ALBERT

Description: BACKGROUND Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficiency of the glycolytic enzyme red cell PK (PK-R). AG-348 is an orally available, small molecule, allosteric activator of PK-R that activates wild-type and a range of mutated PK-R enzymes in vitro, and increases PK-R activity and restores adenosine triphosphate (ATP) levels in red blood cells from patients with PK deficiency ex vivo. AIMS To describe data from the ongoing DRIVE PK study (NCT02476916), an open-label, dose-ranging trial of AG-348 in transfusion-independent adults with PK deficiency. Early data from this study have been reported (Grace et al. EHA 2016, S466). METHODS After providing informed consent, patients are randomized to AG-348 50 mg or 300 mg orally twice daily (BID) for 6 months. Transfusion independence is defined as no more than 3 units of red blood cells transfused in the 12 months preceding first dose and no transfusions in the 4 months preceding first dose. Patients are followed weekly for Weeks 1-3, then every 3 weeks until Week 12, and then monthly until Week 24. Sex hormone levels and iron status are evaluated at Baseline, Week 12 and End of Study. RESULTS As of the data cutoff date of June 20, 2016, 25 patients have been enrolled and treated for ≥3 weeks. Adverse events (AEs) were mild to moderate. The most frequent AEs were nausea (n=11/25), headache, and insomnia (each n=8/25). One serious AE (Grade 2 osteoporosis) has been reported. Grade 3 AEs were hypertension (n=1), hypertriglyceridemia (n=1), insomnia (n=2), and anemia (n=1). One patient was discontinued from treatment because anemia worsened. There were no Grade 4 AEs and no deaths. Three patients had dose reductions because of headache, nausea, or insomnia. Serum levels of sex steroids were measured at baseline, 12 and 24 weeks; increases in free and total testosterone, and decreases in estradiol and estrone, indicate aromatase inhibition by AG-348, consistent with previously reported results. In 25 patients with ≥3 weeks on treatment, 13 patients (52%) showed a hemoglobin (Hgb) increase 〉1.0 g/dL (range: 1.2-4.9 g/dL; median: 3.40 g/dL). Three patients had dose reductions because of Hgb values exceeding the protocol-mandated maximum. Hgb responses were typically observed within 2 weeks of treatment and were stable with continued treatment. Two patients had dose increases because of insufficient response to 50 mg. Maximal change in Hgb from baseline according to genotype is shown in Figure 1. Of the 25 enrolled patients with ≥3 weeks on treatment, 6 (24%) had two non-missense mutations and none of these had an Hgb response 〉1.0 g/dL. Of the 19 patients with at least one missense mutation, 13 (68%) had an Hgb increase exceeding 1.0 g/dL. CONCLUSION AG-348 is a novel, first-in-class, PK-R activator in clinical testing as a disease-altering therapy to improve anemia in patients with PK deficiency. The ongoing DRIVE PK study has now enrolled over 25 patients, and data from additional patients will be available at the time of presentation. Daily dosing with AG-348 is well tolerated and has demonstrated clinically relevant durable increases in Hgb in the majority of the patients with at least 1 missense mutation. Out of the 19 patients with at least one missense mutation, 13 had an Hgb increase of 〉1.0 g/dL. These data highlight the potential of AG-348 as the first disease-altering treatment for patients with PK deficiency. Figure 1 Maximum hemoglobin (Hgb) increase by genotype Figure 1. Maximum hemoglobin (Hgb) increase by genotype Disclosures Grace: Agios Pharmaceuticals: Other: Scientific Advisor, Research Funding. Rose:Novartis: Honoraria; Celgene: Honoraria; Genzyme: Honoraria. Layton:Novartis: Other: Advisory board meeting; Alexion: Other: Advisory board meeting; GSK: Other: Advisory board meeting; Agios: Speakers Bureau. Yaish:Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Consultancy; Octapharma: Consultancy. Barcellini:Agios: Consultancy. Kuo:Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Other: Unrestricted education grant; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Silver:Agios Pharmaceuticals, Inc.: Consultancy. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals, Inc.: Equity Ownership, Other: Employment (former); KSQ Therapeutics: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Dang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yuan:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Barbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Glader:Agios Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Key Points PK deficiency manifests a broad spectrum in anemia severity that moderately improves after splenectomy. Close attention to monitoring for iron overload, gallstones, and other complications is recommended in all patients with PK deficiency.

