Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

DNMT3A mutations are frequent in younger patients with AML and are associated with an unfavorable prognosis.

Mutations in genes of the splicing machinery have been described recently in myelodysplastic syndromes (MDS). In the present study, we examined a cohort of 193 MDS patients for mutations in SRSF2, U2AF1 (synonym U2AF35), ZRSR2, and, as described previously, SF3B1, in the context of other molecular markers, including mutations in ASXL1, RUNX1, NRAS, TP53, IDH1, IDH2, NPM1, and DNMT3A. Mutations in SRSF2, U2AF1, ZRSR2, and SF3B1 were found in 24 (12.4%), 14 (7.3%), 6 (3.1%), and 28 (14.5%) patients, respectively, corresponding to a total of 67 of 193 MDS patients (34.7%). SRSF2 mutations were associated with RUNX1 (P < .001) and IDH1 (P ‫؍‬ .013) mutations, whereas U2AF1 mutations were associated with ASXL1 (P ‫؍‬ .005) and DNMT3A (P ‫؍‬ .004) mutations. In univariate analysis, mutated SRSF2 predicted shorter overall survival and more frequent acute myeloid leukemia progression compared with wildtype SRSF2, whereas mutated U2AF1, ZRSR2, and SF3B1 had no impact on patient outcome. In multivariate analysis, SRSF2 remained an independent poor risk marker for overall survival (hazard ratio ‫؍‬ 2.3; 95% confidence interval, 1.28-4.13; P ‫؍‬ .017) and acute myeloid leukemia progression (hazard ratio ‫؍‬ 2.83; 95% confidence interval, 1.31-6.12; P ‫؍‬ .008). These results show a negative prognostic impact of SRSF2 mutations in MDS. SRSF2 mutations may become useful for clinical risk stratification and treatment decisions in the future.

A cohort of MDS patients was examined for mutations affecting 4 splice genes (SF3B1,SRSF2

CD4 CD25 FOXP3 regulatory T cells (Tregs) are involved in graft-specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft-specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2-CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2-CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2-CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2-CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg-supported weaning after allogeneic transplantation.

Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-␣ promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 ؋ 10 13 particles into skeletal muscles of several mouse strains (C57BL͞6, BALB͞c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 g͞ml in SCID and over 400 g͞ml in C57BL͞6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions. Alpha-1-antitrypsin (AAT) deficiency is the second most common monogenic lung disease, accounting for 3% of all early deaths due to obstructive pulmonary disease. AAT is produced in the liver, secreted into the serum, and circulated to the lung where it protects elastin fibers and other connective tissue components of the alveolar wall from degradation by neutrophil elastase. Current therapy for AAT deficiency includes avoidance of cigarette smoke exposure and weekly i.v. infusions of recombinant human AAT (hAAT) protein (1). Attempts at gene augmentation have been limited by the short duration of expression and by the high circulating levels of AAT, which are required for therapeutic effect (800 g͞ml) (2).Several groups have demonstrated that adeno-associated virus (AAV) vectors are capable of stable in vivo expression (3-5) and are less immunogenic than other viral vectors (6). AAV is a nonpathogenic human parvovirus whose life cycle includes a mechanism for long-term latency. In the case of wild-type AAV (wtAAV), this persistence is due to sitespecific integration into a site on human chromosome 19 (AAVS1) (7), whereas with recombinant AAV (rAAV) vectors, persistence occurs by both episomal persistence and integration into non-chromosome 19 locations (8-9). rAAV latency also differs from that of wtAAV in that wtAAV is rapidly converted to double-stranded DNA in the absence of helper virus (e.g., Ad) infection, whereas rAAV-leading st...

WT1 SNP rs16754 may be a novel independent favorable-risk marker in CN-AML patients that might improve risk and treatment stratification.

IntroductionThe deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells including myeloid leukemias. 1,2 RAS proto-oncogenes (H-RAS, N-RAS, and K-RAS) encode 21-kd G-proteins that play key roles in signal transduction, proliferation, differentiation, and malignant transformation. 3-5 RAS proteins are produced as cytoplasmatic precursors, which require several posttranslational modifications (eg, prenylation, proteolysis, carboxymethylation, and palmitoylation) for membrane binding and full biologic activity. 3-8 RAS functions as a biologic switch that relays signals from ligand-stimulated tyrosine kinase, cytokine, and heterotrimeric G-protein-coupled receptors to cytoplasmatic mitgen-activated protein kinase (MAPK) cascades. In its activated, GTP-bound state, RAS binds to and activates effector molecules such as Rafs, MEKK, PI-3K, and Ral-GEF. [3][4][5][8][9][10][11][12][13][14] Raf kinases (A-Raf, B-Raf, c-Raf-1) selectively phosphorylate and activate MAPK kinases (MAPKK) MEK-1/2 in the MAPK/ERK pathway. [13][14][15][16][17] MEK-1/2 are dual specificity kinases that activate MAPKs (ERK-1/2). 18,19 The best-characterized ERK substrates are cytoplasmic phospholipase A 2 (cPLA 2 ), ribosomal protein S6 kinases (RSKs), and transcription factors Elk-1 and CREB-1. [18][19][20] The importance of deregulation of ERK signaling in the molecular pathogenesis of myeloid leukemias is underscored by the positioning of several oncogene and tumor suppressor gene products on this pathway. 5,[21][22][23] RAS mutations are frequent genetic aberrations found in 20% to 30% of all human tumors, although the incidences in tumor type vary greatly. 1,2 The most commonly observed RAS mutations arise at sites critical for RAS regulation, namely codons 12, 13, and 61. 1,2,5,9 Additionally, mutations occur at codons 15, 16, 18, and 31. 24,25 These mutations result in abrogation of normal intrinsic or GAP-stimulated GTPase activity of RAS, leading to increased half-lives of mutant RAS-GTP. 5,9,26 Transformation results, at least in part, from deregulated stimulation of mitogenic signal transduction pathways. 1,2,5 The highest incidences of RAS mutations were detected in carcinomas of pancreas (90%), thyroid (50%), colon (50%), and lung (30%). RAS mutations are also frequently observed in myelodysplastic syndromes, acute myeloid leukemias (AML), juvenile myelomonocytic myeloid leukemia (JMML), and multiple myelomas (20%-40%). 1,2,5,[21][22][23] N-RAS is mutated in the majority of cases and presence of the mutation is not associated with any particular FAB type, cytogenetic abnormality, or clinical feature including prognosis. 22 In addition to activation by mutation, RAS is also deregulated in myeloid leukemias by constitutive activation of proto-oncogenes such as receptor or nonreceptor tyrosine kinases (RTKs and NRTKs) or inactivation of tumor suppressor genes. 5,[21][22][23] RTKs are constitutively activated by single point mutations (eg, colonystimulating factor-1 [CSF-1] receptor and c...