ALBERT

Description: Key Points The genetic cause of SCID impacts on survival and immune reconstitution and should be considered in tailoring HCT for individual patients. Total and naive CD4+ cell counts in SCID patients 6 and 12 months post-HCT predict long-term survival and sustained immune reconstitution.

Description: Background: CD19-directed chimeric antigen receptor (CAR) T cell therapy is FDA approved for relapsed/refractory diffuse large B cell lymphoma (R/R DLBCL) and variants. Due to active lymphoma, "bridging" therapy may be needed to maintain disease control during the 3 or more weeks required for autologous CAR T manufacturing. Bridging therapy was not allowed in trials of axicabtagene ciloleucel (axi-cel), but could improve outcomes by reducing tumor burden. Bridging chemotherapy is one option, however toxicity and chemo-refractory disease are potential concerns. Here we report a case series of patients receiving bridging radiation therapy (XRT) prior to axi-cel therapy, of which the majority had double hit lymphoma. Patient Characteristics: As of July 31, 2018 eight patients received XRT as part of their bridging therapies. Overall, patients had a median of 3 prior lines of chemotherapy (range 2 to 5). Two were primary refractory to their first two lines of chemotherapy. Six of the 8 patients had double hit lymphoma by FISH. All patients received 3-4 Gy per fraction to a total median dose of 20 Gray (range 9 - 30 Gy). The most commonly used regimen was 30 Gy in 10 fractions (3/7 patients). Four patients received bridging chemotherapy in addition to XRT, mainly oral cytoxan. In 2 patients the XRT was given up until the day of leukapheresis and in 1 of these patients it was continued after leukapheresis. In 6 additional patients the XRT started after leukapheresis. Overall, XRT was completed at a median of 9 days (range 3 - 22 days) prior to starting Flu/Cy conditioning. The median mass diameter receiving XRT was 13.6 cm (range 3 - 30 cm). Outcomes: No significant adverse events were noted at the local site of XRT before or after CAR T infusion. Response assessment within the field of radiation is complicated by a short duration between end of XRT and restaging prior to Flu/Cy conditioning. Nonetheless, only one patient experienced progressive disease in-field after XRT and most had stable disease in-field. Of the 4 cases where the baseline LDH was elevated, only the patient with PD saw their LDH rise after the completion of XRT, while the other 3 of 4 patients had declining LDH at the time of Flu/Cy conditioning. In 7 patients evaluable for toxicity, there was one case of severe cytokine release syndrome (CRS). This event occurred in the patient who progressed through XRT and had rising LDH and declining performance status prior to CAR T infusion (this patient later died of infection). In the other 6 patients the maximum CRS was grade 1 or 2. Three out of the 7 patients had grade 3 or 4 CAR T related encephalopathy syndrome (CRES). For lymphoma outcomes at day 30, 1 patient was in CR, 2 patients PR, 2 patients with SD and 1 patient with PD (Lugano criteria). Two patients are evaluable at day 90 with PR, although one of these patients subsequently relapsed at month 4 (in-field to the prior radiation). Conclusions: Bridging XRT (with or without chemotherapy) can be safely administered and provides disease control in refractory poor risk DLBCL patients prior to axi-cel therapy. All patients were able to receive axi-cel. CAR T-related toxicity occurred, but in this case series was most likely related to disease aggressiveness (6 of 8 patients were double hit) and tumor burden. Further research should focus on the immunologic effects of XRT and its impact on CAR T cell efficacy and toxicity. Table. Table. Disclosures Chavez: Humanigen: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Merck: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Davila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Locke:Cellular BioMedicine Group Inc.: Consultancy; Kite Pharma: Other: Scientific Advisor; Novartis Pharmaceuticals: Other: Scientific Advisor.

Description: Background: The Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) is a complex disorder with a wide range of disease severity and unique hematological and immunological manifestations. Based on this complexity, several approaches are available to these patients, including observation, symptomatic treatment, splenectomy, gene therapy (GT), or allogeneic hematopoietic stem cell transplantation (HSCT). In many instances more than one of these therapeutic options may seem appropriate for any given patient. A prospective, randomized study comparing the pros and cons of these therapeutic options in WAS/XLT would be desirable, but is not feasible due to the rarity and variable severity of the disease, as well as the need for long-term follow-up. Methods and Definitions: We retrospectively assessed via an international, anonymized, file-based survey the consequences of different therapies based on the severity of the disease phenotype and how these therapies affected patients' quality of life as perceived by their treating physician. The frequency of disease- and therapy-related complications with respect to the specific treatment was recorded. "Severe events" were defined as: all fatal events or sepsis, meningitis, pneumonia requiring respiratory support, systemic viral/fungal infections or serious bleeding episodes (intracranial and gastrointestinal) requiring transfusion support. Allogeneic HSCT, splenectomy and GT were defined as "procedures", HSCT and GT as "definitive". Results: A total of 575 patients with a documented WAS gene mutation from 51 centers in 27 countries with a median follow-up of 7.4 years (range: 0.2-75.6), resulting in 5632 patient years, were included in the study. Of these, 240 (42%) carried missense, 67 (12%) nonsense, 90 (16%) splice-site mutations, 77 (13%) deletions, 40 (7%) insertions and 61 (11%) had incomplete or inconclusive mutation information. An allogeneic HSCT was performed in 252 (44%), splenectomy in 78 (14%), GT in 14 (2%) patients, while 264 (46%) patients never had a procedure. At the time of last follow-up or before the first procedure the WAS disease severity score was 1 in 55 (10%), 2 in 144 (25%), 3 in 161 (28%), 4 in 109 (19%) and 5 in 86 (15%) patients. Overall survival of the entire cohort (censored at the time of first definitive procedure, thereby representing the "natural" disease outcome) was 82% (95% confidence interval 78-87) at 15 years and 70% (61-80) at 30 years of age. Ten year overall survival after HSCT was 80 % (74-85). The cumulative incidence (CI) of severe bleeding, severe infection, autoimmunity or malignancy in patients without a procedure at last follow-up or censored before the first procedure was 45% (39-50), 61% (55-66), 46% (40-52) and 31% (25-37) respectively at 15 years of age and 61% (51-69), 70% (62-76), 62% (52-70) and 45% (35-53) at 30 years. The frequency of definitive procedures (HSCT or GT) increased in patients with higher WAS scores, while better natural disease outcomes were associated with lower WAS scores. Overall quality of life (QoL) as perceived by the treating physician was very good, good, limited or unacceptable in 85/457 (19%), 172/457 (38%), 176/457 (39%) and 24/457 (5%) of patients without or before a procedure respectively. QoL was also strongly correlated with the WAS score. At last follow-up after successful HSCT QoL improved to very good in 123/184 (67%), good in 47/184 (26%), limited in 12/184 (7%) and unacceptable in 2/184 (1%). Splenectomy also had a favorable effect on QoL with 16/52 (31%) very good, 24/52 (46%) good, 9/52 (17%) limited and 3/52 (6%) unacceptable. Platelet counts improved from a baseline mean of 36G/l to 91G/l after GT, 159G/l after splenectomy and 204G/l after HSCT. Conclusion: This study presents outcome data of the largest cohort of patients with a WAS gene mutation studied so far and confirms the anticipated spectrum of disease severity and the curative effect of HSCT. The data show that untreated patients with WAS suffer from increasing rates of disease-associated complications over time which correlates well with a significant reduction of QoL. Both HSCT and splenectomy have a positive effect on physician-perceived QoL. Due to the large cohort size this study's data will allow us to assess the influence of specific genotypes on outcome in WAS (analysis ongoing), possibly allowing for more individualized treatment recommendations in the future. Disclosures Albert: GSK: Research Funding.

Description: Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio

Description: Recurrent somatic mutations in core components and modulators of the cohesin ring - a multimeric protein complex that forms a ring structure around DNA and provides spatial genome organization - have been identified across multiple cancer types, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), where they are associated with poor overall survival. Cohesin proteins are involved in sister chromatid cohesion, chromatin organization into loops, transcriptional activation, and DNA damage repair. The mechanisms underlying clonal expansion of these driver mutations are unknown and no therapies have selective efficacy in cohesin-mutant cancers. We sought to determine the effects of mutations in the most frequently mutated cohesin subunit, STAG2, on cohesin complex composition using immunoprecipitation followed by quantitative mass spectrometry (IP-MS), genetic dependencies of STAG2-mutant cells by genome-wide CRISPR screening, and mutant cohesin association with chromatin using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Our goal was to understand how these mutations contribute to cellular transformation and to identify possible therapeutic targets. Applying IP-MS in AML cell lines engineered with different STAG2 mutations, we identified and validated a switch from STAG2- to its paralog STAG1-containing cohesin complexes. In addition, we observed changes in the interaction of the mutant cohesin complex with proteins involved in DNA repair and replication, including PARP1, and RNA-mediated interaction with RNA splicing machinery, including SF3B family members. We next hypothesized that these cohesin-dependent alterations could lead to shifts in genetic dependencies. Using genome-scale CRISPR-Cas9 screens, we identified preferential dependency of STAG2-mutant cells on STAG1, consistent with our proteomics studies. We also found a striking concordance between additional cellular processes highlighted by IP-MS experiments and observed increased dependency of STAG2-mutant cells on DNA damage repair and mRNA processing. Therefore, STAG2 mutations lead to changes in cohesin complex structure and alter interactions with proteins involved in DNA damage, replication, and RNA modification, which become genetic dependencies in this context. Prompted by this concordance, we evaluated DNA replication, DNA damage and splicing in cohesin-mutant cells. We observed a 4-fold increase in replication fork stalling in STAG2-mutant cells, which was associated with accumulation of double strand DNA breaks and activation of the ATR and ATM DNA damage checkpoints. STAG2-mutant cells demonstrated ~100-fold increased sensitivity to the PARP inhibitor talazoparib, which was consistent across models of other cohesin-mutant subunits. In addition, cohesin-mutant cells showed aberrant splicing and increased sensitivity to treatment with SF3B1 inhibitors E7107 and H3B-8800. In aggregate, genetic or pharmacologic perturbation of DNA damage repair or splicing created a synthetic vulnerability for cohesin-mutant cells in vitro and in vivo. Finally, we explored how STAG1-containing complexes alter cohesin-mediated genome compartmentalization in cohesin-mutant cells. Using ChIP-Seq, we observed that STAG2 loss leads to a global decrease in cohesin binding to chromatin, including at sites of insulated neighborhood boundaries, with subsequent gene expression changes. Loss of cohesin binding was associated with increased enhancer activity and super-enhancer expansion in STAG2-mutant cells. In addition, we identified changes in the co-localization of the mutant cohesin complex with super-enhancer enriched factors, DNA damage repair and splicing machinery. These findings are consistent with a model in which wild type and mutant cohesin complexes, defined by their unique composition and patterns of chromatin binding and architecture, have differential abilities to maintain chromatin organization as it relates to spatial organization of super-enhancers, coactivators and transcription factors, as well as DNA damage repair and splicing machinery. Perturbation of any of these components, which have been recently proposed to form phase-separated nuclear bodies, creates vulnerabilities that may be exploited therapeutically with existing drugs in patients with cohesin-mutated malignancies. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. D'Andrea:Intellia Therapeutics: Consultancy; Cedilla Therpeutics: Consultancy, Equity Ownership; EMD-Serono: Consultancy, Research Funding; Sierra: Consultancy, Research Funding; Ideaya: Consultancy, Equity Ownership; Lilly: Consultancy, Research Funding; Formation Biologics: Consultancy. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: BACKGROUND: CD19-specific chimeric antigen receptor (CAR) T-cell therapy has proven to be highly effective in patients with relapsed or refractory large B-cell lymphomas, yielding early complete response (CR) rates of ~40%, which are typically sustained. Unfortunately, most patients will not experience prolonged disease control. Despite this fact, little data exist defining the outcomes and impact of subsequent therapies for such individuals. Limited data also exist on the ability for such patients to pursue further clinical trials or allogeneic hematopoietic stem-cell transplant (HSCT). This project details the specific interventions and outcomes of this population to better inform the management of patients who suffer progressive disease (PD) after CD19-specific CAR T-cell therapy. METHODS: Adults with diffuse large B-cell lymphoma (DLBCL), transformed follicular lymphoma (tFL), primary mediastinal B-cell lymphoma (PMBCL), and high-grade B-cell lymphomas (HGBCL) who received CD19-specific CAR T-cells at the University of Washington/Seattle Cancer Care Alliance were included in this analysis. Patients who received CAR T-cell therapy in conjunction with additional protocol-specified therapy were excluded. Those who exhibited PD or persistent lymphoma after CAR T-cell therapy were the focus of this study. We defined initial PD as patients who had evidence of disease progression on the initial response assessment. Delayed PD was defined as achieving a CR, partial response (PR), or stable disease (SD) on the initial response assessment, but eventually progressed or received subsequent anti-lymphoma therapy. Baseline characteristics and all data were retrieved from the electronic medical record up until date of death or date of last contact in our system, including subsequent interventions and outcomes. Primary endpoint of this analysis was overall survival (OS). RESULTS: Between October 2013 and May 2018, we identified 51 patients with PD following CD19-specific CAR T-cell therapy. Baseline characteristics are listed in the Table 1. Histologies included DLBCL (29), HGBCL (11), tFL (8) and PMBCL (3). Median age was 60 years (range 26-75), 65% were male, median prior regimens was 3 (range 1-8). Median time from CAR T infusion to PD was 42 days (range 11-609), with 27 (53%) patients exhibiting initial PD. Median follow up after time of progression was 4.2 months. Initial PD was associated with a higher risk of death (HR 2.376, 95% CI 1.19-4.75, p=0.0143, Figure 1). The median OS for those with initial PD and delayed PD was 5.1 months (95% CI 2.0-9.3) and 13.6 months (4.1-not reached) respectively. 39 (76%) patients received ≥ 1 subsequent therapies after PD. Initial therapies included: 2nd CAR T infusion (14), targeted therapy (10), chemotherapy +/- rituximab (7), other immunotherapy (3), radiotherapy (3), intrathecal chemotherapy (1) and allogeneic HSCT (1). 12 (24%) patients received no further therapy despite PD. Those who received ≥ 1 subsequent therapies after PD had a lower risk of death (HR 0.344, 95% CI 0.149-0.793, P=0.0122) compared to those who did not. There was no difference in survival if 2nd CAR T infusion was the next line therapy compared to others (p=0.449), targeted therapy compared to others (p=0.417), or chemotherapy compared to others (p=0.565). 5 (10%) patients enrolled onto a clinical trial as next line therapy. 4 (8%) patients eventually received an allogeneic HSCT after PD, 2 of whom are still alive. We identified 8 patients who were alive for ≥ 12 months after progression without evidence of lymphoma. Last line of therapy for these patients included allogeneic HSCT (2), subsequent CD19-specific CAR-T cell infusion (2), ibrutinib (2), lenalidomide/rituximab (1), and radiotherapy (1). CONCLUSIONS: Patients with PD post anti-CD19 CAR T-cell therapy, particularly those exhibiting initial PD, have poor long-term outcomes. Patients receiving at least one anti-lymphoma therapy after PD had improved overall survival, although no single approach appeared to confer a survival benefit. Few enrolled onto a clinical trial or received an allogeneic HSCT. These data reinforce the need to both further improve the durable CR rate after CAR T-cell therapy and to develop effective strategies for those not achieving a CR. Figure 1 Figure 1. Disclosures Gopal: Spectrum: Research Funding; Pfizer: Research Funding; BMS: Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Research Funding; Takeda: Research Funding; Brim: Consultancy; Janssen: Consultancy, Research Funding; Asana: Consultancy; Gilead: Consultancy, Research Funding; Aptevo: Consultancy; Incyte: Consultancy; Teva: Research Funding. Maloney:Juno Therapeutics: Research Funding; Roche/Genentech: Honoraria; Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria; GlaxoSmithKline: Research Funding. Turtle:Caribou Biosciences: Consultancy; Adaptive Biotechnologies: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Bluebird Bio: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptevo: Consultancy; Gilead: Consultancy. Smith:Genentech: Research Funding; Acerta Pharma BV: Research Funding; Incyte Corporation: Research Funding; Merck Sharp and Dohme Corp.: Consultancy, Research Funding; Pharmacyclics: Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding. Shadman:TG Therapeutics: Research Funding; Mustang Biopharma: Research Funding; Acerta Pharma: Research Funding; AstraZeneca: Consultancy; Verastem: Consultancy; Gilead Sciences: Research Funding; AbbVie: Consultancy; Qilu Puget Sound Biotherapeutics: Consultancy; Beigene: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Genentech: Consultancy; Celgene: Research Funding. Cassaday:Seattle Genetics: Other: Spouse Employment, Research Funding; Incyte: Research Funding; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy, Research Funding; Kite Pharma: Research Funding; Merck: Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy. Till:Mustang Bio: Patents & Royalties, Research Funding. Shustov:Seattle Genetics: Research Funding. Acharya:Juno Therapeutics: Research Funding; Teva: Honoraria. Lynch:Takeda Pharmaceuticals: Research Funding; T.G. Therapeutics: Research Funding; Rhizen Pharmaceuticals S.A.: Research Funding; Johnson Graffe Keay Moniz & Wick LLP: Consultancy; Incyte Corporation: Research Funding.

Description: Mitochondrial DNA (mtDNA) replication requires adequate nucleotide pools from the mitochondria and cytoplasm to support DNA biosynthesis. Gene expression profiling of 542 AML patient samples (GSE13159) demonstrated that 55% of AML patients had upregulated mtDNA biosynthesis pathway expression compared to 73 normal hematopoietic cells (mononuclear cells isolated from peripheral blood and bone marrow). We also identified upregulation of pathways which support mitochondrial nucleotide pools, which include mitochondrial nucleotide transporters and a subset of cytoplasmic nucleotide salvage enzymes, which phosphorylate nucleosides to nucleotides. Upregulation of nucleoside kinases in a subset of primary AML samples compared to normal hematopoietic progenitor cells (normal G-CSF (granulocyte-colony stimulating factor) mobilized peripheral blood stem cells (PBSC's)) was confirmed by immunoblotting. These results suggest that AML cells import cytoplasmic nucleotides to support mitochondrial DNA biogenesis. To determine if cytoplasmic nucleoside kinases regulate mtDNA content, we knocked down nucleoside kinases in AML cells. Partial target knockdown of DCK (deoxycytidine kinase) and CMPK1 (cytidine/uridine monophosphate kinase 1) reduced mtDNA content (60+8%, and 62+13%, respectively compared to controls, 5 and 7 days post-shRNA transduction), indicating a role in mtDNA biogenesis. As expected, knockdown of mtDNA replication factors POLG and TFAM reduced mtDNA content in AML cells. The cytidine nucleoside analog, 2'3'-dideoxycytidine (ddC) is activated by DCK and CMPK1 to produce its triphosphate form, ddC-triphosphate (ddC-TP). To assess nucleoside kinase activity, primary AML and normal hematopoietic cells were treated with ddC and total levels of ddC and ddC-TP were measured by mass spectrometry. Levels of ddC did not differ between AML and normal, but ddC-TP levels was increased in AML samples 〉 7-fold compared to normal (p〈 0.05, one-way ANOVA). Previously we and others demonstrated that AML cells and stem cells have increased mitochondrial biogenesis and reliance on oxidative phosphorylation due to decreased spare reserve capacity and an inability to upregulate glycolysis. ddC-TP inhibits the sole mtDNA polymerase POLG, but not nuclear DNA polymerases. Given the increased activity of nucleoside kinases in AML cells over normal, we examined the effects of ddC treatment on mtDNA content and cellular bioenergetics. AML and normal cells were treated with increasing concentrations of ddC. At increasing times after treatment, ddC depleted mtDNA levels 〉 85% at 0.5 uM, 3 day treatment in OCI-AML2 and TEX cells as assessed by qPCR. ddC decreased protein expression of mtDNA encoded electron transport chain (ETC) subunits COXI and COX II, but not nuclear encoded subunit COXIV) and reduced basal oxygen consumption. ddC also decreased proliferation of AML cell lines (OCI-AML2, TEX, HL-60, K562) (〉 95% reduction at 0.5uM, 10 days). Knockdown of DCK abrogated the effects of ddC on AML cell proliferation. We next examined the effects of ddC in primary human leukemia cells (AML = 7, CML blast crisis = 1, CMML-2 = 1) and normal hematopoietic progenitor cells (n=8). ddC preferentially inhibited mtDNA biosynthesis and reduced viability in a subset of primary cells (6 of 9 AML) compared to normal PBSC's (n=8). Sensitivity to ddC positively correlated with mtDNA depletion. Finally, we evaluated the efficacy and toxicity of ddC in mouse models of human AML. ddC (35 mg/kg daily i.p. x 11 days) caused tumor regression in an OCI-AML2 xenograft model without toxicity (changes body weight, behavior, serum chemistries). In OCI-AML2 cells isolated from treated mice, ddC reduced mtDNA by 95% and mtDNA-encoded ETC proteins by 90%. In addition, ddC (75 mg/kg i.p x 6 weeks) significantly reduced human AML bone marrow engraftment in primary AML (n=2, P

Description: Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

Description: Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p

Description: GNPAT (chromosome 1q42.2) encodes the peroxisomal enzyme glyceronephosphate O-acyltransferase. In a previous study, DNA of men with hemochromatosis and HFE p.C282Y homozygosity and either markedly increased iron stores or normal or mildly increased iron stores were evaluated with exome sequencing. Positivity for the GNPAT polymorphism p.D519G (rs11558492) was significantly greater in men with markedly increased iron stores (McLaren CE et al., Hepatology 2015;62:429-39). This result suggests that the p.D519G is a candidate modifier of iron phenotypes in p.C282Y homozygotes. To learn more, we examined associations of p.D519G, age, iron-related variables, and daily alcohol consumption with iron stores in p.C282Y homozygotes classified by extremes of iron overload phenotypes. We defined markedly increased iron stores as serum ferritin 〉1000 µg/L and either hepatic iron 〉236 µmol/g dry weight or mobilizable iron 〉10 g by induction phlebotomy (men and women). Normal or mildly elevated iron stores were defined as serum ferritin