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In HIV-infected individuals, anti-retroviral therapy (ART) is effective in maintaining low viral loads, restoring CD4+ T cell counts, and prolonging life. However, chronic inflammation and immune dysfunction persist even after years of successful ART. Previous studies have suggested that early initiation of ART may result in improved immune reconstitution, but whether more rapid suppression of viral load during early treatment also improves immunological outcomes is unknown. In this study we compare the effects of two regimens initiated during acute infection on T cell phenotype and function. Regimen 1 (n=30) is a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen and Regimen 2 (n=30) combines an integrase strand transfer inhibitor (INSTI) with nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs and NNRTIs inhibit reverse transcription of viral RNA to DNA in the infected cell, while INSTIs block integration of viral DNA into the host genome. Since Regimen 2 targets multiple stages of the viral life cycle, we hypothesize that more rapid viral suppression may be achieved with this treatment schedule. In a cross-sectional study performed 96 weeks after treatment initiation, we are comparing the effects of the two regimens on T cell activation (CD25, CD69, CD38, HLA-DR), exhaustion (PD-1, Tim-3, CD160, T-bet, Eomes), and proliferation using standardized flow cytometry panels. This study will provide valuable insights into the relationship between viral decay kinetics and immune preservation during acute infection, informing the design of future ART regimens.

The aim of this study was to investigate changes in CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) throughout the clinical course of Kawasaki disease (KD) and correlations with response to intravenous immunoglobulin (IVIg) therapy. Participants comprised 18 patients who fulfilled the diagnostic criteria for KD and 20 healthy subjects. Expressions of CD25 and FOXP3 among all CD4(+) T cells in peripheral blood mononuclear cells were analyzed by flow cytometry before and 7 and 30 days after IVIg therapy. Before treatment, percentages of CD4(+)CD25(+)FOXP3(+) Tregs among total CD4(+) Tregs were significantly lower among KD patients (4.19 %; range, 0.16-8.11 %) than among healthy subjects (7.32 %; 4.18-13.42 %; P = 0.0001). Both percentages and absolute numbers of CD4(+)CD25(+)FOXP3(+) Tregs on day 7 after IVIg therapy were significantly increased compared with values before treatment (8.02 % (range, 0.51-12.6 %) vs. 4.19 % (range, 0.16-8.11 %), P = 0.0005; 93.25/ μL (range, 6.67-258.05) vs. 41.85/ μL (range, 0.44-160.62), P 0.0001, respectively). Moreover, percentages and absolute numbers of CD4(+)CD25(+)FOXP3(+) Tregs before treatment were significantly lower in the IVIg-resistant group than in the IVIg-sensitive group (0.18 % (range, 0.16-3.34 %) vs. 4.52 % (range, 2.8-8.11 %), P = 0.0022; 0.68/μL (range, 0.44-53.81) vs. 51.66/μL (range, 2.88-160.62), P = 0.0098, respectively). The frequency of CD4(+)CD25(+)FOXP3(+) Tregs in four of the five IVIg-resistant patients at diagnosis was more than 3 standard deviations below that in healthy subjects. Two of these four patients displayed coronary abnormalities, and one of these two patients developed coronary aneurysm.Lack of CD4(+)CD25(+)FOXP3(+) Tregs before treatment may predict resistance to IVIg therapy in patients with KD.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially "boosting" the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

Juvenile myelomonocytic leukemia (JMML) progenitor cells exhibit in vitro hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Phospho-specific flow cytometry using anti-phosphorylated STAT5 antibody is a new method recently reported to detect GM-CSF hypersensitivity of cells. However, colony assays using methylcellulose medium to measure GM-CSF-hypersensitivity remain as the current gold standard. Interestingly, cytomegalovirus (CMV) infection in infancy often presents with a variety of clinical symptoms that mimic JMML, with CMV giving a positive result by colony assay. We wanted to determine whether aberrant STAT5 activation occurs in CMV infection by using phospho-specific flow cytometry, and to ascertain whether this method is effective at discriminating CMV infection from JMML. Peripheral blood mononuclear cells from patients with JMML and CMV infection displayed an elevated proportion of p-STAT5 cells after low-dose GM-CSF stimulation when compared with cells from normal individuals. However, we found no significant differences in the percentage of p-STAT5 positive cells from patients with CMV infection and JMML at any doses of the GM-CSF doses used. We conclude that patients with CMV infection cannot be discriminated from patients with JMML by this new diagnostic method.

Summary Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML-related mutations.

Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines.We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a.Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method.Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.

A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.

We describe a 4-month-old infant girl with congenital erythroid and myeloid hypoplasia who developed myelodysplastic syndrome. Bone marrow examination showed severe erythroid and myeloid hypoplasia without dysplastic morphology. Flow cytometry detected autoantibodies to myeloid cells, indicating a diagnosis of Diamond-Blackfan anemia with autoimmune neutropenia. The patient was administered prednisolone and rituximab, which brought the neutrophil count and hemoglobin level to within the normal range. However, bicytopenia recurred at the age of 22 months. She was diagnosed with myelodysplastic syndrome because of trilineage dysplasia and the clonal abnormality of 46,XX,dup(1)(q21;q32) in bone marrow. She was transplanted with cord blood from an unrelated human leukocyte antigen-matched donor and has since remained in complete remission for 20 months.

Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs), resulting in peripheral pancytopenia. Several pathogeneses for AA have been hypothesized, and among these, the autoimmune-associated hypothesis is considered to be the most likely [1, 2]. Mesenchymal stem cells (MSCs) have been shown to exert immunosuppressive capacity both in vitro and in vivo. Normal bone marrow (BM)-derived MSCs suppress T cell function and may provide an immunoprotected environment for HSCs [3, 4]. Bacigalupo et al. [5] reported that MSCs derived from the BM of adult AA patients are defective in their ability to suppress T cell proliferation and cytokine release. They hypothesized that these defects may result in increased activation/proliferation of BM T cells, which suppress HSCs in AA patients. The aim of the present study was to investigate whether MSCs from pediatric AA patients also exert decreased suppressive activity on T cells when compared with those derived from normal individuals. Bone marrow samples from 34 AA children (16 males, 18 females) and 15 healthy volunteers were obtained following informed consent from either patients or parents. Median patient age was 10 years (range 2–21 years). The timing of sampling was at diagnosis in 15 patients and after immunosuppressive therapy (IST) with anti-thymocyte globulin and cycrosporin A in 19 patients. Eleven of these were IST responders and eight were non-responders. Aplasia was secondary to acute hepatitis in four patients and the cause was unknown in 30 patients. Seven patients had very severe AA, 19 patients had severe AA and eight patients had moderate AA. Bone marrow mononuclear cells (MNCs) were isolated and cultured as reported previously [5]. Briefly, MNCs were plated in a 25-cm tissue culture flask with complete MesenCult Basal Medium (Stem cell technologies, Vancouver, Canada) containing human MSC supplements. Adherent cells were expanded to the third generation. Characterization of MSCs was determined by flow cytometry and their differentiation into adipogenic and osteoblastic lineages. Surface markers of the MSCs were CD29?, CD44?, CD90?, CD105?, CD73?, CD34-, CD45and CD14-. To demonstrate capacity of MSCs to differentiate to adipocytes, cells were further cultured at 2 9 10 cells/flask with MesenCult basal medium supplemented with human adipocytogenic supplements (Stem cell technologies). Adipogenic differentiation was confirmed by the presence of lipid droplets under optical microscopy after 3 weeks. Similarly, for differentiation to osteoblasts, MSCs were cultured with MesenCult basal medium supplemented with an osteogenic differentiation kit (Stem cell technologies) containing dexamethasone (0.1 lmol/l), ascorbic acid (0.05 mmol/l) and glycerophosphate (10 mmol/l). After 5 weeks of culture, cell layers were stained with silver nitrate (Von Kossa method) in order to detect calcium deposition, which indicates osteogenesis. There were no differences in the expression of MSC surface markers and the differentiation capacity between AA patients and normal individuals. Immunosuppressive activity of MSCs from pediatric patients with AA was tested by their inhibitory effects on PHA-induced T cell proliferation. Peripheral blood MNCs from normal adult volunteers (2 9 10) were dispensed Y. Xu Y. Takahashi A. Yoshimi M. Tanaka H. Yagasaki S. Kojima (&) Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: kojimas@med.nagoya-u.ac.jp

Juvenile myelomonocytic leukemia (JMML), an overlap of myelodysplastic / myeloproliferative neoplasm, is an intractable pediatric myeloid neoplasm. Epigenetic regulation of transcription, particularly by CpG methylation, plays an important role in tumor progression, mainly by repressing tumor-suppressor genes. To clarify the clinical importance of aberrant DNA methylation, we studied the hypermethylation status of 16 target genes in the genomes of 92 patients with JMML by bisulfite conversion and the pryosequencing technique. Among 16 candidate genes, BMP4, CALCA, CDKN2A, and RARB exhibited significant hypermethylation in 72% (67/92) of patients. Based on the number of hypermethylated genes, patients were stratified into three cohorts based on an aberrant methylation score (AMS) of 0, 1–2, or 3–4. In the AMS 0 cohort, the 5-year overall survival (OS) and transplantation-free survival (TFS) were good (69% and 76%, respectively). In the AMS 1–2 cohort, the 5-year OS was comparable to that in the AMS 0 cohort (68%), whereas TFS was poor (6%). In the AMS 3–4 cohort, 5-year OS and TFS were markedly low (8% and 0%, respectively). Epigenetic analysis provides helpful information for clinicians to select treatment strategies for patients with JMML. For patients with AMS 3–4 in whom hematopoietic stem cell transplantation does not improve the prognosis, alternative therapies, including DNA methyltransferase inhibitors and new molecular-targeting agents, should be established as treatment options.

We investigated the relationship between urocortin and the activity of angiotensin-converting enzyme (ACE), which plays a key role in producing the potent vasoconstrictor angiotensin II (Ang II). Urocortin was acutely and subchronically administered to Sprague-Dawley (SD) rats and then the serum and tissue (lung and aorta) ACE levels were evaluated. The tissue ACE mRNA was determined by using reverse transcription and polymerase chain reaction (RT-PCR) analysis. Immunofluorescence studies were also preformed to evaluate the effect of urocortin on ACE in cultured rat aortic endothelial cells (RAECs). Urocortin decreased the serum ACE level 1h after administration, whereas tissue ACE immunoreactivity and mRNA did not change. The prolonged administration of urocortin enhanced tissue ACE activity but the serum ACE level remained low. RT-PCR analysis showed that tissue ACE mRNA was elevated. Immunofluorescence studies also demonstrated an increase of ACE intensity in RAECs exposed to urocortin for 72 h. Corticotropin-releasing factor (CRF) receptor blocker, astressin, abolished the effects of urocortin. Extracellular signal-regulated kinase 1/2 (ERK1/2) pathway blocker, PD98059, also markedly inhibited these effects, suggesting urocortin affects the activity of ACE through the ERK1/2 pathway in rats. These findings support the changes in mean arterial pressure (MAP) following acute and subchronic injections of urocortin in previous studies. Thus, the changes of the ACE activity and its production of Ang II may play a role in the vasodilatory property of urocortin.

