ALBERT

Description: Abstract 3453 Individuals undergoing allogeneic transplantation receive multiple red blood cell transfusions both as part of the transplant procedure and as part of the pre-transplant care of the underlying disease. Therefore these patients may be at risk for complications of transfusional iron overload. Several studies have noted that individuals entering the transplant with baseline elevated serum ferritin values have decreased overall survival and higher rates of disease relapse. Whether the iron is a direct contributor to inferior outcomes or is a marker of more advanced disease (thereby requiring greater transfusions) is unclear. Little is known about the incidence and consequences of iron overload among long-term survivors of allogeneic transplantation. Methods: Using Kaplan-Meier and Cox regression analyses, we performed a single center, retrospective cohort study of consecutive allogeneic transplants performed at Hackensack University Medical Center from January 2002 through June 30, 2009 to determine the association between serum ferritin (measured approximately 1 yr post allogeneic transplant) and overall survival. Results: During the study time frame, 637 allogeneic transplants (Donor Lymphocyte Infusion procedures excluded) were performed at our center and 342 (54%) survived ≥ one year. Among 1-year survivors 240 (70%) had post-transplant serum ferritin values available for review, including 132 (55%) allogeneic sibling, 68 (28%) matched unrelated, and 40 (17%) mismatched unrelated donor transplants. The median post-transplant ferritin value among 1-year survivors of allogeneic transplant was 628 ng/ml (95% CI 17, 5010), with 93 (39%) above 1000 ng/ml and 40 (17%) above 2500 ng/ml. The median post-transplant ferritin levels varied by underlying hematologic disease (aplastic anemia = 1147, acute leukemia = 1067, MDS = 944, CLL = 297, CML = 219, lymphoma = 123, multiple myeloma = 90). The Kaplan-Meier projected 5-year survival rate was 76% for the cohort that had survived one year and had available ferritin values. Fifty late deaths have occurred; causes of late death were disease relapse (n=37, 74%), GVHD (n=7, 14%), infection (n=4, 8%), cardiac (n=1, 2%) and second malignancy (n=1, 2%). The 1-year post-transplant serum ferritin value was a significant predictor of long term survival. Using a cut-off ferritin value of 1000 ng/ml, the 5-year projected survivals were 85% (95 CI 75%-91%) and 64% (95% CI 52–73%) for the low and high ferritin cohorts respectively (Figure, log-rank p

Description: Abstract 2520 INTRODUCTION. Age, cytogenetics, FLT3 and NPM1 mutations are the most significant prognostic factors (PFs) for adult AML treated with standard regimens, but the predictive significance of FLT3 and NPM1 with contemporary treatments is unknown. We examined the clinical significance of NPM1 and FLT3 mutations in adult de novo AML pts enrolled on SWOG study S0106. METHODS. S0106 was a randomized phase III clinical trial for pts of age 18–60 with de novo non-M3 AML, evaluating the effects of adding Gemtuzumab Ozogamicin (GO) to standard induction therapy (Cytosine Arabinoside and Daunomycin, AD), and of post-consolidation GO vs. no additional therapy (ASH, 2009, Abstract 790). Samples from 198 of the 600 eligible pts were evaluated. Analyses for nucleotide insertions in exon 12 of the NPM1 gene and internal tandem duplications (ITD) within exons 14–15 of FLT3 were performed using fragment analyses in diagnostic bone marrow (BM, N=190) and peripheral blood (PB, N=8) samples. Mutant/wild-type (WT) allelic ratios (AR) were computed for all mutations. Effects of mutations and other PFs on complete response (CR), resistant disease (RD), overall survival (OS) and relapse-free survival (RFS) were analyzed by logistic and Cox regression. P-values are 2-sided. RESULTS. Patient characteristics and outcomes are shown in Table 1. In univariate analyses, NPM1-Mut pts had significantly higher CR (81% vs. 58%, P=.0018) and lower RD (13% vs. 28%, P=.028) rates, better OS (64% vs. 47%, P=.045) and RFS (54% vs. 41%, P=.50). FLT3-ITD was not associated with CR or RD, but was associated with poorer OS (hazard ratio [HR] 2.28, P=.0011) and RFS (HR 2.74, P=.0009). FLT3-ITD length (range 18–366, median 46), FLT3 AR (range 0.18–8.2, median 0.98), and NPM1 AR (range 0.2–1.0, median 0.8) were not associated with CR, RD, or OS, but RFS tended to be lower with higher ITD length (P=.076). In multivariate analyses with other PFs, neither NPM1 nor FLT3 was associated with CR or RD rates, however the combined effects of FLT3 and NPM1 identified 3 mutation risk groups for OS (P=.0044, Fig 1A) and