ALBERT

Description: 〈span〉〈div〉ABSTRACT〈/div〉In 2016, students from the geology department at Leicester University used simple low‐frequency geophones and low‐cost seismic dataloggers set up in a primary school and local museum within the city of Leicester to record crowd‐induced vibrations from the King Power Stadium, home of the Leicester City Football Club, a professional soccer team in the English Premier League. Clear signals were detected every time the home team scored a goal, which the students named “Vardyquakes” on social media after the team’s star striker. After a student‐led social media campaign, the story was picked up by the press and turned into a viral news story, leading to hundreds of newspaper articles in papers around the world, together with dozens of TV news stories and interviews with the students. However, the true success of this project was in finding an engaging and reliable tool for encouraging university students to participate in outreach activities with local schools. The football quakes provided a regular and predictable seismic signal that was easy to understand and provided an opportunity to explain to school students how seismic waves are created and can travel through the ground.〈/span〉

Description: Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with

Description: Introduction: Vaso-occlusive pain crisis (VOC) is a significant contributor to the morbidity of Sickle Cell Disease (SCD) and cold exposure has long been associated with increased frequency and intensity of VOC. However, the mechanism by which cold exposure causes the transition from steady state to vaso-occlusion has not been well elucidated. Decreased regional blood flow results in red blood cells spending a longer period of time in the deoxygenated state in the capillaries, increasing the likelihood of hemoglobin S polymerization in the microvasculature and subsequent vaso-occlusion. Regional blood flow is primarily regulated by the autonomic nervous system (ANS) and recent literature shows evidence of cardiovascular autonomic dysfunction in SCD and of its role as a disease modulating factor. We hypothesized that cold exposure triggers a central autonomic response leading to vasoconstriction in the microvasculature and that SCD subjects will have a stronger response when compared to controls. Methods: 17 SCD and 16 control (healthy or sickle trait) subjects aged 13 to 39 years were exposed to thermal stimuli via a computer-controlled thermode (TSA-II) placed on the thenar eminence of the right hand. Predetermined individual threshold temperatures for heat detection, cold detection, heat pain and cold pain were applied, and changes in the microvascular blood flow (MBF) were measured at a sub-second sampling rate on the contralateral thumb using photo-plethysmography (PPG). Mean MBF was derived from the amplitude of the PPG signal, a greater decrease in mean MBF from baseline signifies stronger vasoconstriction. The vasoconstriction response within the PPG signal, and the time (in sec) to vasoconstriction were determined from the cross-correlation function of the pain stimulus signal and the vascular response. The R-to-R interval (RRI) derived from electrocardiogram was used to evaluate cardiac autonomic balance. Standard deviation of RRI represents total heart rate variability, Spectral indices of the RRI represent parasympathetic activity (high frequency power; HFP) and sympatho-vagal balance (Low to high Ratio; LHR = low frequency power(LFP)/HFP). Prior to the study, subjects were administered measures of current anxiety and general level of anxiety (STAI Y1 and Y2, respectively) as well as a measure of pain related anxiety (PASS). Results: All the thermal stimulation tasks caused a significant decrease in MBF from baseline (p values for cold detection, heat detection, heat pain and cold pain=0.027,