Introduction: Acquired aplastic anemia (AA) is an immune mediated disease characterized by severe quantitative defects in stem cell number resulting in a hypocellular marrow and peripheral blood cytopenias. Immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine A (CyA) is the standard therapy for patients with AA who lack a human leukocyte antigen (HLA)-matched sibling donor. Previous studies identified minor paroxysmal nocturnal hemoglobinuria (PNH) clones and short telomere length (TL) as predictive markers of the response to IST. However, the likelihood ratio of these markers was insufficient to influence clinical decisions. Here we assessed whether the combination of these markers could more accurately predict the response to IST. Methods: Between July 2001 and November 2013, 116 patients (65 boys and 51 girls) diagnosed with AA were examined to identify a minor population of PNH-type cells and TL of peripheral blood lymphocytes. Fifty-three patients received horse ATG (Lymphoglobulin, 15mg/kg/day for 5 days) and 63 received rabbit ATG (Thymoglobulin, 2.5−3.75 mg/kg/day for 5 days). CyA (6mg/kg/day) was started on day 1 and continued to at least day 180. The dose of CyA was adjusted to maintain trough levels between 100 and 200 ng/ml. Written informed consent was obtained from all patients’ parents. The ethics committee of each participating hospital approved the study. We used a flow cytometric assay to detect PNH-type granulocytes and red blood cells (RBCs). Presence of >0.007% CD13+ CD55− CD59− granulocytes and/or >0.009% glycophorin A+ CD55− CD59−RBCs was defined as PNH positive (PNH+), based on the results from healthy individuals. The average relative TL of peripheral lymphocytes was measured by flow-fluorescence in situ hybridization (flow-FISH) using a Telomere PNA Kit (Dako Cytomation). Based on our previous study, we defined −1.0 SD of age-adjusted controls as the cut-off value. Results: The median age at IST was 9.8 years (0.9−16.0). Disease severity of patients was assessed as moderate in 39, severe in 37, and very severe in 40, respectively. The causes of AA were idiopathic in 104 patients and hepatitis-associated in 12 patients. The median follow-up period from the time of IST was 36 months (range 1−134). Thirty-nine patients (34%) carried a minor PNH clone at diagnosis. Compared with healthy individuals, their median of TL was −1.08 SD (−4.01 to +3.01 SD). Overall, 60 of 116 patients (51.7%) responded to IST at 6 months after administration of ATG. In univariate analysis, the response rate at 6 months was higher in PNH+ patients than in PNH- patients (74.4% vs. 40.3%, respectively, p = 0.001). Longer TLs (≥−1.0 SD of age-adjusted control) were also associated with a favorable response to IST at 6 months (73.2% vs. 31.7%, p Conclusion: Combination of the absence of a minor PNH clone and a short TL was an efficient predictor of poor response to IST. These patients had low chances to respond to IST, which challenges the current standard of treatment. Prospective clinical trials are required to confirm these findings. Disclosures No relevant conflicts of interest to declare.

Introduction: The appropriate classification of bone marrow (BM) failure syndromes in children is challenging, particularly in relation to histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study is to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 children with three diseases using whole-exome sequencing. Patients and Methods: Wediagnosed AA, RCC, or RCMD on the basis of morphology and histological findings of bone marrow (BM) according to the 2008 World Health Organization (WHO) classification criteria. Patients with AA exhibited hypocellular BM and no morphologically dysplastic changes in any of three hematopoietic cell lineages, while patients with RCC had 10% in one cell lineage. Patients with RCMD exhibited >10% dysplastic changes in two or more cell lineages. We obtained peripheral blood and BM samples from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). We performed exome capture from paired DNA (non-T cells/CD3+ lymphocytes) using SureSelect® Human All Exon V4 kit (Agilent Technologies, Santa Clara, CA), which covered all part of the coding exons, followed by massively-parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations and germline variants were detected through our pipeline for whole-exome sequencing (Genomon-exome). All candidate somatic nucleotide changes were validated by Sanger sequencing. The ethics committee of Nagoya University Graduate School of Medicine approved this study. Results: Whole-exome sequencing pipeline identified a total of 14 non-synonymous somatic (one nonsense, 11 missense, and two frameshift) changes among the 29 patients, which resulted in only 0.48 mutations per patient. The average numbers of somatic mutations per sample were not significantly different among these groups (0.50 in AA, 0.36 in RCC, and 0.60 in RCMD). As a whole, childhood AA, RCC, and RCMD were characterized by a paucity of somatic mutations compared with adult myelodysplastic syndromes (MDS) in which 10 or more mutations per exome were detected on average. Among the mutated genes, BCOR-inactivating mutations in two patients (p.S158fs in AA and p.E1286X in RCMD) were considered significant genetic events based on previous reports that it is a driver gene in MDS. With regard to germline events, we did not detect any germline mutations of inherited BM failure syndromes. Moreover, we did not identifiy significantly frequent germline events in the entire cohort or any genetic hallmarks to be able to discriminate between these three diseases. When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0%) vs. 6% (0%–26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100%–100%) vs. 95% (70%–99%)] were not significantly different. Conclusion: We usedwhole-exome sequencing analysis for gene mutational profiling of children with AA, RCC, and RCMD. Idiopathic bone marrow failure syndromes in children are characterized by a paucity of somatic gene mutations, irrespective of histological diagnosis. These findings suggest that histological diagnosis based on the WHO classification system does not discriminate the mutational profile of idiopathic BM failure syndromes in children. Disclosures No relevant conflicts of interest to declare.

