ALBERT

Description: Copper deficiency has long been recognized as cause of hematopoietic dysfunction, and diagnosis is straightforward if it is pursued. Over a three year period, we diagnosed copper deficiency in seven pts referred to our university-based hematology and BMT outpatient clinics for evaluation and treatment of myelodysplastic syndrome (MDS) or neutropenia/anemia. This represents approximately 3% of new outpatient referrals carrying an ICD-9 code corresponding to MDS or neutropenia. Patient characteristics are shown in Table 1. Three of the seven pts carried a presumptive diagnosis of MDS, and one had received several months of decitabine therapy. Two patients were actually referred for consideration of stem cell transplantation; the others were referred for evaluation of cytopenias. Six of the seven patients had been evaluated by a hematologist prior to referral. Only 3 of 7 pts in our series had recognized risk factors for copper deficiency such as prior gastric bypass or other GI dysfunction; postulated risk factors in the other pts are included in Table1. All pts in our series were anemic and leukopenic, with absolute neutropenia at presentation. In contrast, platelet counts were normal or elevated in all pts, consistent with most previous reports. Five of the seven pts in our series had neuropathy or myelopathy of variable severity. The hematologic abnormalities responded rapidly and completely to oral and/or IV copper replacement in all pts, although neurologic recovery was slow and incomplete. Conclusions: Our experience indicates that copper deficiency is a relatively common cause of neutropenia and anemia, and the consequences of missing the diagnosis may be substantial, including performance of unnecessary transfusion, chemotherapy, and even stem cell transplantation. Normal or elevated platelet counts and concomitant neuropathy or myelopathy are important clues to the diagnosis. Furthermore, copper deficiency must be considered even in pts without obvious GI absorption abnormalities. Serum copper and ceruloplasmin assays should be incorporated into the routine evaluation of most pts with cytopenias and suspected MDS. Age/gender Ref. diagnosis Risk factor Associated symptoms Serum copper (80–155 mcg/dl) Ceruloplasmin (21–53 mg/dl 53F MDS–RAEB unknown Severe sensory-motor neuropathy 29 7 43F MDS–RA Excess carbonated soda intake? Severe sensory-motor neuropathy 2

Description: Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype.

Description: The second-generation TKIs (2G-TKIs) Dasatinib (DAS) and Nilotinib (NIL) yield faster responses in newly diagnosed chronic phase (CP) CML as compared to Imatinib (IM) however, long-term safety of these agents is a growing concern. We identified 10 patients with CP CML diagnosed between 08/2013 and 06/2015 who initiated 2G-TKIs and were then switched after optimal response at 3 months to IM. DAS was administered to 8 patients at 100 mg/d and NIL in two patients at 300 mg twice a day. Response to TKI was assessed by quantitative reverse transcriptase polymerase chain reaction (qPCR) for BCR-ABL1. Response to 2G-TKIs after 3 months was as follows: CHR (n=10), 1 log (

Description: Abstract 2576 Cerebrospinal fluid (CSF) involvement by leukemic blasts occurs in fewer than 10 % of adult patients with newly diagnosed acute lymphoblastic leukemia/lymphoma (ALL). Leukemic meningitis is diagnosed by microscopic detection of blasts in the CSF. Flow cytometry is a highly sensitive tool for detection of aberrant cells. We sought to analyze the additional benefit flow cytometry might provide for the diagnosis of leukemic meningitis. Between 11/2007 and 8/2011, 80 patients were diagnosed with ALL and treated at Emory University. 800 CSF samples were available for analysis 80 of which were collected from a diagnostic lumbar puncture (LP), 689 from follow-up LPs and 31 from LPs obtained at the time of relapse. As shown in the table, flow cytometry confirmed the presence of leukemic blasts in one, four and five samples diagnosed with leukemic meningitis by cytology at diagnosis, different stages of treatment and relapse, respectively. One and three samples were positive for leukemic blasts by cytology but negative by flow cytometry during different treatment stages and relapse respectively. We conclude that flow cytometry provided no additional benefit to cytology in the diagnosis of leukemic meningitis. Table: CSF Cytology and Flow Cytometry in 80 Adult ALL patients: CSF samples New Diagnosis N=80 Induction/Consolidation/Intensification/Maintenance/Remission/Post-transplant N = 689 Systemic relapse N = 31 N Cytology N = 80 Flow cytometry N = 66 Cytology N = 689 Flow cytometry N = 188 Cytology N = 31 Flow cytometry N = 13 Negative 79/80 65/66 684/689 184/188 23/31 8/13 Positive 1/80 1/66∼ 5/689 4/188∼* 8/31 5/13∼** ∼ CSF samples positive by flow cytometry were also positive by cytology * One CSF sample was positive by cytology but negative by flow cytometry ** CSF flow cytometry was not done in 3/8 positive CSF samples by cytology Disclosures: No relevant conflicts of interest to declare.

