral blood monocytes and dendritic cells (DCs) of myeloid and plasmacytoid phenotypes. Its expression was maintained during in vitro monocyte differentiation to dendritic cells (moDC) and was further increased after maturation with bacterial lipopolysaccharide (LPS), CD40L, and TNFalpha. But PTPgamma was absent when monocytes from the same donor were induced to differentiate in macrophages. B and T lymphocytes did not express PTPgamma. Rather, PTPgamma mRNA was expressed in primary and secondary lymphoid tissues, and the highest expression was in the spleen. PTPgamma was detected by immunohistochemistry in subsets of myeloid-derived DCs and specialized macrophages (tingible bodies, sinus and alveolar macrophages). Classic macrophages in infective or reactive granulomatous reactions did not express PTPgamma. Increased PTPgamma expression was associated with a decreased ability to induce proliferation and interferon-gamma secretion in T cells by moDCs from patients with advanced pancreatic cancer. Taken together, these results indicate that PTPgamma is a finely regulated protein in DC and macrophage subsets in vitro and in vivo.