Introduction Recent advances in cancer genetics have led to the identification of somatic mutations in SET-binding protein 1 (SETBP1) in myeloid malignancies categorized as myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). Heterozygous point mutations in SETBP1 are essentially found at a genomic level in myeloid malignancies, and the frequency of the mutated allele in cDNA suggests somatic heterozygosity without substantial imbalance in allelic expression. Thus, mutant SETBP1 presumably has a dominant altered biological activity. Most mutations in SETBP1 are located in the SKI homologous region. This region is suggested to include regions critical for ubiquitin binding and SETBP1 degradation. SETBP1 binds to SET, which protects against protease cleavage, and thus may result in PP2A inhibition and cell proliferation. Overexpression of SETBP1 resulting from a p.G870S alteration showed higher levels of the protein compared with wild-type (WT), indicating a prolonged halftime of SETBP1, which led to reduced PP2A activity and a higher cell proliferation rate. To date, however, our molecular biological understanding of SETBP1 mutations has been obtained only through observations of exogenous overexpression in cell lines. This may result in bias, considering the predicted dominant-negative function of SETBP1 mutations. Therefore, we used an RNA-guided endonuclease (RGEN), the CRISPR/Cas9 system, to generate a cell line harboring point mutations resulting in only relevant single amino acid substitutions in SETBP1. We analyzed cell signaling using the cell line thus established. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and gRNAs of interest. The gRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate gRNAs were gRNA#1, 5′-TAGGGAGCCAATCTCGCAC-3′; gRNA#2, 5′-TGTCCCAATGCCGCTGTCGC-3′; gRNA#4, 5′-GTCCCAATGCCGCTGTCGCT-3′; and gRNA#7, 5′-GAGACGATCCCCAGCGACAG-3′. pCAG-EGxxFP harboring the 500 bp target region of WT SETBP1 was constructed for gRNA selection. For homology-dependent repair (HDR), we synthesized 70 mer single-stranded oligonucleotides (ssODN) having both the SETBP1 c.2602G>A, p.D868N mutation and synonymous mutation in the PAM. HEK293T cells were cultured in DMEM with 10% FBS. For cell signaling analysis, the cells were serum-depleted for 16 h prior to western blotting. Anti-SETBP1 antibody (ab98222), anti-phospho-Y307 PP2A antibody (E155), and anti phospho-p44/42 MAPK antibody (CST#4370) were used for cell signaling analysis. Results To validate an efficient sgRNA for DNA scission, we cotransfected pCAG-EGxxFP-SETBP1 and pSpCas9(BB)-SETBP1-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR, was observed 48 h later, and we determined that gRNA#2 was the most efficient. Next we cotransfected 293T cells with pCAG-EGxxFP-SETBP1, pSpCas9(BB)-SETBP1-gRNA#2, and ssODN for mutagenesis. Five days after transfection, single EGFP-positive clones were isolated using the FACSAria cell sorting system. Sanger sequencing confirmed that 293T cells harboring the SETBP1 p.D868N homozygous mutation were established. A clone with WT SETBP1 was also maintained as a control. To elucidate the effects of the SETBP1 mutation in 293T cells, we performed cell signaling analysis by western blotting. 293T-SETBP1 p.D868N cells showed higher levels of SETBP1 protein with lower molecular weight compared with WT, indicating a prolonged halftime, possibly due to loss of ubiquitination. In addition, 293T-SETBP1 p.D868N cells showed a higher phosphorylation level of PP2A (Y307, C subunit), a marker of PP2A inactivation. Finally, the phosphorylation level of p44/42 MAPK (ERK1/2) was increased in 293T-SETBP1 p.D868N cells. Conclusions We confirmed that the SETBP1 p.D868N mutation caused a prolonged halftime, resulting in PP2A inactivation and p44/42 MAPK activation in 293T cell lines. Our data suggest a potential therapy target for malignancies harboring SETBP1 mutations. More generally, this work illustrates the utility of RGEN technology for studying hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests. Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies. Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes. Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted. Disclosures No relevant conflicts of interest to declare.

Recent studies suggest that aberrant methylation plays a fundamental role in the development of a variety of cancers, including myeloid malignancies. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is characterized by both excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor. It is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) according to the World Health Organization classification. We recently reported that somatic mutations in SETBP1 and JAK3 were identified in JMML patients and were associated with poor outcomes (Nat Genet 2013;45:937–41). The goal of this study was to clarify the clinical significance of aberrant DNA methylation in JMML. We studied 92 children (61 boys and 31 girls) who were diagnosed with JMML in institutions throughout Japan. A diagnosis of JMML was made based on internationally accepted criteria. We quantitatively evaluated the CpG methylation pattern in the promoter regions of 16 candidate genes (APC, BMP4, CALCA, CDH13, CDKN2A, CDKN2B, CHFR, DAPK, DMR-H19, ER, IGF2, MGMT, MLH1, RARB, RASSF1, TP73) from genomic DNA derived from bone marrow specimens at the time of diagnosis. This was accomplished by bisulfite conversion and the pryosequencing technique. We defined aberrant methylation as >3 standard deviations from the mean methylation level derived from 8 healthy individuals. The median age at diagnosis was 16 months (range, 0.3–160). By genetic analysis, PTPN11, NF1, NRAS, KRAS, and CBL mutations were found in 39 (42%), 7 (8%), 12 (13%), 13 (14%), and 11 (12%) patients, respectively. In addition, 16 patients had SETBP1 or JAK3 mutations. Karyotypic abnormalities were detected in 15 patients, including 8 with monosomy 7. The median monocyte count, percentage of hemoglobin F, and platelet count at the time of diagnosis were 4.6x109/L (range, 0.2–31.6), 21% (range, 0–68), and 61.0x109/L (range, 1.4–483), respectively. The median observation period was 18 months (range, 1–287). During observation, 56 of the 92 patients received allogeneic hematopoietic stem cell transplantation (HSCT), and 30 of 92 patients died. Outcomes were assessed according to transplantation-free survival (TFS), in which HSCT and death were censored, and overall survival (OS) by the Kaplan-Meier method. Aberrant methylation of BMP4, CALCA, CDKN2A, CDKN2B, DMR-H19 and RARB were detected, of which hypermethylation of BMP4, CALCA, CDKN2A, and RARB were associated with poor TFS according to univariate analyses (P The present study shows that aberrant methylation of four genes (BMP4, CALCA, CDKN2A, and RARB) is associated with poor outcomes in JMML patients. Patients with SETBP1/JAK3 mutations frequently show the hypermethylation of these genes. Further, allogeneic HSCT is associated with improved outcomes for patients with AMS = 1 and 2. Therefore, these results suggest that examination of the methylation pattern of these four genes may help guide clinical decisions for the management of patients with JMML. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

Abstract 170 Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods: We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation. Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers' protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results: For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases. Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion: Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