Description: Background: APL is a highly curable malignancy with reported survival above 90% in many co-operative group studies. However these spectacular results are not evident in the general population. US SEER data and other population based studies from Swedish Cancer Registry and Brazil showed that early deaths (ED) can be as high as 30%, leading to a considerably lower survival compared to clinical trials where ED is around 5-10%. Decreasing ED remains a global challenge and the highest priority at all leukemia treatment centers and will result in population wide survival in this most curable leukemia. We report results of our prospective trial using a set of simplified treatment guidelines along with expert support designed to decrease ED. Methods: A network of leukemia treatment centers was established in Georgia, South Carolina and neighboring states. An aggressive outreach effort was made by visiting most of the leukemia treatment centers to publicize the concept and educate treating physicians in the community about ED in APL. The protocol provides a simplified two page treatment algorithm that emphasizes quick diagnosis, prompt initiation of therapy and proactive and aggressive management of the major causes of death during induction. Expert and treating physician communication was established very early when a diagnosis of APL was suspected and was maintained until the completion of induction. Study was approved by local IRBs (if applicable) and funded by the Lymphoma Leukemia Society (LLS). Informed consent was obtained upon confirmation of a diagnosis of APL and there were no exclusion criteria. Patient accrual was initiated in July 2013 and continued till May 2016 when the accrual goal of 120 was met on an intent to treat basis. Statistics are descriptive. Results: Between 7/2013 and 5/2016, 120 patients were enrolled at 5 large leukemia centers (n=54, 45%) and 24 community hospitals (n=66, 55%). Only 3 hospitals treated more than 3 APL patients/year. Median age was 54 years (range 21-84 years). 68 were male. 84% were low risk (WBC 〈 10,000/mm3) and median WBC count was 4.3 (range 0.3-170,000/mm3). ATRA was initiated at suspicion of APL diagnosis in 100% of patients and was the only treatment in 2(1.5%) patients. Arsenic was combined with ATRA in 93 (81.5%) patients while the other 17% received chemotherapy. 15(13%) had bleeding complications at presentation. Treatment course was complicated by infection and differentiation syndrome (DS) in 31(28%) and 40(34%) patients respectively. There were 12 early deaths, of which 1 was a Jehovah's Witness who declined transfusions and 1 who enrolled 12 days after diagnosis while in multi-organ failure. Incidence of ED was 10/118 (8.5%). The cause of death was disseminated intravascular coagulation (DIC) (n=4), DS (n=2), infection (n=1), anemia (n=1), multi-organ failure (n=4). With a median follow-up of 10.6 months, 2 low risk patients relapsed: I due to non-compliance 1 year after diagnosis and 1 with CNS relapse 3 months after completing consolidation. With a median follow up of 320 days (range 1-965) overall survival (Figure 1) was 87%. There were four late deaths; relapse (n=1), second cancer (n=1) and non-APL related comorbidities (n=2). Conclusions: Results of this prospective trial showed that a simplified treatment algorithm along with support from experts and co-management with treating physicians in the community decreased induction mortality (8.5%) and improved survival (87%) compared to SEER data (1 year relative survival of 71%). We believe our experience warrants large scale implementation and is presently approved as an ECOG/ACRIN trial (EA9131). This model can be applicable to other cancers and life-threatening diseases. Figure Overall Survival Figure. Overall Survival Disclosures Jillella: Leukemia Lymphoma Society: Research Funding. Heffner:AbbVie: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Stuart:Astellas: Research Funding; Celator: Research Funding; Sunesis: Consultancy, Honoraria, Other: Travel, Accomodations, Expenses, Research Funding; Agios: Research Funding; Bayer: Research Funding; Incyte: Research Funding. Gerber:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Grunwald:Medtronic: Equity Ownership; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; Amgen: Research Funding; Janssen: Research Funding. Kota:Pfizer: Membership on an entity's Board of Directors or advisory committees; Ariad Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Pain is the hallmark of sickle cell disease (SCD), and the most common reason for emergency department (ED) visits and hospitalizations in adults and children with SCD. The Emory University Sickle Cell Center at Grady Memorial Hospital has been providing specialized services for adult SCD patients for 30 years. In addition to daily sickle cell specialized clinics, the center is set up with a 24/7 acute care unit (ACU) that is staffed by specialized SCD providers and manages vaso-occlusive episodes (VOE). The patients are started on intravenous narcotics, fluids and antibiotics within 30 minutes of presenting to the ACU. After eight hours of management, the patient is then either discharged home if the VOE is controlled or admitted to the hospital for continued management. Annually, around 3000 ACU visits are recorded with 17% hospital admission rate (over the last 5 years). Little information is known about the clinical characteristics of adults with SCD and chronic opioid use in the setting of VOE pain. Methods: Clinical data from adult SCD patient visits for VOE presenting to the ACU was prospectively collected over a 4-month period (March 1, 2015- July 1 2015) as part of a screening process of an ongoing clinical trial. Results: 214 patients were evaluated for VOE requiring parenteral opioids, with an admission rate of 18%. Mean age was 31+/-14 years, 53% were male and the majority (80%) had hemoglobin (Hb) SS. See Table 1 for clinical characteristics. Over 80% of patients treated in the ACU were ultimately discharged home. Interestingly, the rate of chronic opioid use was around 50% in both admitted and discharged patients from the ACU. Conclusions: The admission rate for VOE in adults with SCD is low at our center through the utilization of the ACU. 51% of patients requiring admission for continued pain therapy were on daily opioids and most of these patients had Hb-SS. 50% of the patients discharged from the ACU were also on daily opioids which represents a significant finding when trying to recruit opioid-na•ve patients on clinical trials to manage VOE. Additionally, this high percentage of daily opioid users reflects a group of patients with possibly more severe disease, who are at risk of tolerance, hyperanalgesia and difficulty in managing VOE in the outpatient setting. This information is valuable for clinical trial design, as chronicopioid use is often an exclusion criterion for enrollment into novel pain therapy trials in SCD. More research is needed on this important topic. Table 1. Clinical Characteristics of Adults with SCD and VOE presenting to Grady Acute Care Center Characteristics Total(N=214) Admitted(N=38) Discharged (N=176) Patient Visits (%) 100% 18% 82% Male n (%) 114 (53%) 19 (50%) 95 (54%) Female n (%) 100 (47%) 19 (50%) 81 (46%) Age ± SD 31±14 29±10 31±9 Hb SS 170 (80%) 34 (89%) 136 (77%) Hb SC 37 (17%) 4 (11%) 33 (19%) Hb S beta+ thalassemia 7 (3%) 0 7 (4%) Chronic Opioids n (%) 108 (51%) 19 (50%) 89 (51%) Disclosures No relevant conflicts of interest to declare.