RFS (P=.0003, Fig 1B), since NPM1 did not significantly affect outcomes within the FLT3-ITD pts. These risk groups are FLT3-WT/NPM1-Mut (Good Risk: 3-yr OS 82%, RFS 69%), FLT3-WT/NPM1-WT (Intermediate Risk: OS 49%, RFS 43%), and FLT3-ITD (Poor Risk: OS 29%, RFS 14%). The impact of adding GO to induction therapy was examined within each risk group. In each risk group, CR rates were higher in the AD+GO arm, though not significantly so. Likewise, the RD rates were lower in the AD+GO arm, but this difference was significant only in the largest group: Intermediate Risk, FLT3-WT/NPM1-WT, 17% vs. 34% (P=.026). Treatment arm did not significantly affect OS and RFS in any mutation risk group. CONCLUSION. This study confirmed prognostic effects of FLT3 and NPM1 mutations in de novo AML pts treated with AD or AD+GO. Analyses of the joint impact of NPM1 and FLT3 mutations do not rule out the possibility that they act independently. With the small numbers of pts in the “good” and “poor” risk groups, there was no clear evidence that mutation status predicts clinical benefit from adding GO to therapy. We are evaluating additional samples and will update these results as data matures. Disclosures: No relevant conflicts of interest to declare.

Description: We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Description: Abstract 743 We have previously identified a subset of National Cancer Institute (NCI)-HR B-cell precursor (BCP) ALL patients with a gene expression profile similar to that of BCR-ABL1 ALL (BCR-ABL1-like ALL (Mullighan, N Engl J Med 2009; den Boer, Lancet Oncology 2009; Harvey, Blood, 2010, and unpublished data) and poor outcome on the COG P9906 trial, which was limited to a selected subset of HR BCP ALL patients. These cases are BCR-ABL1-negative but commonly have deletion or mutation of IKZF1. Up to half of these cases harbor rearrangements, deletions and/or mutations activating cytokine receptors and tyrosine kinase signaling (e.g. CRLF2 and activating JAK1/2 mutations), although the kinase-activating mutations in many cases remain unknown. In this analysis, we have assessed the prognostic significance of this BCR-ABL1-like signature in an unselected cohort of BCR-ABL1 negative BCP ALL patients consecutively enrolled on COG AALL0232. This phase 3 trial utilized a 2×2 factorial design comparing dexamethasone (DEX) versus prednisone (PRED) during induction, and high dose methotrexate (HD-MTX) versus Capizzi methotrexate (C-MTX) during interim maintenance 1 (IM-1). We recently reported improved event free survival (EFS) for patients receiving HD-MTX versus C-MTX (Larsen, J Clin Oncol 29: 6s, 2011) and for DEX versus PRED among patients

Description: Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Thrombotic thrombocytopenic purpura (TTP) is a microvascular occlusive disorder characterized by systemic aggregation of platelets, thrombocytopenia, and mechanical injury to erythrocytes. Between 30% and 80% of TTP cases are associated with ADAMTS13 deficiency. Thienopyridine-derivative anti-platelet agents, ticlopidine and clopidogrel, are the drugs most commonly associated with TTP. The structures differ only by a carboxymethyl side-chain and have no common metabolites. Since 2002, our R01 research project has focused on evaluating thienopyridine-associated TTP. Herein, we present the final results. Clinical and laboratory data were obtained from case reports, the FDA’s MedWatch program, a Japanese national reference laboratory for ADAMTS13 assays, and apheresis centers at Duke University, University of North Carolina, Northwestern University, and the Mayo Clinic. Epidemiologic data for rate estimation for thienopyridine-associated TTP among persons who receive cardiac stents were obtained from international cardiology laboratories. Pharmacovigilance information was obtained from package inserts for the drugs. Most thienopyridine-associated TTP cases are associated with two weeks or more of ticlopidine rather than clopidogrel, are immune-mediated involving neutralizing antibodies to ADAMTS13, resolve with therapeutic plasma exchange (TPE), and have spontaneous relapses. Less frequently, cases are associated with clopidogrel, occur within days of drug initiation, may be a direct result of endothelial cell damage, are less responsive to TPE, and are less likely to recur. Thienopyridine-associated TTP patients with severe deficiency of ADAMTS13 activity have a different profile than those with normal ADAMTS13 levels. Among thienopyridine-associated TTP patients who have ADAMTS13 deficiency, TPE is usually performed for a few days and patients recover without detectable organ damage. In contrast, among thienopyridine-associated TTP patients who do not have ADAMTS13 deficiency, several weeks of TPE is required for recovery, and 30% mortality rates have been reported. Despite similar chemical structures, ticlopidine- and clopidogrel-associated TTP probably occur by different mechanisms and have different clinical presentations and expected outcomes. Clinical Characteristics Onset Platelet Count