Abstract 352 Background: Autologous stem cell transplantation (ASCT) for MM leads to complete responses in ∼20–40% of patients but rare cures. Patients with rapid recovery of T cells post ASCT may have improved outcomes suggesting possible immune mediated tumor control. We have shown that adoptive T-cell transfers after ASCT for MM using vaccine-primed and ex-vivo costimulated autologous T-cells in combination with pre- and post-transplant immunizations using a tumor antigen vaccine (+ GM-CSF and montanide) led to vaccine-directed T-cell responses in about 1/3 of patients and enhanced recovery of polyclonal T and B cell counts and function. We hypothesized that addition of Poly-ICLC (Hiltonol®) – a TLR-3 agonistic vaccine adjuvant could increase tumor antigen vaccine responses through better priming and boosting. Methods: We report interim results of a Phase II clinical trial (NCT01245673) evaluating safety and activity of autologous T cells primed in-vivo with a MAGE-A3 multipeptide vaccine (Orphan Drug Designation: GL-0817) mixed with GM-CSF and Poly-ICLC (Hiltonol®) +/− montanide. Inclusion criteria included measurable disease or high-risk cytogenetics. MAGE-A3 is expressed in ∼50% MM cases and more frequently in relapsed/extramedullary/proliferative disease. The MAGE-A3 vaccine has 2 HLA-A2-restricted class I peptides and 1 promiscuous class II peptide linked to an HIV-1-TAT membrane translocation sequence (Trojan peptide) to enhance peptide presentation. Vaccine-primed T cells were collected by leukapheresis, costimulated and expanded ex-vivo using anti-CD3/anti-CD28 mAb conjugated beads. T-cells were infused at day +2 after ASCT followed by booster immunizations at days 14, 42, 90, 120 and 150 post-transplant. Lenalidomide maintenance was started at day +100. Patients were evaluated for MM responses in accordance with IMWG criteria at days 60, 100, 180 post-transplant and Q3 months thereafter. T-cell and B-cell responses to the vaccine were evaluated by IFN-g or IL-2 cytokine production (all patients), dextramer binding to CD8 cells (HLA-A2 positive patients) and ELISA antibody assays at days 14, 60, 100 and 180 post-transplant. Results: 25 patients were transplanted on study. At a median followup of 6 months (range 2–18 months), 24 patients are surviving while 1 patient relapsed at about day +60 and died. Four additional patients have relapsed at 7,9,18 and 18 months post-transplant, yielding a 1-year Kaplan-Meier EFS of 77%. Of the 16 patients evaluable for response at day 100, 7/16 (44%) had CR/nCR using the study enrollment (post-induction) myeloma markers as a baseline while at day 180, 7/13 (53%) had CR/nCR. T-cell infusions were well-tolerated with no probable/definite grade 3 or higher toxicities. Vaccinations were associated with > 50 mm injection site reactions (redness, induration or both) after 1 or more immunizations in the majority of patients. Two patients developed large and prolonged inflammatory reactions which evolved into sterile abscesses. These resolved over 2–3 months with conservative management but as a result montanide was eliminated from the vaccine formulation for patients 11–25. Thereafter vaccine reactogenicity was decreased with no additional sterile abscesses. Of 16 patients tested for immune responses to date, 2 patients were unevaluable due to poor sample viability. Dextramer staining demonstrated MAGE-A3-specific CD8 T-cells in 4/4 (100%) of evaluable HLA-A2+ patients. Cytokine production in response to MAGE-A3 stimulation was seen in 11/14 (79%) patients; responses usually peaked at day 100 or day 180. Robust MAGE-A3 antibody responses were detected in 7/9 patients who received montanide in the vaccine formulation but in 0/7 patients who did not. Conclusions: Combination immunotherapy using a MAGE-A3 multipeptide tumor antigen vaccine plus vaccine-primed and costimulated autologous T-cells after ASCT for MM is well-tolerated and associated with encouraging early clinical responses. The addition of Poly-ICLC (Hiltonol®) to the vaccine formulation was associated with a high frequency of post-ASCT T-cell functional responses. The combination of Poly-ICLC +GM-CSF + Montanide led to robust injection site reactions that were occasionally severe and prolonged. Elimination of the montanide reduced injection site reactogenicity but may also have compromised the B-cell responses to the tumor antigen vaccine. Disclosures: Rapoport: Gliknik, Inc: Research Funding. Stadtmauer:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Vogl:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Research Funding; Otsuka: Consultancy; Acetylon: Research Funding. Strome:Gliknik, Inc: Equity Ownership, Patents & Royalties, Research Funding. Salazar:Oncovir, Inc: Employment. Levine:TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.

Abstract 266 Introduction: Lenalidomide is an immunomodulatory drug with effects on the innate immune system that may enhance antibody-dependent cell mediated cytotoxicity as well as the development of specific anti-tumor immune responses. These immunologic effects may synergize with the action of rituximab. To test the efficacy of lenalidomide combined with rituximab, we are conducting a single center, open label phase II clinical trial in patients (pts) with indolent B-cell or mantle cell lymphomas previously resistant to rituximab. Patients and Methods: Eligible pts must have relapsed/refractory indolent B-cell or mantle cell lymphoma with measurable disease that has failed to respond or has progressed within six months of a standard course of rituximab monotherapy (375 mg/m2 weekly for at least four weeks) or a prior rituximab-containing chemotherapy regimen. Thus, all pts enrolled are considered rituximab refractory. Patients were enrolled sequentially into two treatment cohorts. In Cohort 1, pts received two 28-day treatment cycles of lenalidomide 10 mg every day and dexamethasone 8 mg once weekly (Part I: lenalidomide + dexamethasone). After assessment of response to Part I, all pts received a single course of rituximab 375 mg/m2, consisting of four weekly doses during cycle 3 (Part II: lenalidomide + dexamethasone + rituximab). Treatment with lenalidomide + dexamethasone continued during and subsequent to rituximab; stable and responding pts continued on lenalidomide + dexamethasone until disease progression or development of clinically unacceptable toxicity. Response assessment after Part II was performed three months after the first dose of rituximab. In Cohort 2, dexamethasone was eliminated; otherwise, eligibility and treatment were the same. Results: As of August 9, 2011, 45 pts have started therapy (Cohort 1, n = 27; Cohort 2, n = 18); diagnoses include: follicular (n = 28), mantle cell (n = 11), small lymphocytic (n = 4), and marginal zone (n = 2) lymphomas; median age is 58 years (range: 35–85); male: female ratio is 31:14; median number of prior therapies is 3 (range: 1 – 7); LDH is increased in 18%. For Cohort 1, 24 pts completed Part II and are evaluable for response; 3 pts are not evaluable, 1 due to death (myocarditis) and 2 due to removal from study (1 thrombocytopenia attributed to myelodysplasia; 1 rash attributed to lenalidomide). For Cohort 2, 11 pts are evaluable for response assessment after Part II; 2 pts are not evaluable due to discontinuation of protocol therapy (1 rash attributed to lenalidomide; 1 with early symptomatic progression of lymphoma); 5 pts have not completed Part II. For 35 pts completing Parts I and II, at a median follow-up of 11.8 months, PFS is 73% (95%CI: 53 – 86%). Overall response rate (ORR) after Part I is 37% (6 CR; 7 PR); ORR after Part II is 60% (12 CR; 9 PR). Overall response rates after Part II do not differ between Cohort 1 (with dexamethasone) and Cohort 2 (without dexamethasone) [58% vs. 64%, respectively; p = 0.5]. There were fewer dose interruptions during Part I in Cohort 1 (15%) versus Cohort 2 (50%) [p = 0.02], reflecting reduced episodes of tumor flare and rash in Cohort 1. Conclusions: For rituximab-resistant pts with indolent B-cell or mantle cell lymphomas, the combination of continuous daily lenalidomide and a single four week course of rituximab, with or without low-dose weekly dexamethasone, achieves a high overall response rate with relatively durable responses. The addition of low-dose weekly dexamethasone may improve tolerance without decreasing the efficacy of the lenalidomide-rituximab combination. Disclosures: No relevant conflicts of interest to declare.