Description: Background: PKD causes a defect in glycolysis resulting in a hereditary non-spherocytic hemolytic anemia. The prevalence of iron overload is not well described for PKD. Aim: We aim to describe the demographic features and prevalence of iron overload in transfusion dependent and transfusion independent patients with PKD. Methods: Between March 2014 and April 2016, 203 patients enrolled on the PKD Natural History Study at 29 IRB approved sites. All patients were confirmed to have two compound heterozygous or homozygous mutations in the PKLR gene. Children 〈 1 year of age (n=9) were excluded from this analysis, because elevated ferritin levels are less reliably related to iron overload. Patients were designated with iron overload at the time of enrollment if the plasma ferritin was 〉1000 ng/mL or the patient was on chelation therapy at any time during the prior 12 months. Patients were designated with having had iron overload if a MRI ever showed liver iron content (LIC) 〉3 mg/g dry weight or if they had ever received chelation therapy. Tests of association were performed using Fisher's exact test (categorical) and Wilcoxon rank sum test (continuous). Linear associations between variables were measured by Pearson correlation coefficient. P-values

Description: Background: An international, multicenter registry was established to collect clinical data on patients with PKD, the most common glycolytic defect causing congenital non-spherocytic hemolytic anemia. Aim: To describe and categorize the phenotypic spectrum of PKD, including the range of lab parameters, management, and complications. Methods: 144 patients enrolled on the PKD NHS at 23 sites from 3/2014 to 6/2015. For this report, baseline and retrospective data were included. PKD patients were categorized into 4 severity groups (Gp1-Gp4), with increasing severity of the disease: Gp1. Never regularly transfused +/- prior acute transfusions; Gp2. Regular transfusions prior to splenectomy, post-splenectomy baseline hemoglobin (hb) 〉8.7 g/dl; Gp3. Regular transfusions prior to splenectomy, post-splenectomy baseline hb ≤8.7 g/dl; and Gp4. Post-splenectomy and currently regularly transfused. To reduce the risk of chance associations when dividing the cohort by phenotype, the cohort was divided into a test set (n=73) to define the categories and a subsequent validation set (n=71). Results: Median age at enrollment was 19.9 years (0.1-70.7) with 47% males. This cohort was 95% Caucasian and 38% Amish. Perinatal complications were frequently reported including preterm birth (33%), perinatal transfusions (35%), and hydrops (10%). Newborn jaundice was common, requiring phototherapy (91%) and/or exchange transfusion (46%). 68% (98/144) had undergone splenectomy at a median age of 3.1 years (0.6-28.1). Common indications for splenectomy included reducing transfusion burden, improving anemia, and enhancing quality of life. The median pre-splenectomy hb was 7.0 g/dl (4.5-12.5). Splenectomy reduced the transfusion burden in 91%. Triggers for hemolysis included: pregnancy (59%), infections (61%), stress (35%), and medications (6%). In the 37% (53/144) who required a cholecystectomy, the median age was 14 years (2.6-60.4) and 35 (66%) occurred post-splenectomy. In both the test and validation cohorts, increased severity was associated with a younger age at diagnosis (p

Description: Background: Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia. The spectrum of disease in PKD is broad, ranging from an incidentally discovered mild anemia to a severe transfusion-dependent anemia. Splenectomy partially ameliorates the anemia and reduces the transfusion burden in the majority of patients. Because hemoglobin poorly correlates with symptoms in PKD, transfusion requirements are typically used clinically to classify disease severity with those who are regularly transfused despite splenectomy recognized as the most severely affected subgroup. Aim: To compare demographics, complications, and laboratory results between the most severely-affected PKD patients (those that are splenectomized and regularly transfused) with non-regularly transfused splenectomized PKD patients. Methods: After ethics committee approval, patients were enrolled on the PKD Natural History Study, a prospective 30 site international study. All patients had molecularly confirmed PKD. Only splenectomized patients were included in the analysis. Transfusion frequency was observed over a 3-year period. Patients were divided into two groups based on transfusion frequency: the severe phenotype group was defined as those who receive regular transfusions (≥6 discrete red cell transfusion episodes per year) and the control group did not receive regular transfusions. Phenotype stability over the 3-year period was also assessed. Results: 154 splenectomized patients with PKD were included: 30 patients in the severe PKD phenotype group and 124 patients in the comparison PKD group. Results of the analysis comparing the two groups are described in the Table. Severely affected patients were more likely to be female (77% versus 51%, p=0.013), older at the time of splenectomy (median age: 5 versus 3.6, p=0.011), have iron overload (93% vs. 51%, p

Description: Background: Pyruvate Kinase (PK) deficiency is caused by mutations in the PKLR gene leading to chronic hereditary non-spherocytic hemolytic anemia. Diagnostic evaluation can be challenging with falsely normal PK enzyme activity levels in chronically transfused patients, in those with profound reticulocytosis, or in patients with mutations with unusual biochemical properties. Genetic testing can also be complex, as up to 20% of affected patients have new PKLR variants and up to 10% of patients have variants not routinely detected through exon sequencing. The phenotypic spectrum is broad, and biomarkers of clinical severity would be helpful for both prognosis and monitoring. Aims: To describe the correlation of PK enzyme activity, red cell PK (PK-R) protein level, and red cell metabolites [adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG)] with clinical phenotype. To estimate the sensitivity of PK enzyme activity to diagnose PK deficiency in a cohort of patients with genetically confirmed PK deficiency. Methods: Blood samples were collected from a subset of 41 patients enrolled in the PK Deficiency Natural History Study, all with two confirmed PKLR mutations and no red cell transfusions for ≥3 months prior to the blood sample. PK and hexokinase (HK) enzyme activity testing were performed using standard biochemical assays (Beutler 1985) at the Stanford Red Blood Cell Special Studies Laboratory (normal ranges: PK activity: 3.2-6.5 EU/gm Hb, HK activity: 0.14-0.37 EU/gm Hb). Baseline PK-R protein was quantitated by antibody-based capture and detection using a Meso Scale assay and the signal normalized to a control sample without PK deficiency. ATP and 2,3-DPG concentrations (μg/ml) in blood were analyzed using qualified tandem mass spectrometry methods and then normalized for individual hemoglobin (Hb) values (ATP and 2,3-DPG concentrations were converted to g/dL and divided by Hb (g/dL)). Spearman correlation coefficients were calculated to estimate the correlation between continuous variables, and Wilcoxon rank-sum testing was used to assess the association of continuous and binary variables. Results: The median age was 25.3 years (range 1.4-60.4) and 80% of patients were splenectomized (Table). The mean PK enzyme activity level was 1.1 EU/gm Hb with 90% (37/41) of patients with low PK activity. Normal PK activity was observed in 4 patients, 3 of whom had high HK activity and a low PK/HK ratio (normal mean PK/HK ratio 15.6, range: 8.7-22.5), with a sensitivity of 98% (40/41) when both PK activity and PK/HK ratio were used together. There were no correlations between PK enzyme activity and clinical severity indicators, including transfusion status (p=0.3), splenectomy status (p=0.4), or post-splenectomy Hb (r=0.109). Normalized ATP levels were significantly correlated with normalized 2,3-DPG levels (r=0.93) and higher in ever transfused (median=0.0022, range=0.0011-0.0029) compared with never transfused patients (median=0.0012 , range=0.0008-0.0020), (p=0.004). PK-R protein level was inversely correlated with the total number of transfusions prior to enrollment (r= -0.53) and was higher in never transfused (median=40.1%, range=9.8-73.9%) compared with ever transfused patients (median=7.7%, range=0.4%-15.1%), (p=0.0014). Conclusions: In this cohort with a molecularly confirmed diagnosis, PK deficiency was detected by enzyme testing with 98% sensitivity by utilizing both the PK activity and the PK/HK ratio. Although these assays cannot distinguish between heterozygotes and affected patients, the combination of PK activity and the PK/HK ratio is useful as a screening test to determine which patients require genetic testing. There was no correlation between PK enzyme activity and clinical phenotype. PK-R protein and ATP levels were inversely correlated with clinical severity and may be a helpful marker of disease phenotype. Disclosures Al-Samkari: Agios: Consultancy, Research Funding; Moderna: Consultancy; Dova: Consultancy, Research Funding. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Thompson:bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Kuo:Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Kwiatkowski:Celgene: Consultancy; Terumo: Research Funding; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Background: Pyruvate Kinase (PK) deficiency is the most common enzyme defect of the glycolytic pathway causing hereditary non-spherocytic hemolytic anemia. Patients have a broad phenotypic spectrum, ranging from mild anemia to transfusion dependence, and there is wide variation in transfusion practices and decisions about splenectomy. No prior studies have reported on the use of validated health related quality of life (HRQoL) measures in this population. Aim: To describe patient reported outcomes including general HRQoL and fatigue in adults with PK deficiency and the correlation with clinical and laboratory features. Methods: Patients were enrolled on the PK Deficiency Natural History Study, a prospective 30 site international study. All patients had molecularly confirmed PK deficiency. Adults (n=132), ages ≥18 years, completed the EuroQol-5D (high index equivalent to better QoL), PROMIS Fatigue Short Form 7a (high scores equivalent to higher fatigue), and the Functional Assessment of Cancer Therapy-Anemia (FACT-An, high scores equivalent to less fatigue) surveys at enrollment and annually. Timing of administration was convenience based. Survey data were analyzed according to proprietary scoring guidelines. Tests of association were performed using Fisher's exact test (categorical) and Wilcoxon rank sum test (continuous). Regular transfusions were defined as ≥6 transfusions in 12 months. Genotypes were grouped as two missense mutations (M/M), one missense/one non-missense (M/NM), or two non-missense mutations (NM/NM)); non-missense included deletions or other drastic variants. The minimal important difference (MID) for the FACT-An has been reported as 7 points (Cella et al, J Pain Symptom Manage 2002). P-values

Description: Background: PKD is the most common enzyme defect of the glycolytic pathway causing hereditary non-spherocytic chronic hemolytic anemia. PKD is transmitted as an autosomal recessive trait, caused by both homozygous and compound heterozygote mutations in the PKLR gene, and is characterized by molecular heterogeneity with 〉 200 different mutations reported. Aim: To describe the PKLR genotypes in the PKD NHS with an in depth characterization of 20 newly reported mutations. Methods: Participants (pts) were enrolled in the PKD NHS, a prospective international study open at 23 sites in North America and Europe. Pts with prior PKLR gene sequencing were not resequenced. DNA from all other pts was extracted and the PKLR gene analyzed by Sanger sequencing at 1 of 2 central labs. All new missense mutations affected highly conserved residues in multiple domains of the PKLR gene, were not detected in 1000 genomes and LOVD database, and were considered pathogenic by NCBI and/or UniProtKB and by Polyphen analysis. Results: Genotype information was available on 140 enrolled pts. Of these, 66 (47%) were related to other subjects enrolled in the study. Molecular characterization confirmed the wide heterogeneity of PKD with 65 different mutations identified, including: 42 missense, 20 disruptive mutations (7 splicing, 6 frameshift, 3 stop codons, and 4 large deletions), 2 inframe insertion/deletions, and 1 promoter variant. Sixty-six pts were homozygous, of whom 55 were of Amish origin carrying the p.R479H mutation. Of the 55, 46 had been transfusion dependent prior to splenectomy and 9 had only received transfusions for acute stressors; 93% had been splenectomized, and all were transfusion independent post-splenectomy. Thirty-nine cases had 2 different missense mutations; 18 had one missense and one disruptive mutation, and 16 had 2 disruptive mutations; 1 patient with 17% residual PK activity displayed 3 different mutations (R510W, E241X and V276WXfs45). Besides R479H, the most common mutations were: R510W (16% of the mutated alleles), R486W (12%), and G241X (9%). Frequencies of R510W and R486W were less than those reported in Europe (41% and 30%, respectively). Twenty mutations, all affecting the PK structural domains, have not been previously described: 14 missense, 3 splicing (c.966(-9) a〉g; c.1116(+2) t〉c; c.375(+1) g〉a), 1 frameshift (R40R fsX7), 1 inframe insertion of 2 amino acids, and 1 large deletion spanning intron 2 to intron 3 (Table). The 3 new splice site mutations were predicted to affect normal splicing when analyzed by HSF3.0, using both HSF and MaxEnt algorithms; in particular, homozygous c.966(-9) a〉g was detected in a patient with moderate anemia, reticulocytosis, and mental retardation of unclear etiology. The two new missense mutations detected at a homozygous level (A137V and N156G) were associated with moderate or severe anemia and need for regular transfusions. The latter is located in the Aβ3 catalytic domain/K binding site and probably affects the catalytic efficiency of the enzyme. All the remaining new variants were detected in compound heterozygosity making it difficult to predict their effect on clinical phenotype. Intra-family clinical variability was observed; no correlation was found among the kinds of mutations and the residual PK activity. Conclusion: The molecular features of the largest international cohort of PKD pts are described, including a report of 20 new mutations, thus confirming the wide heterogeneity of the molecular genotype in PKD. Figure 1. Figure 1. Disclosures Morton: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Eber:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yaish:Agios: Membership on an entity's Board of Directors or advisory committees. Nottage:Janssen Pharmaceuticals: Employment. Kuo:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees. Grace:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.