Description: Introduction Over the last two decades, sickle cell disease has become a model of diffuse endotheliopathy. In 2004, Gladwin and colleagues demonstrated that elevated tricuspid regurgitation jet velocity (TRJ) was an independent predictor of mortality and that chronic red blood cell hemolysis underlies pulmonary vascular disease (Gladwin et. al. NEJM, PMID:14985486). Belhassen and colleagues demonstrated that flow-mediated dilation of the brachial artery (FMD) was decreased in patients with sickle cell disease, specifically shear-mediated vasodilation (Belhassen et. al. Blood, PMID:11238095). Our group demonstrated that plasma free hemoglobin links decreased FMD and elevated TRJ in patients with sickle cell disease and that chronic transfusion therapy improves FMD (Detterich et. al. Blood, PMID:26036801). We aimed to determine whether markers of vascular function predict the risk of mortality in a cohort of patients with sickle cell disease. Methods This was a single center, prospective cohort study of non-transfused and chronically transfused sickle cell anemia patients at Children's Hospital of Los Angeles between 2009 and 2012 aiming to assess vascular function in sickle cell disease. This study was approved by the Children's Hospital Los Angeles Institutional Review Board. We used FMD, a test of shear-mediated endothelial nitric oxide release as the model for vascular function. We simultaneously measured the regional hemoglobin oxygen saturation in the hand using near infrared spectroscopy (NIRS). We also measured microcirculatory perfusion using laser doppler flowmetry (LDF) over the skin of the nailbed. Echocardiography was performed on the same day to assess tricuspid regurgitation jet velocity and cardiac function. Markers of inflammation, cell count and hemolysis were also measured on the day of the study. We prospectively followed them until 2018 to assess our primary outcome, survival. All of the measurements were made pre-transfusion for chronically transfused subjects. Kaplan Meier and Cox-Proportional Hazards multivariate model was used to determine independent predictors of survival. All statistics were performed using Stata/IC version 14.0. Results We enrolled 26 chronically transfused patients with SCD and 63 non-transfused SCD patients in a study assessing the effects of hemolysis on endothelial function. Overall mortality in our cohort was 20% with a median follow up time of 7.8 years (7.1-8.3 IQR). Median age was 24 years at the time of study (17-36 IQR). Median age at the time of death was 36 years(28-51 IQR) and at the last follow up for the alive patients was 30 years (25-41 IQR), p=0.01. In the patients who died, they had higher TRJ (median 270 vs 235, p=0.03) and had elevated plasma Hgb, median 26 vs 14, p=0.01. FMD, regional oxygen saturation and microcirculatory perfusion were not different between alive and deceased patients. There was no difference for inflammatory markers (white blood cell count, platelet count, high sensitivity C-reactive protein), hemoglobin S%/transfusion, or anemia (hemoglobin/hematocrit/reticulocytes) between alive or deceased patients. By Kaplan Meier analysis individual factors associated with mortality were TRJ〉250 (p=0.04) and age〉21 (p=0.02) (Fig 1 and Fig 2). By Cox-Proportional Hazards Model, both TR jet velocity and plasma hemoglobin remain significant after correcting for age (Table 1). Conclusions In our cohort, there are multiple abnormal markers of vascular function and elevated markers of hemolysis; however, the strongest predictors of mortality were age, plasma free hemoglobin and tricuspid regurgitation jet velocity. These findings support the association between hemolysis, pulmonary vascular disease and mortality, a controversial topic in sickle cell disease. Disclosures Coates: Vifor Pharma: Consultancy; Sangamo: Consultancy, Honoraria; Celgene Corp.: Consultancy; ApoPharma: Consultancy, Honoraria.

