ALBERT

Description: Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and 〉60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 692 Congenital neutropenia syndromes comprise a heterogeneous group of disorders, whose genetic etiology remains often unknown. We describe a consanguineous pedigree with several affected individuals characterized by predisposition to recurrent and chronic bacterial and viral infections. Affected patients had chronic bronchitis/bronchiectasis, recurrent bacterial and herpes simplex skin infections, and disseminated warts associated with human papillomavirus and molluscum contagiosum virus. One patient developed a lymphoproliferative disorder associated with EBV-infection. Patients had congenital neutropenia with fluctuating absolute neutrophil granulocyte counts (180-4000/μl), yet no evidence of cyclic neutropenia. Immunophenotyping of peripheral blood revealed a paucity of peripheral T- and B-cells. Interestingly all patients showed evidences of autoimmunity. In addition all affected patients had subtle and hemodynamically not relevant cardiac defects such as ASD-II (P1), patent foramen ovale (P2) and patent foramen ovale associated with mitral, tricuspid and pulmonary insufficiency (P3). Genome-wide genotyping and linkage analysis of the index family yielded a LOD score of 4.3 on a linkage interval on chromosome 20. Candidate gene sequencing revealed a homozygous nonsense mutation in exon 7 of the gene STK4 (formerly MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apoptosis. Isolated STK4-deficient lymphocytes and neutrophils of these patients exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis in response to various proapoptotic stimuli. Lymphopenia and congenital neutropenia may therefore be a consequence of increased loss of peripheral lymphocytes and neutrophils, similar to other well defined monogenetic diseases of the immune system. STK4 deficiency is a novel human primary immunodeficiency syndrome and highlights the role of the HIPPO pathway for the development of the human immune and cardiac systems. Disclosures: No relevant conflicts of interest to declare.

Description: Clinical and laboratory evidence support an immune pathogenesis in most cases of idiopathic aplastic anemia (AA) and closely related disorders such as paroxysmal nocturnal hemoglobinuria (PNH). While external triggers are likely necessary, a complex constellation of immunogenetic factors may determine disease susceptibility. Many immunogenetic factors can influence the quality of immune response and affect the propensity to immune-mediated attack on hematopoietic stem cells in AA. Here we investigated whether KIR and KIR-L (HLA-A) genotype and cytokine/receptor gene variants are over-represented in AA and PNH. We studied a cohort of 77 patients with AA (23 AA, 20 AA/PNH and 34 PNH), 10 with hypocellular MDS and 175 healthy controls. The following SNPs in immunoregulatory genes were analyzed: IL-1α (−889 T/C), IL-2 (−330 T/G +166 G/T), IL-4 (−1098 T/G −590 T/C −33 T/C), IL-1R (−1970 C/T), IL-1Rα (mspa111100 T/C), IL-4RA (+ 190 G/A), IL-1β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, −819 C/T, −592 C/A), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF-γ (+874 A/T), TNF-α (−308 G/A, −238 G/A) and immunomodulatory receptor genes including CTLA-4 exon 6 (+49 G/A), FcRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of NK and cytotoxic T cells, we examined the combined impact of KIR/KIR-L genotypes on the risk of AA and PNH syndrome. In AA we found a decreased frequency of inhibitory KIR-2DL3 genes (68% vs. 89%, p=.0002); analysis of the KIR genotype in correlation with the corresponding KIR-L profile, revealed a decreased frequency of stimulatory 2DS1/C2 mismatch resulting in a potentially enhanced cytotoxic activity (14% vs.44%, p=.003). No association was found for most of the SNPs tested. However, when we examined the frequency TGF-β genotypes, increased frequency of GG variant in codon 25 (61% vs. 35% in controls, p=.03), associated with the “high secretor” phenotype, was found in AA. This relationship was also present in hypocellular MDS (82% vs. 32%, p=.007). Additionally, we found a lower incidence of TT genotypes for the IL-1Rα gene (33% vs. 62% p=.02). We confirm that the hypersecretor genotype T/T of INF-γ was over-represented in AA (28% vs. 10% in controls, p=.02). Subgroup analysis revealed that the T/T genotype of IFN-γ (35% vs. 14% p=.01) correlated with presence of a PNH clone. Previously, we have shown the association of HLA-DR15 with responsiveness to immunosuppression. When AA patients were subgrouped according to response to ATG/CsA, therapy refractoriness correlated with the presence of the C2/C2 haplotype (30% vs. 0% p=.02) and inhibitory KIR-2DL3/C1 mismatch (70% vs. 0%, p=.01) which may result in a greater propensity to breach of self-tolerance. In comparison, in the total AA group, C2/C2 haplotype and KIR-2DL3/C1 mismatch were present in 17% vs. 24% and 8% vs. 16% of controls, respectively. An increase in the frequency of 2DL3 and a decrease in 2DS1 mismatch may result in imbalance between cytotoxicity and KIR inhibition. In sum, our findings demonstrate that complex inherited traits involving immunogenetic factors may genetically determine propensity to bone marrow failure syndromes.

Description: Introduction: CD34+-selected peripheral blood stem cell infusions without conditioning have recently been utilized for poor graft function (PGF) with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this pilot trial utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected peripheral blood stem cell infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response we included a cohort of donors mobilized with plerixafor in addition to the standard G-CSF. Methods: Poor graft function (PGF) was defined as ANC 〈 0.5k/μL, platelets 〈 30k/μL, or platelet or RBC transfusion dependence despite full donor chimerism and in the absence of GVHD more than 60 days following allogeneic stem cell transplant (allo-SCT). Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only (SC at a dose of 10 μg/kg daily for 5 days), (2) fresh mobilized products using G-CSF (SC at a dose of 10 μg/kg daily for 5 days, days-4 through day 0) and plerixafor (IV at a dose of 320 μg/kg on day 0), and (3) cryopreserved cells mobilized with G-CSF (SC at a dose of 10 μg/kg daily for 5 days). Seventeen donor-recipient pairs were enrolled onto this prospective study. The original allo-SCT donor either underwent an additional peripheral blood stem cell (PBSC) mobilization and collection with G-CSF only (n=5) or G-CSF plus plerixafor (n=9) or a cryopreserved product (n=3) from a previous collection (using G-CSF only) was used to create the CD34+ cells for infusion. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. Neutrophil improvement was defined as neutrophil count 〉 500/μl without growth factor support for 〉7 days; platelet improvement was defined as platelet count ≥ 50,000/µl without platelet transfusion support for 〉 7 days; and RBC improvement was defined as hemoglobin 〉 9 g/dL and transfusion independence. Complete response was defined as improvement of all involved cells lines; partial response was defined as improvement of platelets and/or neutrophils with continuing RBC transfusion dependence. Results: The mean post-selection CD34+ count per kg of recipient weight was 3.7x106, 12x106, and 1.7x106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. Mean CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells prior to CD34 selection) was 58%, 67%, and 31% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively (Table 1 and Figure 1). The mean number of CD3+ T cells/kg infused in each product was 0.004 x 106, 0.032 x 106, and 0.015 x 106 for G-CSF only, G-CSF plus plerixafore and cryopreserved groups respectively (Table 1). Following the CD34+-selected stem cell infusion, 12 of the 17 recipients (71%) had a complete response including all 3 who received cryopreserved products; 3 had a partial response (17%) and 2 patients (12%) did not respond. One year relapse-free and overall survival was 71% and 65%. There was no treatment related toxicity reported other than graft-versus-host-disease (GVHD); three (18%) developed acute GVHD (1 grade I localized to the skin, 2 grade II with gut involvement), 6 chronic GVHD (2 limited and 4 extensive) (Table 1). Conclusion: Cryopreserved products seem to be a viable alternative to create CD34+ -selected peripheral blood stem cell infusions for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone. Disclosures Abboud: Teva Phamaceutical: Research Funding. Uy:Novartis: Research Funding.