Description: Abstract 2529 Genomic alterations involving the CRLF2 gene lead to over-expression of intact CRLF2 and have significant prognostic value in pediatric BCP-ALL. Not only do patients with these lesions have inferior outcomes, they also have a very high frequency of JAK1 and JAK2 mutations and may be candidates for targeted therapies. The two major CRLF2 lesions include cryptic translocations that produce IgH@-CRLF2 and interstitial deletions of the pseudoautosomal region of X/Y causing P2RY8-CRLF2 fusion. Both lesions can be detected by fluorescence in situ hybridization (FISH), and genomic PCR or RT-PCR can identify P2RY8-CRLF2. To develop rapid and inexpensive assays for detection/screening of these events, we developed a flow cytometry based method to measure CRLF2 expression and compared this assay to quantitative RT-PCR (qPCR) measurement of CRLF2 expression by evaluating their performance in an unselected cohort of 279 newly diagnosed pediatric BCP-ALL patients consecutively enrolled on the COG AALL03B1 biology/classification study between 10/30/09-5/1/10. Flow cytometry was performed first in real time on diagnostic specimens shipped to a central COG reference laboratory and then residual diagnostic material was shipped to a separate laboratory for RNA isolation and qPCR analysis. Of the 279 cases analyzed by flow, 257 (92%) yielded sufficient RNA quality and quantity for qPCR analysis. In our previous studies with qPCR and CRLF2 it was shown that CRLF2 lesions occurred only among those cases with the highest expression (ΔCt 〈 8). In order to assure that we identified all cases with CRLF2 lesions, we performed FISH and P2RY8-CRLF2 PCR on all cases with qPCR expression ΔCt 〈 10 (n = 109) and an additional 14 cases with a flow blast/lymph CRLF2 mean fluorescence intensity (MFI) ratio 〉1.15. Of these 123 cases, 11 were determined by FISH to have the IGH@-CRLF2 translocation and 15 were shown to have P2RY8-CRLF2 fusions by PCR. Figure 1 shows the locations of these genomic lesion-positive cases among the qPCR (panel A) and flow cytometry (panel B) CRLF2 expression data. The overall frequency of CRLF2 lesions among these patients is 10.1% (assuming all lesions were identified among the highest expressing cases) and, surprisingly, the frequencies of IgH@-CRLF2 and P2RY8-CRLF2 were very similar (4.3% and 5.8%, respectively). With both methods, the 11 IgH@-CRLF2 cases were found to be the highest expressing (among the top 12 cases by qPCR and 16 cases by flow). Receiver operating curve analysis of each method identified cutoffs with excellent performance: qPCR cutoff CRLF2 ΔCt = 5.47 with 96.9% specificity and 88.5% sensitivity; flow cutoff MFI CRLF2 ratio of 2.04 with 95.9% specificity and 92.3% sensitivity. The broader dynamic range of the qPCR assay may be necessary for the identification of poor risk cases with high CRLF2 expression that lack genomic lesions, however both methods are rapid, highly effective and very comparable for finding ALL cases that harbor CRLF2 genomic lesions, and suitable for incorporation in large scale clinical trials. Figure 1. qPCR and Flow Cytometry Results for CRLF2 Expression. Panel A (qPCR ΔCt values) and Panel B (log2 blast/lymphocyte ratios) plot the expression values for the 123 patients with the highest CRLF2 expression. Panel B plots the log2 blast/lymphocyte ratio for CRLF2 expression determined by flow cytometry. Small dots show the expression for each patient while the large diamonds highlight cases proven to have CRLF2 lesions either by FISH (IGH@-CRLF2) or PCR (P2RY8-CRLF2). Each unit of expression represents a two-fold difference in intensity. Figure 1. qPCR and Flow Cytometry Results for CRLF2 Expression. Panel A (qPCR ΔCt values) and Panel B (log2 blast/lymphocyte ratios) plot the expression values for the 123 patients with the highest CRLF2 expression. Panel B plots the log2 blast/lymphocyte ratio for CRLF2 expression determined by flow cytometry. Small dots show the expression for each patient while the large diamonds highlight cases proven to have CRLF2 lesions either by FISH (IGH@-CRLF2) or PCR (P2RY8-CRLF2). Each unit of expression represents a two-fold difference in intensity. Disclosures: No relevant conflicts of interest to declare.