Description: Red blood cell pyruvate kinase deficiency (PKD) is the most frequent enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Its incidence has been estimated to be 1 in 20,000, especially in regions where consanguinity is common. It is inherited in autosomal recessive manner occurring as result of homozygous or compound heterozygous mutations affecting both alleles in PKLR gene. Over 200 mutations have been described in patients with PKD causing various severity of hemolysis. Most cases are due to missense mutations, but small deletions, insertions, splice site alterations, frame shifts, disruption of erythroid specific promoters, and nonsense mutations have been reported. Alu elements are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. These mobile elements have contributed significantly to evolution and human diversity by causing gene rearrangements. Alu insertions particularly have been associated with a number of human diseases either by disrupting a coding region or a splice signal. In this abstract, we report an Alu-element insertion in the coding region of PKLR gene as a novel cause of PKD causing severe hereditary nonspherocytic hemolytic anemia. The proband was a 16-month-old middle eastern male born to consanguineous parents. He was diagnosed with severe chronic hemolytic anemia and neonatal jaundice requiring regular blood transfusion since birth. There were no known family members with anemia requiring transfusions. The patient's RBC phenotypic analysis was challenging since most of the circulating RBCs were donor cells. Using a Next-Generation sequencing panel for 27 hemolytic anemia associated genes, we identified and characterized a novel homozygous insertional mutation in exon 6 of PKLR gene along with heterozygosity for ANK1 c.1404+15C〉T variant. The mutation in exon 6 was identified as Alu element insertion. The inserted Alu element (~370 bp in size), belonging to the youngest Yb8 subfamily, disrupts the reading frame at isoleucine 314 in exon 6 of PKLR gene, leading to a premature stop codon within the inserted sequence. Quantitative reverse transcription-PCR (qRT-PCR) in the patient's reticulocyte RNA using primers for the sequence before the Alu insertion revealed a transcript present but decreased by 80% compared to the average of controls' reticulocyte RNA. This truncated transcript even if translated is expected to cause severe enzyme deficiency. Both parents had a normal hemoglobin level with mild reticulocytosis. The mother had a PK enzyme activity of 4.5 units/g Hb (normal range 6.7-14.3 units/g Hb) indicating that she is heterozygous for PKD. Paternal PK activity level was not determined due to insufficient sample. Carrier testing for mutation in PKLR gene in both parents is currently in process at the time of this submission. The variant in ANK1 gene was predicted to be damaging by affecting the splice site in ankyrin gene. However the father was also heterozygous for this variant and his ektacytometry was normal suggesting a benign nature of ANK1 c.1404+15C〉T. This case represents, to our knowledge, the first report of a pathogenic PKLR mutation due to an Alu element insertion and demonstrates a novel mechanism causing severe hereditary nonspherocytic hemolytic anemia. This report also illustrates the challenge of diagnosing congenital hemolytic anemias in chronically transfused patients. The patients often present with a complex clinical picture and ambiguous laboratory findings and may have several potentially damaging genetics variants identified by next generation sequencing. A critical evaluation of the clinical symptoms, laboratory findings and genetic data from other family members and bioinformatics analysis are required to clarify the contribution of these variants and to arrive at an accurate diagnosis, which will guide the institution of targeted interventions and appropriate genetic counseling. Disclosures No relevant conflicts of interest to declare.

Description: Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Previous preclinical studies demonstrated potent anti-proliferative and apoptotic effects of FF-10501-01 on AML cell lines, including HMA-resistant derivatives, through inhibition of de novo guanine nucleotide synthesis. Therefore, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ≥ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. A total of 29 patients, 15M and 14F (23 AML, 6 MDS) have been treated in 7 dose cohorts (50 - 500 mg/m2 PO BID) for 14 days on and 14 days off, and 400 mg/m2 for 21 days on and 7 days off, each 28-day cycle. Median (range) values: age 75 yrs (59 - 88); baseline bone marrow blast counts for AML 34% (12 - 82), for MDS 10% (5 - 16), and overall 30% (5 - 82); and prior treatment regimens 2 (1 - 7). All patients relapsed from, or progressed on, prior HMAs. At baseline, mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A, NOTCH1, JAK2, IDH2, PTPN11, KRA, TP53, RUNX1, EZH2 and/or MDM2 were present in 13 of 29 (45%) of patients. Atrial fibrillation (Gr 2) was reported in 2 subjects at a dose of 500 mg/m2 BID. This met the definition of dose-limiting toxicity (DLT) and no further enrollment was made at this dose level. The maximally tolerated dose (MTD) was declared at 1 dose level lower, 400 mg/m2 BID, and this cohort was expanded to 6 subjects. No DLTs have been observed in N=7 total subjects treated at 400 mg/m2 BID x 14 days. FF-10501-01 has been very well tolerated through 24 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related thrombocytopenia, neutropenia and bone marrow aplasia (all Gr 4) were reported in 1 patient at 200 mg/m2 BID. The median number of FF-10501-01 cycles received to date is 2 (range 1 - 24). Partial remissions have occurred in 2 AML patients (50 and 100 mg/m2 BID) after 3 cycles, lasting for 5 and 24 cycles, respectively, with the higher dose patient still on study after 24 cycles. A total of 8/23 (34.8%) AML patients, including the 2 PRs, have attained stable disease (SD) control with no disease progression over 3 - 24 cycles. Three AML patients remain on study through 3, 23 and 24 cycles, respectively. A bone marrow complete response was achieved in 1 MDS patient treated at 400 mg/m2 BID after 1 cycle. Although the bone marrow blast counts have increased since, this patient remains stable and is still on therapy through 14 cycles. Three of 6 MDS patients (50%), including the marrow CR, attained SD control with no disease progression over 3, 14 and 14 cycles, and 2 remain on study through 3 and 14 cycles, respectively. FF-10501-01 was rapidly absorbed with mean Tmax of 2.74 hours and mean t1/2 of 4.05 hours. Drug exposure (AUC0-24 and AUCcourse) increased with dose in a near linear manner. Potent suppression of circulating xanthine monophosphate (XMP), a marker of IMPDH activity, has been observed following FF-10501-01 administration on Day 1 of Cycles 1 and 2 at dose levels of 50 mg/m2 BID and above. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS in patients who have failed or progressed on HMAs and with one or more baseline mutations in pathways known to be affected in AML and MDS. Preclinical activity was seen in multiple leukemia cell lines, including HMA-resistant derivatives. In a Phase 1 trial, clinical activity with a marrow CR, PRs, long-term disease stabilization (≥ 5 cycles) and a highly tolerable safety profile were observed. The Phase 2a expansion phase of the study is soon to begin. Disclosures DiNardo: Agios: Research Funding; Daiichi Sankyo: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Abbvie: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Daver:BMS: Research Funding; Kiromic: Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; Sunesis: Consultancy, Research Funding. Denton:Westat Corporation: Employment. Smith:Westat Corporation: Employment. Tiefenwerth:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

Description: Introduction Kidney iron deposition has been described in hemolytic disorders including mechanical valves, paroxysmal nocturnal hematuria, and sickle cell disease (Roberts & Morrow, Circulation 1966; Leonardi & Ruol, Blood 1960). Circulating plasma hemoglobin is filtered at the glomerulus and reabsorbed via the megalin cubulin system in the proximal and distal tubules (Gburek et al, J Am Soc Nephrol 2002). On MRI, this manifests as signal loss on gradient and spin echo sequences in the cortex of the kidney with complete sparing of the medulla (Jeong et al, Radiographics 2002). The signal darkening is quantified by the parameter R2*, which has been shown to be directly proportional to tissue iron in the liver and heart. Kidney R2* has previously been demonstrated to rise proportionally to lactate dehydrogenase (LDH) in chronically transfused sickle cell disease (SCD) patients (Wood et al, Br J Haematol 2016; Schein et al, J Magn Reson Imaging 2008), but LDH is not a specific marker of hemolysis, and chronically transfused patients could potentially deposit iron in the kidney through other mechanisms. Therefore, we characterized the relationship between kidney R2*, urinary iron and markers of hemolysis in non-transfused SCD patients. Methods Sixty-five non-transfused SCD patients were recruited to the study, which was approved by the Institutional Review Board of Children's Hospital Los Angeles. Following medical history and physical exam, subjects completed blood and urine testing, and then abdominal MRI for assessment of somatic iron stores. R2* measurements were collected using multiple gradient echo pulse sequences on 1.5 Tesla magnets. Statistical analysis was performed using JMP® Pro, Version 14.0.0 (SAS Institute Inc., Cary, NC, 2018). Results Subjects were generally adults with a mean age of 32 years. Nearly three quarters of subjects had homozygous sickle cell disease, while a quarter had SC disease or S-Beta thalassemia, and one subject had sickle cell trait. Most subjects were anemic, and all subjects had elevated markers of hemolysis. Fifty-four percent of subjects had an elevated kidney R2* level (≥34 Hz). On univariate analysis, kidney R2* was associated with urinary iron (R2=0.52, p