Description: Background: Acute chest syndrome (ACS) is one of the most feared complications of sickle cell disease. This syndrome is defined by a clinical constellation of symptoms including fever, infiltrate on lung imaging and hypoxia. ACS is a well-known complication to occur early in the hospitalization of patients for sickle cell vaso-occlusive pain crises (VOP). It is hard to differentiate from pneumonia and its rate of occurrence has not been well characterized. Management of ACS includes exchange or simple transfusion, depending on the severity of its presentation, along with critical care support and pain control. Early recognition of this clinical syndrome can lead to better management and outcomes for patients with sickle cell disease. Methods: The Emory University Georgia Comprehensive Sickle Cell Center at Grady Health System has 1073 sickle cell patients and provides a 24/7 acute care unit (ACU) for the evaluation of patients presenting with pain crises. We conducted a retrospective review of all the ACS cases reported at the Center for the last 5 years. Results: The number of sickle cell patient-visits in the ACC was 4409. 30 cases of ACS were confirmed on retrospective review. The rate of ACS was 6.8 per 1000 patient visits. The median age was 39, and the patients' phenotypes 89% SS and 11% SC. Of the patients evaluated, 18% were smokers, 43% were taking hydroxyurea and 89% were taking folic acid. Seventy five percent of the patients were successfully managed with supportive care only, while the remaining 25% required red cell exchanges. On admission, the median white cell count (WBC), platelet count, LDH and reticulocyte count were increased above their upper limits of normal. Eighteen percent of the patients had a d-dimer checked and the median was well above the range of normal (6097). Table 1 shows the patient characteristics and data. Discussion: The rate of ACS over the last 5 years at our sickle cell center is well below historic reports of ACS. We believe that several factors explain our observation. Because our 24/7 ACU is staffed with providers who know their patients' histories and treatment plans and are experienced in the management of VOP, we postulate that our comprehensive management approach involving the rapid and appropriate initiation of intravenous fluids, the administration of pain medications along with antibiotics, and the use of anti-inflammatory medications contribute to the truncation of the sickle cell-related inflammatory state and the prevention of serious complications such as ACS. The fact that inflammatory markers (WBC, Platelet count, LDH) were initially elevated is to be expected in the setting of VOP. What is remarkable is the high percentage of good patient outcomes associated with our comprehensive management approach. This retrospective report should be validated in a prospective study that compares the results of the management of all acute care VOP visits delivered in a 24/7 ACU with the management of acute care VOP delivered in a general emergency room. Such a study should include the collection of laboratory and clinical data that is typically abnormal in patients with VOP. If a significantly decreased rate of ACS rate is observed when 24/7 ACU care is provided, then the institution of 24/7 acute care units would be warranted on a national level to improve the survival of patients with sickle cell disease. Table 1. Characteristics of patients studied Age median (range) 39 (19-58) Gender Females 14/ males 14 Genotype n (%) SC 3 (11%)/ SS 25 (89%) Smoking (%) 18 % Hydrea use (%) 43% Folic acid use (%) 89% Management n (%) Exchanged 7 (25%)/ supportive 21 (75%) Hemoglobin median (range) 10.4 (4.9-11.1) White count median (range) 18.6 (6.1-47) Platelet median (range) 492 (79-646) Reticuolcyte count median (range) 300.6 (151-597) LDH median (range) 677 (238-2287) D-dimer % patients/ median (range) 18%/ 6097 (609-33370) Ferritin % patients/ median (range) 11%/ 2844 (335.2-6060) Total/direct bilirubin median (range) 3.7/1.3 (1-19/0.2-4.9) Creatinine median (range) 1.3 (0.5-3.5) AST/ALT median (range) 59/49 (21-110/12-118) Disclosures No relevant conflicts of interest to declare.

Description: Three genes, SFPQ, NONO, and PSPC1 encode for a small family of tandem RRM domain-containing proteins with diverse functions in RNA processing, transcription, and DNA repair. Our previous work has shown that an SFPQ•NONO complex promotes a distinct sub-pathway of non-homologous end joining (NHEJ) in vitro, implicating it as a novel double-strand break (DSB) repair factor. Consistently, attenuation of NONO function in human fibroblasts leads to partial radiosensitivity, delayed resolution of DNA repair foci, and an increase in radiation-dependent chromosome aberrations. To investigate the in vivo function of this complex, we knocked out Nono, the mouse homolog of the human NONO gene. We hypothesized that a DSB repair deficiency would lead to stem cell exhaustion, sensitivity to DNA damaging agents, or both. First, we investigated hematopoietic stem cells (HSCs), which are known to be sensitive to impairment of DNA repair. Nono-deficient (gt/0) mice showed reduced bone marrow and spleen cellularity, potentially due to reduced body size. The proportion of HSCs (LSK-SLAM) in relation to total bone marrow cells was similar in Nono-deficient and wild type (WT) mice, and bone marrow cell cultures yielded similar numbers and types of colonies. However, HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from gt/0 mice also displayed significantly higher levels of ROS and proliferation (accessed via BrdU incorporation), as well as higher percentage of Ki-67 positivity when compared to HSCs from WT mice. Together, these results indicate that Nono-deficient HSCs exhibit impaired capacity for self-renewal that may be caused by excessive HSC proliferation, a primary defect in response to genotoxic stress or a combination of both. To further elucidate this phenomenon, we investigated potential differences in sensitivity to the DNA crosslinking agent mitomycin C (MMC) and radiation. As previously observed in other genomic instability models (e.g., Fanconi anemia), colony-forming-cells from Nono-deficient mice were highly sensitive to MMC and X-rays (2 Gγ). Utilizing FACS-sorted HSCs, we determined that NONO protein levels are induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. We also evaluated changes in the testis, another organ frequently affected by DNA repair gene mutations. Accordingly, testes of Nono-deficient mice showed growth retardation that became apparent between 18 and 26 days of postnatal development. The co-occurrence of hematopoietic and germ cell defects is reminiscent of other DNA repair gene defects, including those seen in Fanconi anemia. Collectively, our data identify NONO as a novel and important regulator of both HSC maintenance and germ cell function. Its potential effect in other organ systems and mechanism of action are under investigation. Disclosures No relevant conflicts of interest to declare.

Description: Congenital dyserythropoietic anemias (CDA) are rare hereditary diseases of abnormal erythropoiesis. The CDA Registry of North America (CDAR) (NCT02964494) was opened in 2016 to investigate the natural history and molecular biology of CDA. CDA type I (CDA-I) is a recessive form of CDA characterized by macrocytic anemia, hemolysis with inadequate reticulocytosis, and iron overload. The bone marrow shows binucleated erythroblasts with chromatin bridges by light microscopy and spongy heterochromatin in erythroblasts by electron microscopy. The phenotypic heterogeneity in presentation and course of CDA-I is remarkable. Most CDA-I cases are caused by biallelic mutations in CDAN1or C15orf41, and 10-20% do not have an identifiable mutation. Non-hematological features, especially skeletal features, were historically reported in 10-20% of patients (Wickramasinghe, 1998). Due to the rarity of CDA-I and its clinical overlap with several disorders, the diagnosis is often missed or delayed by up to 17 yrs (median) (Roy, 2019). We describe in this study the characteristics and clinical course of CDA-I patients due to CDAN1 mutations enrolled in CDAR. Patients with a phenotypic diagnosis of CDA and their family members were enrolled in CDAR. Clinical and demographic data were gathered from participants at study entry and updated periodically thereafter. Participants elect to give blood, bone marrow, and DNA samples to the biorepository associated with CDAR. Participants with a phenotypic diagnosis of CDA-I and confirmed mutations in CDAN1 were included in this study. Six participants had a diagnosis of CDA-I due to biallelic CDAN1 mutations, comprising 18% (6/33) of affected CDAR participants. CDAN1 mutations were found in 75% of cases diagnosed phenotypically as CDA-I. All six participants presented early in life with a variable degree of non-immune hemolysis, and the diagnosis was confirmed within a median of 2 years from presentation. The characteristics of participants are summarized in table 1. Two had family history of stillbirth or fetal demise in older siblings due to hydrops fetalis. One participant presented prenatally with fetal anemia and started intrauterine transfusions at 24 weeks of gestation; 2 presented with severe anemia and signs of hydrops, pulmonary hypertension, transaminitis, severe hyperbilirubinemia, and thrombocytopenia at birth; and 3 presented with neonatal jaundice and moderate anemia. All participants required blood transfusions in the neonatal period. Three had spontaneous improvement and did not require transfusions after the first year of life. One remained transfusion-dependent at last follow up at the age of 4 yrs. One became transfusion-independent after starting interferon-alpha at 1 yr of age and did not need further transfusions even after discontinuation at 3 yrs of age. One had splenectomy at 11 y.o because he was misdiagnosed to have a membrane disorder but presented in adulthood with hemolytic anemia and pulmonary hypertension and was diagnosed at that time with CDA-I by genetic sequencing. All participants had one or more non-hematological manifestations, including hypertrophic skin folds, onychocryptosis, curved toenails, syndactyly, café-au-lait spots, macrocephaly, spinal fusion, scoliosis, and short stature. One participant suffered a thalamic stroke in the postnatal period, 2 had transient neonatal pulmonary hypertension in the setting of severe anemia, and one had pulmonary hypertension post-splenectomy in adulthood. Ferritin was high in all participants at last follow up, and 4 received chelation therapy. In summary, mutations in CDAN1 are the most common identified mutations in CDAR. CDA-I causes early-onset macrocytic anemia, which may present prenatally, with variable severity of hemolysis ranging from hydrops to mild neonatal jaundice and anemia. Non-hematological manifestations, mainly skeletal, nail and skin abnormalities are more common in CDA-I than previously reported, and their presence in infants with unexplained anemia should raise suspicion for the diagnosis. The availability of molecular testing has significantly accelerated the diagnosis. Management of patients with CDA-I requires multidisciplinary approach from an early age to improve outcome. Collaboration between clinicians, scientists, patients, and families is needed to advance the understanding and treatment of this rare disease. Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Kalfa:Agios: Other: local PI of clinical research trial; FORMA: Other: sponsored research agreement.

Description: Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of theirEla2 gene (“V72M”) reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.