Description: Background: EPO and its derivative darbepoietin alfa (DAR) are important treatments of anemia in lower risk MDS. Prognostic factors of response and of its duration have been recently updated (Blood, 2005, 106, 803–11) and we reanalyzed them in a large series of patients (pts) treated in France and Belgium. Patients: 419 MDS pts were treated with EPO (≥30000UI/wk for at least 12 wks) or DAR (300μg/wk)± GCSF in 25 GFM centers between 1998 and 2006 (160 prospectively analyzed in 3 consecutive trials, and 259 retrospectively analyzed). Median follow-up was 54 months, median age: 73.5 years. WHO classification: RA (14%), RCMD (16%), 5q- syndrome (4%), RARS (21%) RCMD-RS (13%), RAEB-1 (22%), RAEB-2 (6%), and also 4%CMML (FAB); karyotype: 64% FAV, 16% INT, and 4% UNFAV (16% failure or not done). IPSS: 34% LOW, 40% INT-1, 8% INT-2, 2% HIGH (16% unavailable). 185, 126, 80 and 28 pts received EPO alone (alfa or beta), DAR, EPO+G and DAR+G respectively. Median pre-treatment EPO level was 76 UI/l (only 7% pts〉500 UI/l). All pts had Hb2 RBC units/month). Results: 63% pts responded (IWG criteria: 43%HI-E major and 20% HI-E minor), including 57%, 63%, 57%, 66%, 63% with EPO alfa alone, beta alone, EPO+G, DAR alone, DAR+G response (p=ns). Median response duration was 20 mos (range 3–74 mos), 25 and 14 mos for major and minor responses (p= 0.001). Relapse was associated with treatment discontinuation (45%), progression to higher grade MDS (12%) or AML (13%), but without evident cause in 30% cases. In univariate analysis, significantly higher response rates were observed in pts with

Description: Correlative laboratory studies were developed in a phase I trial to evaluate the safety of intracoronary injection of escalating doses of bone marrow (BM) CD133+ cells in patients with chronic coronary ischemia. Concurrent with patient cellular therapy, CD133+ cells were phenotyped and tested functionally with endothelial cell colony formation and in vitro and in vivo transmigration. BM (194 ± 11 ml) was isolated from patients meeting study inclusion criteria. CD133+ cells (20 ± 13 x 106, 84 ± 7% purity and 76 ± 7% viability (7AAD)) were isolated using the CliniMACS device (Miltenyi). Contaminating cells following the CliniMACS selection were: 〈 5% of CD3, CD3neg/CD56, CD19 (immature/mature), CD14, and CD71 cells with 5% CD61, 8% CD13+ SSChigh. BM, PB (peripheral blood), cord blood (CB)-derived endothelial progenitor cells (EPC) were assessed by a culture assay (StemCell Technologies) scoring early outgrowth CFU-EC. SEACOAST patients yielded significantly less colonies compared to controls of matched PB and BM (donors 28–48 yrs) and CB: normal donor (ND) PB, 65; ND BM, 40; CB, 43; SEACOAST patient PB, 2, SEACOAST patient BM, 1. Transmigration assays were used to evaluate the functionality of selected CD133+ cells to chemotactic agents stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF). Selected CD133+ cells were recovered, resuspended in DMEM/1% HSA media and after a 37°C incubation for 16–20 hrs, 5 x 104 CD133+ cells were added to transwells (5 mm) for 3 hours. Transmigrated cells were quantitated by flow cytometry using anti-CD45, anti-CD133 antibodies, and Fluorosphere beads. Surface expression on ND BM CD133+ cells of CXCR4 and VEGF-R2 was 0–16.4% and 1.2–4.3%, respectively. Transmigration was effected by 200 ng/ml (range of 16–62%) but not to 10 ng/ml VEGF. For CD133+ cells devoid of the expression of CXCR4, SDF-1-induced transmigration was absent. Expression of CXCR4 and VEGF-R2 on clinical trial patient-selected CD133+ cells was 0–5% and 0–2%, respectively, and transmigration was 5–19% to 200 ng/ml SDF-1 but not to 10 ng/ml VEGF. Patient selected CD133+ cells or PB mononuclear cells (PBMC), ND CD133+ cells, or a vehicle control were injected via a left intraventricular route into NOD/SCID mice with a femoral artery ligation immediately after injury. Doppler flow measurements