8060 Background: Lenalidomide may enhance anti-tumor immune responses by inhibiting Tregs and by augmenting NK-cell mediated antibody-dependent cytotoxicity. These immunologic effects may synergize...

Background Fludarabine-based therapies prolong survival of patients (pts) with chronic lymphocytic leukemia (CLL), but deplete T-cells causing immunosuppression which increases the risk of infection and may limit control of minimal residual disease by the immune system. Adoptive immunotherapy using autologous CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) enhances immune reconstitution after fludarabine-based therapy of lymphoma (Schuster Blood abstract 2007). Methods We conducted a multicenter phase I/II trial of ACTC following fludarabine- or alemtuzumab-based therapy of CLL. Eligibility required IWCLL 2008 indications for treatment of CLL. After leukapheresis, pts received fludarabine- or alemtuzumab-based therapy. Eight - 12 weeks after last immuno-chemotherapy, responding pts (CR, PR) received a single dose of ACTC prepared from autologous T-cells collected before therapy. Primary study endpoints were feasibility, defined as the ability to generate a T-cell dose of 1.0 E+10+/- 20%, and safety, defined as Results Thirty-four pts with CLL were enrolled (median age: 62). Eighteen patients were previously untreated and 16 patients had relapsed or refractory CLL. Four pts (12%) had del(17p), 11 pts (33%) del(11q), and 3 (9%) both del(17p) and del(11q). For previously untreated patients, 17 received fludarabine-cyclophosphamide-rituximab (FCR) and 1 pt received fludarabine-rituximab (FR); for relapsed patients, 10 received FCR, 3 FCR-bevacizumab, 2 alemtuzumab, and 1 oxaliplatin-fludarabine-cytarabine-rituximab (OFAR). Ten patients did not receive ACTC infusion for the following reasons: 6 pts (including 1 receiving alemtuzumab) had progressive disease prior to ACTC, 1 pt developed autoimmune hemolyic anemia during FCR, 1 pt developed ITP during FCR, 1 pt developed pure red cell aplasia during FCR, and 1 pt receiving alemtuzumab was removed from study per physician preference. Median ACTC dose was 1.0 E+10 CD3 cells (range: 3.94 E+08 -1.03 E+10). Two pts had progressive disease less than 90 days after ACTC and 1 pt was lost to follow-up. Twenty-one pts (14 CR; 2 CRi; and 5 PR at the time of ACTC infusion) were evaluable for studies of immune reconstitution at ≥90 days after ACTC. After chemotherapy prior to ACTC (D0), median CD4 count was 120 (range: 19-573); 90 days after ACTC (D+90), median CD4 count was 399 (range 62-1818) (p = 0.0003; median fold CD4 increase 2.3, range -0.5–20.9) [figure 1]. Median CD8 count also increased significantly (p = 0.004; median D0 = 86, range: 4-682; D+90 = 267, range: 18-2876; median fold CD8 increase 1.1, range: -0.8-18.4) [figure 2]. The increases in CD4 and CD8 counts between D0 and D+90 were not dependent on ACTC dose. There were no SAEs or grade 3 or higher non-hematologic toxicities related to T-cell infusion. For all 21 patients evaluable for immune reconstitution at D+90, median follow-up is 33 months with median PFS 30 months (not shown). PFS for previously untreated patients is 42 months, whereas PFS for relapsed patients is 12 months (figure 3). There was no correlation between magnitude of increase in post-infusion CD4 and CD8 counts and PFS at 1 year. Conclusions For CLL patients, ACTC production is feasible. ACTC infusion is well tolerated and results in a dose-independent acceleration of CD4+ and CD8+ cell recovery after fludarabine-based chemotherapy compared to historical controls. The application of adoptive immunotherapy with ACTC to accelerate immune reconstitution after immunochemotherapy could potentially provide an optimal immunologic milieu for early application of additional immunotherapeutic interventions. Disclosures: Levine: Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. June: Novartis: Patents & Royalties, Research Funding.

Abstract 674 Immune recovery is an important determinant in multiple outcomes following allogeneic hematopoietic stem cell transplant (allo-HSCT). Delays in B and T cell reconstitution are associated with an increased risk of infection, relapse and secondary malignancy. Strategies to enhance post-transplant T-cell reconstitution could therefore improve morbidity and mortality after allo-HSCT. The cytokine Interleukin-7 (IL-7) is a unique therapeutic candidate to promote immune reconstitution because it has a central role in T cell development and survival. Murine models of allo-HSCT have demonstrated that IL-7 can enhance thymopoiesis as well as promote peripheral T cell survival and expansion. Initial clinical trials performed with recombinant human IL-7 (rhIL-7) have demonstrated a dose-dependent expansion of CD4+ and CD8+ T cells with an acceptable toxicity profile in patients with solid tumors or HIV infection. Hence we are conducting a phase I trial of post-transplant administration of rhIL-7 (CYT107, Cytheris Inc) in recipients of a T cell depleted (TCD) allo-HSCT to determine the safety, toxicity and biological activity on T cell reconstitution. To date, 9 patients (AML=7, MDS=2), with a median age of 59.3 years (range 27–67 years) have been treated with escalating doses of rhIL-7 (3 at 10 mcg/kg, 6 at 20 mcg/kg) administered subcutaneously weekly for 3 weeks following TCD allo-HSCT from an HLA compatible donor. Accrual is ongoing in the final cohort (30 mcg/kg). Recombinant hIL-7 was started at a median of 96 days post allo-HSCT (range 61–244 days). Most patients experienced transient minor injection site reactions. One patient (20 mcg/kg) developed a biopsy proven hypersensitivity drug rash a week after the first injection and was removed from the study (evaluable for toxicity but not immune recovery endpoints). No other significant injection-related toxicities have occurred, and no patients have developed GVHD. No anti-IL-7 antibodies or neutralizing antibodies have developed following rhIL-7 injection. Two of 9 patients with high-risk AML have relapsed (4 and 9 months post rhIL-7), an incidence consistent with published data in patients undergoing allo-HSCT for AML in CR, irrespective of T-cell depletion. Eight patients remain alive with a median follow-up of 14.5 months post rhIL-7 administration. At baseline, the median T cell counts were 91/mm3 (range 5 – 219 /mm3), 43/mm3 (range 9 – 299 /mm3) and 0 (range 0 – 17 /mm3) for CD4+, CD8+ and CD45RA+ T cells, respectively. Preliminary assessment of the immunological effects of rhIL-7 in 8 evaluable patients has demonstrated an increase in CD4+ T cells exhibiting a naïve or central memory phenotype (69% median increase over baseline at day 21 – range 8% to 35-fold increase), and CD8+ T cells exhibiting a naïve or effector memory phenotype (94% median increase over baseline at day 28 – range 0 to 11-fold increase). There was no observed effect on the frequency of CD4+CD25+FoxP3+ T cells or CD19+ B cells. TCR excision circles (TREC) analysis performed on CD4+ and CD8+ subsets in the first 6 patients, using absolute quantification real-time PCR, demonstrated increases in TRECs in 5/6 patients indicating enhanced T cell production. Finally, all 3 CMV-seropositive patients developed CD8+ T cell CMV-specific responses detected by intracellular IFNγ production to overlapping CMV-pp65 pentadecapeptides peptide pools after administration of rhIL-7. In one patient, we also analyzed CMV-specific T-cell frequency using HLA-A*0201 restricted MHC-tetramers. The highest CMV-specific response levels were noted in this patient with a history of CMV viremia and low-level CMV-specific CD8+ T cells prior to rhIL-7 (5.3-fold increase to the A0201-restricted immunodominant NLV peptide by tetramer assay after rhIL-7). Our pre-clinical data and early clinical results suggest that administration of rhIL-7 in recipients of a TCD allo-HSCT has minimal toxicity and can enhance post-transplant immune recovery without causing GVHD. Disclosures: Perales: Cytheris: Research Funding. Croughs:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morre:Cytheris: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van den Brink:Cytheris: Research Funding.

Abstract 420 Juvenile myelomonocytic leukemia (JMML) is a distinct subtype of myelodysplastic syndrome/myeloproliferative disorder (MDS/MPD) which, in analogy to chronic myelomonocytic leukemia (CMML), is characterized by excessive proliferation of myelomonocytic cells, but unlike CMML it occurs in young children and shows characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Chromosomal defect are present in 22% of patients in particular involving del7/7q. Mutations of genes involved in GM-CSF signal transduction, including RAS and PTPN11, can be identified in a majority of children with JMML, constitutional mutations of NF1 can be found in another 10% of patients with JMML, but in significant proportion of patients no molecular lesions were identified. To further clarify the molecular pathogenesis of JMML we have applied a high density SNP array-based karyptyping and screened for associated mutations including established defected in RAS, PTPN11 and NF1 but also c-Cbl and TET genes, recently identified in patients with CMML and MDS/MPD. We studied 49 children with JMML diagnosed between 1988 and 2008 in 28 institutions throughout Japan. The median age at diagnosis was 28 months (range, 1-75 months). Karyotypic abnormalities were detected in 11 patients, including 7 patients with monosomy 7. Two children had clinical evidence of NF1 mutations. Out of 49 patients, 32 received hematopoietic stem cell transplantation (HSCT). We performed mutational analysis of the genes known to be affected by mutations in JMML. PTPN11 mutations were found in 26/49 (53%) while NRAS and KRAS mutations were found in 2/49 (4%) and 1/49 (2%), respectively. None of the patients screened showed the presence of TET2 mutations, previously shown to be present in a significant proportion of patients with MDS/MPD, including CMML. High-density Affymetrix 250K single nucleotide polymorphism array (SNP-A) were applied as a karyotyping platform to identify LOH and submicroscopic copy number changes. Signal intensity was analyzed and SNP calls determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number and areas of UPD were investigated using Hidden Markov Model and CNAG v3.0 software. Compared to the results of conventional metaphase cytogenetics (MC), SNP-A identified significantly more genetic abnormalities (25% vs 49%; p=.02). In 1 patient UPD17q was present ivolving NF1 locus. In 4 patients UPD11q involving c-cbl locus (11q23.1) was found. Sequencing of c-Cbl gene family revealed mutations of c-Cbl in 5/49 (10%), and no Cbl-b mutations. All but 1 mutations were homozygous and were located in the RFD (exon 8 and intron 8). C-Cbl mutations were mutually exclusive with PTPN11, NRAS, and KRAS mutations or had clinical diagnosis of NF1. Unlike in CMML, no UPD4q24 or homo- or heterozygous TET2 mutations were found. Histomorphologic analysis did not reveal any distinct c-Cbl mutation-associated features or differences in count between patients grouped based on the presence of specific mutations. Similarly, there were no differences in gender, or the presence of cytogenetic abnormalities and the probability of 2 year overall survival of c-Cbl mutant cases between patients grouped according to mutational status. All patients with c-Cbl mutations displayed GM-CSF hypersensitivity at initial presentation but did not differ in this feature from the remaining JMML patients. However, mutant c-Cbl cases showed earlier presentation (median age 12 months vs. 29 months, p = .037) and lower median hemoglobin F fraction (3.5 % vs. 25%, p=.02). In sum, c-Cbl mutations constitute a novel important pathogenic lesion in JMML. While their presence suggests functional similarity to CMML, absence of TET2 mutation in JMML and rarity of PTPN11 mutations in CMML constitute important distinctive features of both diseases. Disclosures: No relevant conflicts of interest to declare.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by specific hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Mutations in RAS, NF1, or PTPN11 positioned in the GM-CSF signal transduction pathway, which contribute to myeloid proliferation, have been recognized in the pathogenesis of JMML. Recently a multi-step model for leukemogenesis has been proposed. In this model, the pathogenesis of leukemia requires at least two classes of mutations:primary mutations of genes implicated in cell differentiation such as AML1 and PU.1 andadditional mutations of genes contributing to myeloid proliferation such as FLT3, RAS, and PTPN11. We hypothesized that in patients with JMML, in addition to known mutations of genes in the GM-CSF pathway involved in myeloid proliferation, potential causative mutations of other classes might be acquired. AML1 encodes a transcription factor that is essential for definitive hematopoiesis, and its mutations have recently been found in adults with acute myeloid leukemia and myelodysplastic syndrome. However, no information is available on the profiles of mutations in these genes and the relationship between these mutations and clinical features of JMML in children. We analyzed mutations of N-RAS, K-RAS, and PTPN11 in 50, and of AML1 in 30 Japanese children with JMML by direct sequencing. Correlation between the mutational status and clinical and laboratory findings, including age at diagnosis, sex, fetal hemoglobin (HbF), platelets count, cytogenetic abnormalities, and hepatomegaly, all which are suggested prognostic factors for JMML, were also assessed. Of the 50 patients with JMML, cytogenetic abnormalities were detected in 14, including 8 with monosomy 7. PTPN11 and N-/K-RAS mutations were found in 24 (48%) and 7 (14%) patients, respectively, and a novel mutation in AML1 was identified in one patient. No simultaneous mutations in these genes were found. Age at diagnosis was older (median 36 vs 11 months, p=0.0005) and HbF level was higher (31.0% vs 5.1%, p=0.033) in patients with the PTPN11 mutation than those with the RAS mutation. No difference was observed between patients with PTPN11 and RAS mutations in sex ratio, white blood cell count, platelets count, and the incidence of cytogenetic abnormalities and hepatomegaly. Our results suggest that the clinical outcome of patients with PTPN11 might differ from those with RAS mutations because a higher level of HbF and older age have been reported to be poor prognostic factors. In one patient with JMML, we identified a novel mutation in the AML1 gene that belongs to the class of genes contributing to cell differentiation instead of the class of genes in the GM-CSF pathway involved in myeloid proliferation.

Juvenile myelomonocytic leukemia (JMML) is a rare, clonal myeloproliferative disorder afflicting young children and is characterized by specific hypersensitivity of JMML cells to GM-CSF in vitro. The pathogenesis of JMML seems to arise from dysregulation of GM-CSF signal transduction. Approximately, 75% of patients with JMML exhibit dysregulation due to mutations in RAS, NF1 or PTPN11, all of which are positioned in the GM-CSF pathway. Potential causative mutations in remaining patients are anticipated to be in signaling components downstream of the GM-CSF receptor. Recently, a somatic point mutation (JAK2 V617F) in the JAK2 gene, which plays a major role in the development of hematopoietic cells by transducing signals from growth factor receptors including GM-CSF receptor, was found commonly in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. All these myeloproliferative disorders are also characterized by hypersensitivity of hematopoietic progenitor cells to several growth factors and cytokines. Because the frequency of JAK2 V617F mutation in JMML remains unknown, we performed mutational analysis of JAK2 in 39 Japanese children (aged 1 to 69 months) with JMML to assess the pathogenetic relevance of this mutation in JMML, in addition to mutational analysis of PTPN11 and N-RAS. Genomic DNA were prepared from bone marrow or peripheral blood samples taken at initial diagnosis. Mutations of JAK2 (exon 12), PTPN11 (exon 3 and 13) and N-RAS (exon 1 and 2) were screened by direct sequencing. PTPN11 or N-RAS mutations were found in 19 (49%) or 3 (8%) patients respectively, and this is compatible with previous reports. In this study, no JAK2 V617F mutations were detected in any of the 39 JMML patients. In conclusion, we confirmed the presence of PTPN11 and N-RAS mutations in JMML. However, the JAK2 V617F mutation appears to be uncommon in patients with JMML, while it is extraordinarily common in classic MPD. Future investigation should focus on other signaling components in the same signaling pathway as JAK2, or on other parallel signal transduction cascades of the GM-CSF receptor.

Jevenile myelomonocytic leukemia (JMML) is a rare myeloproferative disorder of early childhood, arising from pluripotent stem cells. Previous studies indicate the clonal nature of JMML, involving myeloid, erythroid, megakaryocyte and B-lymphoid lineage. However, it is unclear whether T lymphocyte lineage is involved or not. In one previous report, a patient with JMML and cytogenetic evidence of monosomy 7, a combination of fluorescence activated cell sorting and fluorescence in situ hybridization (FISH) analysis confirmed no T cell lineage involvement. One explanation of sparing T cell lymphocyte involvement in JMML is that the majority of T-lymphocytes are long-lived and borne before the occurrence of the disease. We demonstrated that cells from six patients of JMML repopurate in NOD/SCIDγcnull mice and differenciate into granulocyte, monocyte, erythroid, B-lymphocyte, T-lymphocyte and natural killer(NK) cells. The diagnosis of JMML was based on the internationally accepted criteria. 0.01 to 1×107mononuclear cells were injected through the tail vein of five week old NOD/SCIDγcnull mice. 12 week after transplantation mice were sacrificed and the cells are collected from the femoral bone and spleen and subjected to flow cytometry. The percentage of human CD45 antigen positive cells ranged from 41 to 73%. To examine the involvement of lymphocyte subpopulations, we purified human CD3+,CD19+and CD56+ cells from a murine bone marrow and spleen cells transplanted from a patient with monosomy 7. The purity of cells were more than 95%. The percentage of monosomy 7 cells determined by FISH analysis ranged from 96 to 100% in purified CD3+, CD19+and CD56+ cells. These findings support the concept that JMML originate in transplantable multilineage hematopoietic stem cells. This novel murine xenotransplant model should be useful to investigate the nature of stem cells and test new therapies for patients with JMML.