Description: Abstract 2759 MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and diverse cellular processes. Expression profiling of miRNAs has identified dysregulated miRNAs in many cancers, including acute myeloid leukemia (AML). However, the mechanism for altered miRNA gene expression and the frequency of miRNA gene mutations in AML is largely unknown. We performed next-generation sequencing and high-resolution comparative genomic hybridization (CGH) to determine the frequency of miRNA gene mutations in 30 patients with therapy-related AML (t-AML). The sequencing is completed, and final analysis is underway. Herein, we report the results of the CGH. We designed custom CGH arrays for all 835 miRNA genes in miRBase (version 14) and 44 genes involved in miRNA processing. Average probe spacing was 30 bp for miRNA genes and 80 bp for miRNA processing genes and 10 kb flanking regions were interrogated for all genes. In each case, genomic DNA from leukemic blasts was compared with DNA from a skin biopsy from that patient. The median age of the 30 patients with t-AML was 49.2 years (25-77) with M:F ratio 13:17. The mean blast % was 81% (31-100). Consistent with previous reports, many of these patients had an abnormal karyotype with abnormalities of chromosome 5 and 7, 13% and 17% respectively. As expected, CGH analysis confirmed copy number alterations already identified by cytogenetics. In addition, we identified a single t-AML sample (from a male patient) that carried a small (435 kb) hemizygous deletion of miR-223 on chromosome × that was not apparent by cytogenetics. Quantitative PCR of genomic DNA confirmed the loss of the miR-223 gene, and real time RT-PCR demonstrated loss of miR-223 expression. Of note, miR-223 has been implicated in the regulation of granulopoiesis, and mice lacking miR-223 display a myeloproliferative phenotype (Johnnidis, Nature 2008). We screened an additional 27 AML patients for miR-223 expression and identified 3 other samples with miR-223 expression 2 standard deviations below the normal (based on CD34+ cells from healthy donors). No copy number alterations in miR-223 were detected in these patients, suggesting epigenetic silencing of miR-223. Consistent with this possibility, one of these patients carried a t(8;21), which has been shown to epigenetically silence miR-223 (Fazi, Cancer Cell 2007). The mechanism by which miR-223 is silenced in the other two AML samples is under investigation. In summary, cytogenetically silent deletions of miRNA genes are uncommon in t-AML. Loss of miR-223 expression can occur through somatic mutation or epigenetic silencing and is likely to contribute to leukemic transformation in a subset of patients with AML. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Akt, a serine/threonine protein kinase, is a critical regulator of cellular apoptosis, proliferation, and differentiation. Constitutive activation (phosphorylation) of Akt is frequently observed in various types of cancer, and may represent a therapeutic target. Akt inhibitors, such as perifosine, are in clinical development. We evaluated the expression of phosphorylated Akt (pAkt) in patients with newly diagnosed ALL. Methods: Between 1998 and 2005, patients with newly diagnosed ALL and an available diagnostic bone marrow biopsy performed at the Cleveland Clinic Foundation were evaluated. B5-fixed bone marrow core biopsies were reviewed for areas with the highest concentration of blasts. A tissue microarray was constructed using 1 mm tissue cores. The cores were arrayed in duplicate in the majority of samples, with a few samples having only 1 core. Immunohistochemistry was performed for pAkt with a monoclonal antibody to pAkt (Rockland Immunochemicals), using automated stainers and heat induced epitope retrieval. pAkt staining of blasts was estimated in 10% increments. The pattern of staining for pAkt was classified as positive (activated) if nuclear or cytoplasmic staining occurred in more than 10% of the blasts. Results: Eighty-eight patients with newly diagnosed ALL were treated at our institution during this time. Forty-five patients were evaluable. Thirty-three cases were not evaluable for the following reasons: bone marrow cores not available (9), bone marrow aspirate performed at an outside hospital (20), bone marrow aspirate sample not sufficient (1), and diagnostic bone marrow aspirate not performed (3). The median age was 37 [range 18–81]. Sixteen patients (35%) had poor risk cytogenetics, 12 (27%) normal karyotype, 9(20%) miscellaneous abnormalities, and 8 (18%) unknown cytogenetics as defined by CALGB criteria. Forty-one patients (91%) had precursor B-cell ALL, and 4 patients (9%) precursor T cell ALL. The median white count was 6.6 × 109/L [range 0.93–278], and median LDH 772 U/L [range 155–4608]. The expression of pAkt was characterized predominately by cytoplasmic staining with 8 cases exhibiting nuclear staining. Akt was activated (phosphorylated) in 32 out of 45 patients (71%) with newly diagnosed ALL. The median percentage of blasts with pAkt in the cytoplasm was 30%, with 19 patients (42%) having ≥50% blasts with cytoplasmic pAkt. There was a trend towards more patients with Philadelphia chromosome (Ph) positive ALL having ≥50% blasts with pAkt in the cytoplasm (7/10 patients) versus patients with other cytogenetic abnormalities (12/33) (p=0.079). Conclusions: Akt was activated in the majority of patients with newly diagnosed ALL. Akt may be a therapeutic target in ALL, and especially for patients with Ph+ ALL. Larger studies will be needed to define the prognostic significance of Akt activation in ALL.

Description: Background: This clinical trial addressed feasibility and safety of microtransplantation by HLA-mismatched allogeneic cellular therapy (HMMACT) in poor risk acute myeloid leukemia patients. A secondary objective was to estimate complete response rate, infections, GVHD incidence, and induction mortality. Methods: Patients with high risk AML were enrolled: 4 were 〉 75 yo, 3 with MLL+ ((t(6;11), 11+ and t(10;11)), 3 complex/del7, one FLT3+. Patients received induction chemotherapy with mitoxantrone (10 mg/m2 IV for 3 days) and cytarabine (150 mg/m2 IV for 7 days) and received HLA-mismatched GCSF mobilized PBSCs on day 9 or 10. Family donors were concurrently HLA typed and best available donor underwent GCSF mobilization (5 mg/kg SQ BID x 4 days) and leukapheresis after medical evaluation and safety testing. HLA partial mismatch patients (4- 5/10 antigen) were chosen. Apheresis cells were counted and analyzed by flow cytometry for CD34+ cells; CD3+, CD4+CD25+ and CD8 + cells; and CD3-CD56+ NK cells. Target CD3 cell dose was 1 x 108/kg per cycle; cells cryopreserved as for standard stem cell donors. Family donor HLA-mismatched GCSF mobilized peripheral blood cells were infused fresh on day 9 or 10 (up to day 16), 36-50 hours after end of cytarabine. Bone marrow biopsy was evaluated on day 14 and 28 for AML remission status, and T/NK cell number. Patients achieving a complete response proceeded to consolidation with cytarabine 0.5 -1.0 gram/m2 x 6 doses with fresh or cryopreserved HMMACT cells from their donor for 2 courses a month apart. Patients: 10 patients age 31 – 80 yo consented: 8 received allo cells; 1 screen failure (no haplo family member identified); 1 patient too ill after initiating chemo to collect donor. Eight patients received at least one course of HMMACT; 1 withdrew early due to AML progression, a second for poor performance status. Three patients had de novo AML; 5 patients had secondary AML. Six Patients had2 or more cycles of HMMACT: four achieved CR: Pt 1 and 4 received 3 cycles, Pt 2 and 6 received 2 cycles of cell infusions and all achieved CR. Pt 9 received 2 inductions with cells, and sustained a partial clinical remission for 6 months. Two Patients had 1 cycle of HMMACT:Pt 3: received 1 cycle cells, but response not assessed; Pt 8: received 1 cycle cells, and achieved CR 3+mos but declined further therapy. Safety: Acute reactions to cell infusions were minimal. Delayed reactions attributed to cell infusions included fever grade 1-2, rash grade 1-2, diarrhea grade 1-2 resolving in 7-10 days. Patients with prior MDS or refractory leukemia requiring 2 inductions had a longer time to ANC and platelet recovery. Feasibility: Two patients had no donor; 2 donors only collected enough for 2 cell infusions. Response: 5/8 pts receiving cells had CR/CRi (62.5%) lasting 3 to 10+ months. Conclusions: Although there were the usual significant complications of treating leukemia and infections, the cellular therapy itself was well-tolerated. Patients often had fevers in first week after cell infusions, or experienced transient rash and diarrhea in the same time period, which were self resolving in all cases and may reflect elimination of allogeneic cells by the patient’s immune system. No patient developed GVHD. Feasibility of obtaining family donors was as expected for this diverse US population. Infectious complications were low by AML treatment standards. Complete remission rates are encouraging, but not as durable as hoped, likely reflecting the fact that all of our patients had high risk cytogenetics in contrast to the prior published studies by Chinese investigators. The majority were elderly who would not otherwise be offered intensive therapy. Family donors achieved expected cell targets and tolerated mobilization/collection procedures well. All but one patient receiving 2 to 3 cycles achieved complete remission. Two elderly patients who received one or two cell infusions during induction sustained prolonged survival of 6 months in spite of no further therapy, one achieving a CR and the other a sustained PR. Neutrophil and platelet recoveries tended to follow the pattern of the patient's prior secondary leukemia or myelodysplastic disorder. Once patients achieved complete remission however, recovery of blood counts was relatively rapid on further cycles of consolidation. We are now assessing Treg, T and NK cell phenotype of patient and donor cells by flow cyAtometry as biological correlates. Disclosures Chaudhary: University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

Description: Only 30% of adults with ALL are cured. The identification of modifiable prognostic factors is important in designing future treatment paradigms, and improving the outcome of patients. PRT is integral in the treatment of ALL, and eradicates minimal residual disease (MRD) after complete remission (CR) has been achieved with induction chemotherapy (IC). Decreasing the time from the start of IC to the initiation of PRT may improve prognosis by eradicating MRD at an earlier stage, and preventing the development of resistant leukemic clones. The goal of this study was to retrospectively evaluate the impact of time from the start of IC to PRT, and determine whether or not this affects progression-free survival (PFS) and overall survival (OS). Methods: We retrospectively evaluated adults with newly diagnosed ALL treated at CCF between the years 1996–2005. 116 patients with ALL were seen. 57 patients were newly diagnosed and received IC and PRT. Cox proportional hazards analysis was used to identify univariate and multivariate correlates of CR, OS, relapse after remission, and PFS. Only variables significant in the univariate setting were included in multivariate analysis. Results were summarized as the hazard ratio (HR) with 95% confidence intervals (CI). The following variables were used in the analysis: pre-treatment characteristics (age, WBC, cytogenetic (CG) risk group, LDH), time to WBC recovery (time from the initiation of IC to an ANC of 500 after IC), CD20 expression, and time from the initiation of IC to start of PRT. CG risk was defined by CALGB criteria. Results: Characteristics at diagnosis: median age of 38 years [range 16–72], median LDH 673 U/L [112–5753], and median WBC 17.8 × 103/L [1.2–364]. 33.6% of patients had poor risk CG, 22.4% normal CG, 15% miscellaneous, and 29% unknown. Most patients received a vincristine/prednisone based IC (88%). However, 12% received high dose cytarabine/mitoxantrone IC. The CR rate was 75.3% for patients age 〈 60, and 60% for age ≥ 60. Median disease-free survival was 20.2% at 5 years. The median time from IC to PRT was 7.0 weeks [4.1–17.0]. On univariate analysis, increased age and increased time to WBC recovery were associated with a lower CR rate. Age and time from IC to PRT (per week increase (PWI)) [HR 1.17(CI 1.04–1.32), p=0.009] were significant predictors for relapse after remission. Increased age, poor risk CG, and time from IC to PRT (PWI) [HR 1.13(1.02–1.25), p=0.024] predicted decreased PFS. All of these factors (including time to WBC recovery) predicted decreased OS, with time from IC to PRT (PWI) having a HR of 1.11[(1.01–1.23), p=0.039]. On multivariate analysis, there was a trend for longer time from IC to PRT (PWI) [HR 1.53(0.92–2.54, p=0.10] predicting decreased OS and increased chance of relapse (PWI) [HR 1.12(0.98–1.29), p=0.09]. When patients age 〉 60 and poor risk CG were excluded, time from IC to PRT (PWI) was the only factor associated with decreased OS [HR 1.34(1.08–1.67), p=0.009], PFS [HR 1.27(1.04–1.55, p=0.019)], and an increased chance of relapse [HR 1.26(0.99–1.61), p=0.06]. Conclusions: Strategies to improve the prognosis of ALL are needed. Time from IC to PRT is an independent prognostic factor for treatment outcome, and administering PRT on time may improve our outcomes in adult patients with ALL.

Description: Abstract 271 MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have been implicated in the pathogenesis of human cancer. Most current studies utilize array-based or quantitative reverse-transcription-polymerase chain reaction (RT-PCR) approaches to measure miRNA expression. However, these approaches do not interrogate all known (or predicted) miRNAs and are unable to detect mutations in miRNAs. Herein, we use “next generation” sequencing approaches to comprehensively assess miRNA expression and to identify genetic variants of miRNA genes in a patient with AML. This patient (UPN 933124) was a 56 year old female with FAB M1 AML. Routine cytogenetics revealed a normal 46 XY karyotype, and high-resolution comparative genomic hybridization studies revealed no somatic copy number alterations at a resolution of ∼5kb. We previously reported the sequence of genic regions in the cancer genome of this patient (Nature 456:66, 2008). Massively-parallel sequencing of small RNAs isolated from the myeloblasts of patient 933124 was performed using the ABI SOLiD sequencing platform. As a control, we also analyzed pooled RNA isolated from CD34+ bone marrow cells of 4 healthy volunteers. In each case, RNA was size fractionated (corresponding to RNAs of 19-26 nucleotides in length) to enrich for miRNAs prior to sequencing. A total of 28 ×106 sequence reads from AML 933124 and 20×106 reads from the pooled normal CD34+ cells were obtained. Expression of 498 and 458 known miRNAs were detected in AML 933124 and CD34 cells, respectively. MiR-233 was the most highly expressed miRNA in both AML 933124 and CD34; remarkably, it represented 47.3% of all miRNA reads in AML 933124. To determine if extremely high miR-223 expression is a consistent feature of AML, we performed real time RT-PCR for miR-223 in an additional 23 AML samples and in CD34+ cells from 4 healthy donors. Of note, to avoid underestimating miR-233 expression, significant dilution of the RNA samples was required to ensure that the RT-PCR assay for miR-223 was in the linear range. Compared with normal CD34+ cells, miR-223 expression in AML 933124 was increased 6.8-fold. However, increased miR-223 expression was not a common feature of AML, with only 2 of 23 AML samples (both therapy-related AML) showing increased miR-223 expression. A large number of sequence reads mapped to unannotated regions of the genome. Using an in-house program developed to view predicted RNA structure, more than 10 novel putative miRNAs were identified, some of which were differentially expressed in AML 933124 compared with normal CD34+ cells. To detect genetic variants of miRNA genes, we designed 454-amplification and sequencing primers to sequence all 695 miRNA genes that were in the Sanger miR database (version 12.0). We sequenced approximately 200 bp flanking the mature miRNA (total ∼400 bp per miRNA gene) to ensure that mutations affecting the primary miRNA were detected. To differentiate germline polymorphisms from somatic mutations, genomic DNA from both leukemic blasts and a skin biopsy from this patient were sequenced. Average sequence coverage depth was 52.2X, and 95% of miRNAs had at least one supporting read. Thirteen single nucleotide variants and 3 Indels in miRNA genes were detected. All were present in the skin DNA sample, suggesting that they represent germline polymorphisms. Finally, we analyzed the previously generated whole genome sequence for this AML genome for genetic variants in the 3'-untranslated region of all coding genes. A single somatic mutation in the 3'-UTR of TNFAIP2 was detected. This mutation is predicted to disrupt the binding of several expressed miRNAs. However, no recurrent mutations in the 3'-UTR of TNFAIP2 were detected in an additional 180 patients with AML. Thus, the contribution of this somatic mutation to the pathogenesis of AML is unclear. These data demonstrate the feasibility of ‘next generation' sequencing technologies to identify novel miRNAs, accurately measure mature miRNA expression, and identify both somatic and germline genetic variants of miRNA genes in primary cancer. Using this platform, studies are underway to comprehensively characterize miRNAs in additional human AML samples. Disclosures: No relevant conflicts of interest to declare.

Description: Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophil granulocytes. Mutations in ELANE, GFI1, HAX1, G6PC3, WAS, and VPS45 are known to cause SCN. We here describe a new monogenetic SCN variant with biallelic mutations in the gene encoding Jagunal homolog 1 (JAGN1). We studied an index family from Northern Africa with a total of 5 children suffering from SCN. An Affymetrix SNP array-based genetic linkage analysis was performed and identified a single interval of perfect segregation with highly significant multi-marker LOD scores of at least 4.5spanning approximately 1.5Mbp from 9.52Mb to 11.04Mb on chromosome 3 of NCBI’s human genome build 36.3. This interval contained a total of 30 genes, including JAGN1 which encodes an ER-resident protein. Sanger sequencing revealed a homozygous mutation c.3G〉A in exon 1 of the JAGN1 gene; this mutation leads to disruption of the defined start of translation. Systematic analysis of a cohort of 90 SCN patients identified 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients, thus accounting for approximately 10% of SCN patients. The clinical phenotype was variable and included failure to thrive, developmental delay and bone skeletal abnormalities. The only consistent finding in all JAGN1-deficient patients was SCN and partial or complete refractoriness to therapy with rh-GCSF. JAGN1 is the human ortholog of a gene originally identified in Drosophila melanogaster. Jagunal-deficient oocytes are characterized by defective ER reorganization and aberrant membrane trafficking during vitellogenesis. We found that JAGN1-mutant human granulocytes showed aberrantly enlarged ER structures and paucity of secretory vesicles. We hypothesized that that ER aberrations may be associated with defective N-glycosylation of multiple proteins in neutrophil granulocytes and found that JAGN1-mutant neutrophil granulocytes exhibited anomalous N-glycomic profiles characterized by a marked reduction in fucosylation of all their multi-antennary glycans. JAGN1-deficient neutrophil granulocytes showed increased apoptosis in response to TNFa and staurosporine, likely accounting for the lack of mature neutrophils in these patients. Additional studies in JAGN1-knockdown cells indicate that JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. Global proteomic analysis of the JAGN1-interactome identified a limited number of interaction partners including members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) which suggest a role for JAGN1 in vesicular trafficking from Golgi to ER. Taken together, JAGN1 emerges as a hitherto unrecognized factor necessary in differentiation and survival of neutrophil granulocytes and a novel gene implicated in SCN. Disclosures: No relevant conflicts of interest to declare.