were obtained weekly for 6 weeks comparing the perfusion ratio of ischemic/healthy limbs. At 28 days, perfusion ratios were statistically higher in study groups receiving ND CD133+ cells (0.51 ± 0.06) compared to controls (0.37 ± 0.03, p=0.025). Mice receiving patient CD133+ cells (0.46 ± 0.04) or PBMC (0.37 ± 0.08) did not show statistically significant improvement over control animals (p= 0.07, p= 0.94, respectively). BM was harvested to assess human engraftment by cytometric analysis. Mice injected with 0.5 x 106 patient BM CD133+ cells showed 70% purity and 〉70% viability) to chronic ischemic patients via an intracoronary route, important correlative in vitro and in vivo assays has demonstrated the diminished potency of BM-derived CD133+ cells as compared to CB and ND PB and BM-derived cells.

Description: Abstract 2752 Introduction: We recently identified a poor prognostic subgroup of pediatric BCR-ABL1 negative ALL patients characterized by deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) and a gene expression signature similar to BCR-ABL1 positive ALL, raising the possibility of activated tyrosine kinase signaling within this leukemia subtype. Targeted sequencing revealed activating sequence mutations in the Janus tyrosine kinases (JAK1 (N=3), JAK2 (N=17) and JAK3 (N=1)) in 21 of 187 (11.2%) BCR-ABL1 negative, high-risk pediatric ALL cases. All 21 cases with JAK mutations had the BCR-ABL1-like expression profile, accounting for about 50% of the cases with this phenotype, suggesting that mutations in JAK kinases account for some, but not all, cases with this distinctive profile. To determine whether mutations in other kinases might also be associated with this distinctive gene expression profile, we sequenced 126 genes encoding tyrosine kinases and mediators of kinase signaling in an additional 46 high-risk ALL cases with a BCR-ABL1-like expression profile. The genes sequenced included the entire tyrosine kinome. Methods: The 46 leukemia specimens studied were from patients enrolled on COG clinical trials for high risk ALL (P9906, n=23 and AALL0232, n=23), with risk defined primarily by elevated WBC and/or age 〉 10 years. All 46 cases had a BCR-ABL1 like expression profile. The 23 P9906 cases all lacked JAK mutations, while 3 of the 23 AALL0232 cases were found to have activating JAK mutations (JAK1 (N=1), JAK2 (N=2)). The entire coding region and UTRs of each gene was amplified by PCR of whole genome amplified genomic DNA, and subjected to Sanger sequencing. A CEPH sample (NA19085) was also included as a normal control DNA. Results: A total of 1,149,117 bases were sequenced bi-directionally for each sample; 96% of the targeted bases were covered with high-quality sequencing data. We identified a total of 2,302 variations predicted to change protein sequences, 173 of which are novel, putative variations after removing germline variations found in dbSNP, The Cancer Genome Atlas Project (TCGA) and the normal CEPH sample NA19085 in this study. For each novel variation, the tumor DNA was resequenced and matching normal DNA was sequenced to validate the original observation and to distinguish somatic from inherited variants. The results show that 105 variations are germline, 20 are false positives while the remaining markers failed in validation assay. Aside from 1 FLT3 mutation (23aainsN609), there are no confirmed somatic mutations in any other tyrosine kinase genes. Conclusion: Aside from JAK mutations, somatically acquired sequence mutations in tyrosine kinase genes are rare in children with high risk ALL and BCR-ABL1 like gene expression profiles. We are pursuing the identification of alternative mechanisms for kinase activation that might explain the distinctive expression profile observed in these cases. Disclosures: Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding. Hunger:bristol myers squibb: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees.