ALBERT

Description: Background: Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by mutations in the PKLR gene, leading to a deficiency of the glycolytic enzyme red cell PK (PK-R). Current treatments for PK deficiency are supportive only. Mitapivat (AG-348) is an oral, small-molecule, allosteric PK-R activator in clinical trials for PK deficiency. We previously described results from DRIVE PK, a phase 2, randomized, open-label, dose-ranging study in adults with PK deficiency (N=52) treated with mitapivat for a median of 6 months. Aim: To report long-term safety and efficacy of mitapivat in patients who continue treatment in the ongoing Extension period of the DRIVE PK study (ClinicalTrials.gov NCT02476916). Methods: Patients were eligible to participate if ≥18 years of age with a confirmed diagnosis of PK deficiency (enzyme and molecular testing); baseline hemoglobin (Hb) levels ≤12.0 g/dL (males) or ≤11.0 g/dL (females); and if they had not received more than 3 units of red blood cells in the prior 12 months, with no transfusions in the prior 4 months. Patients were initially randomized 1:1 to receive mitapivat 50 mg twice daily (BID) or 300 mg BID for a 6-month Core period. Dose adjustment was allowed during the Core period based on safety and efficacy. Patients experiencing clinical benefit without concerning safety issues related to mitapivat (investigator discretion) could opt to enter the Extension period, with follow-up visits every 3 months. Safety (adverse events [AEs]) and efficacy (hematologic parameters including Hb) were assessed. Protocol amendments during the Extension period required that (1) patients who did not have an increase from baseline Hb of ≥1.0 g/dL for ≥3 of the prior 4 measurements withdraw from the study, and (2) patients treated with mitapivat doses 〉25 mg BID undergo a dose taper and continue on the dose that maintained their Hb level no lower than 1.0 g/dL below their pre-taper Hb level. Results: Fifty-two patients enrolled in this study and were treated in the 24-week Core period; 43 (83%) patients completed the Core period and 36 (69%) entered the Extension period. Eighteen patients discontinued from the Extension period: investigator decision (n=8), AEs (n=1), consent withdrawal (n=1), noncompliance (n=1), or other (n=7). Thus, 18 patients, all of whom received ≥29 months of treatment with mitapivat (median 35.6, range 28.7-41.9) have continued treatment. Ten of these 18 patients were male, 11 had a prior splenectomy, and 5 had a history of iron chelation. Median age was 33.5 (range 19-61) years; mean baseline Hb was 9.7 (range 7.9-12.0) g/dL. All patients had ≥1 missense PKLR mutation. The doses (post-taper) at which treatment was continued were (BID): ≤25 mg (n=12), 50 mg (n=5), and 200 mg (n=1). Improvements in Hb levels and markers of hemolysis (reticulocytes, indirect bilirubin, haptoglobin) were sustained (Figure). Among the 18 patients, headache was the most commonly reported AE during both the Extension (n=7, 38.9%) and Core (n=10, 55.6%) periods. Reports of insomnia and fatigue during the Extension period (n=5, 27.8% each) were the same as or similar to those during the Core period. There were fewer reports of nausea (2 vs 6) and hot flush (0 vs 5) in the Extension period. Nasopharyngitis was reported in 5 patients in the Extension period vs 1 patient in the Core period. These data are consistent with the AE profile for the 52 patients treated overall in the Core period, in that headache (44%), insomnia (40%), and nausea (38%) were the most commonly reported AEs and were transient (generally resolved within 7 days without intervention). Conclusion: Chronic daily dosing with mitapivat for a median of 3 years was well tolerated, with no new safety signals reported. Increased Hb levels and improvements in hemolysis markers were sustained at the optimized individual doses. These long-term data support the potential of mitapivat as the first disease-altering therapy for PK deficiency. Two phase 3 trials are underway to further study the effect of mitapivat in patients with PK deficiency. Disclosures Grace: Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Layton:Novartis: Membership on an entity's Board of Directors or advisory committees; Cerus Corporation: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Galactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Barcellini:Novartis: Research Funding, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau; Apellis: Consultancy; Incyte: Consultancy, Other: Advisory board; Agios: Consultancy, Other: Advisory board; Bioverativ: Consultancy, Other: Advisory board. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, 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. Sheth:Apopharma: Other: Clinical trial DSMB; CRSPR/Vertex: Other: Clinical Trial Steering committee; Celgene: Consultancy. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Apopharma: Research Funding; Novartis: Research Funding; Terumo: Research Funding; Celgene: Consultancy; Imara: Consultancy; Agios: Consultancy. Hua:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Hawkins:Bristol Myers Squibb: Equity Ownership; Infinity Pharma: Equity Ownership; Agios: Employment, Equity Ownership; Jazz Pharmaceuticals: Equity Ownership. Mix:Agios: Employment, Equity Ownership. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Key Points Mixed, atypical, and warm immunoglobulin G plus C AIHA (∼30% of cases) more frequently have a severe onset (Hb ≤6 g/dL) and require multiple therapy lines. Infections, particularly after splenectomy, acute renal failure, Evans syndrome, and multitreatment, were predictors of fatal outcome.

Description: The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count 〉600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count 〉600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

Description: Abstract 4597 Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well established that B-chronic lymphocytic leukemia (B-CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. The aim of this study was to evaluate the gene expression of TLRs in 97 B-CLL patients (median age 74 yrs, range 41–89, 41 female and 57 male) and to relate it with the clinical course of the disease (median follow-up 107 months, range 36–336), in particular with infections and autoimmunity, and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). The gene expression of TLR4 and TLR9 was evaluated in total RNA of B cell from B-CLL patients and controls (pool of 10 healthy donors) by real-time PCR performed with a model 7300 real-time PCR system (Applied Biosystems). The TLR4 and TLR9 relative quantification expression (RQ) was normalized according to GAPDH (internal control gene) and to control mRNAs. We found that TLR4 gene expression was decreased (RQ=16.1+/−1.56) and TLR9 increased (RQ=2725+/−165) in B-CLL patients vs controls (RQ=100). TLR4 gene expression was reduced in: a) stage Binet B-C/Rai II-III-IV (n=20) versus low risk cases (Binet A/Rai 0-I) (10.8+/−2.2 vs 17.4+/−1.9, p=0.048); b) patients treated with I line therapy (n=25) and II line therapy (n=14) vs untreated patients (n=58) (13.1+/−2 vs 19.2+/−2.3, p=0.05, and 8.3+/−2.1 vs 19.2+/−2.3, p=0.03, respectively); c) patients untreated but with ITT and treated with stable disease or progressive disease (n=28) vs untreated and treated with DFS〉24 months at the time of the investigation (n=69) (12+/−2 vs 17.8+/−2, p=0.048); d) patients with grade 2–4 infections (n=52), according to Common Terminology Criteria for Adverse Events, vs patients with grade 0–1 infections (13.2+/−1.5 vs 19.51+/−2.9, p=0.04); e) patients with autoimmune phenomena (n=12) vs cases without (8.8+/−2.5 vs 17.12+/−1.7, p=0.04). As far as TLR9 is concerned, we found that it was significantly reduced in untreated and treated with II line therapy patients vs patients treated with I line therapy (2549+/−183 vs 3528+/−356, p=0.01, and 1893+/−277 vs 3528+/−356, p=0.01, respectively). Finally, we found that patients with an unmutated IgVH region status (n=15) showed TLR4 gene expression significantly reduced compared with patients with mutated IgVH region status (n=29) (9.57+/−2.1 vs 18.8+/−3.2, p=0.028). (Data are expressed as mean+/−SE for all data). These results show that in B-CLL patients the reduced expression of TLR4 is consistent with a reduced ability to mount a proper immune response and it is even more pronounced in “active” patients that show high prevalence of infectious episodes and autoimmune phenomena. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Myelodysplatic syndromes (MDS) are characterized by ineffective and dysplastic hematopoiesis and peripheral cytopenias. Moreover, autoimmune phenomena, mainly directed against RBC, are described in early MDS, i.e. refractory anemia (RA) and RA with ringed sideroblasts (RARS). We already reported an autoimmune reactivity against erythroblasts in 50% of RA and RARS adult patients, along with peripheral signs of hemolysis, increased BM erythroblasts; and efficacy of steroid therapy. It has become evident that there are significant differences between MDS in children and adults. Most importantly, the cure is the aim of therapy in all children with MDS, while therapeutic possibilities are often limited in adults. MDS and myeloproliferative disorders are rare in childhood and there is no widely accepted system for their diagnosis and classification. Aims of this study were: To evaluate BM autoimmunity in 9 pediatric patients with MDS by the same method used in adults, named mitogen-stimulated-direct antiglobulin test (MS-DAT), to correlate BM MS-DAT positivity with clinical parameters and therapy. Methods: MS-DAT was performed by stimulating BM with PMA and PHA and antibodies were detected in supernatants by competitive solid phase ELISA. Results: We demonstrated the presence of anti-erythroblast autoimmunity in roughly half of pediatric MDS patients, as already reported in adult MDS. More precisely, 4 out of 9 patients showed positive MS-DAT in BM. At variance with adult MDS, positive patients do not show increased erythroblast counts and peripheral signs of hemolysis (i.e higher reticulocytes, indirect bilirubin, and LDH, and lower haptoglobin) compared with MS-DAT negative ones. One of the four patients with anti-erithroblasts autoimmunity has RA, the other three show peripheral and medullary signs of refractory cytopenia with multilineage dysplasia (RCMD). Two out of four were successfully treated with steroid therapy (with a follow up respectively of 6 and 1 months). Discussion: Although the short follow up and the limited number of patients does not allow definitive conclusions, we retain useful to investigate anti-erythroid autoimmunity in all pediatric MDS in order to identify selected patients with low risk MDS who display autoimmune phenomena. In these cases immunosoppressive therapy may be an effective treatment option.

Description: Abstract 5091 Background The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed by various methods with different sensitivity. Recently mitogen stimulated (MS)-DAT was suggested able to disclose a latent anti-erythrocyte autoimmunity Aims: To compare different traditional (tube, microcolumn and solid phase) and new (mitogen stimulated, MS)-DAT methods in different diagnostic conditions: 54 consecutive cases of hemolytic anemia of suspected autoimmune origin, 28 idiopathic AIHA in clinical remission, and 12 difficult cases of DAT-negative AIHA, and 2) to correlate results with hematologic and hemolytic parameters. Methods DAT tube, microcolumn, solid phase, and eluates were performed by standard techniques. MS-DAT was performed by stimulating whole blood with PHA, PMA and PWM and antibodies were detected by competitive solid phase ELISA. Results Forty out of 54 consecutive cases of suspected AIHA were positive by one or more tests namely 14 DAT-tube, 19 microcolumn, 19 solid phase and 35 MS-DAT. Among the 14 DAT-tube positive cases, 11 were confirmed by all test, and 3 by one or more (1 microcolumn, 1 solid phase, and 1 MS-DAT), eluates were positive in 11, and the majority of patients (10/14) showed hemolytic anemia. As regards the 26 DAT-tube negative cases, 7 were positive in microcolumn and solid-phase (eluates positive in 2/8, panreactive), and 16 in MS-DAT, although in both groups anemia and hemolytic signs were less clear. Mitogen stimulation increased the amount of RBC-bound IgG in all groups, suggesting that MS-DAT could disclose a latent autoimmunity. Tube-negative/other methods positive cases included patients with B-CLL, myelodysplasia/aplasia, and thalassemia intermedia, in which autoimmune phenomena are more frequently observed than overt clinical autoimmune diseases. MS-DAT failed to detect anti-erythrocyte antibodies in half cases AIHA in clinical remission which still were tube-positive. Finally, MS-DAT was the only positive test in 10 cases of AIHA of difficult diagnosis, and mitogen stimulation allowed the identification of autoantibody specificity in culture supernatants of 2 cases which gave weak positive results in microcolumn/solid phase only. Conclusion We concluded that a battery of tests rather than a single test is recommended for the diagnosis of AIHA, depending on the specific clinical context: tube is still a good choice for first screening, microcolumn and solid-phase, which are the automated routine, should be confirmed by a positive eluate to diagnose AIHA, although positivity without a clinical equivalent may be taken into account as part of an autoimmune habitus. MS-DAT could be considered as an additional test for selected cases of difficult diagnosis. Disclosures No relevant conflicts of interest to declare.

Description: We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin c. Mutation −73g〉c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a〉g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.

Description: Autoimmune phenomena, particularly directed against RBC, and alteration of apoptosis have been reported in MDS. We recently described a new method for the detection of anti-RBC antibodies in mitogen-stimulated whole blood cultures, named mitogen-stimulated-DAT (MS-DAT), which is able to disclose a latent anti-RBC autoimmunity in different diseases (autoimmune hemolytic anemia in clinical remission and in B-CLL). We investigated MS-DAT positivity and apoptosis in peripheral blood and bone marrow cultures from 14 patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS), compared with controls (healthy volunteers and miscellaneous haematological conditions). MS-DAT was performed by stimulating whole blood and marrow cultures with PMA, and anti-RBC or anti-erythroblast antibodies were detected by competitive solid phase ELISA. As apoptotic markers we evaluated NF-kB and Caspase-3 activity by ELISA and enzymatic assay, respectively. As shown in table, the amount of anti-erythroblast antibodies was significantly greater in BM cultures of MDS versus controls, and the test was clearly positive in 8/14 patients (not shown). The anti-apoptotic marker NF-kB was significantly increased in MDS versus controls, and consistently, caspase-3 activity decreased, although not significantly. On the contrary, PB of MDS displayed no MS-DAT positivity and no significant alterations of apoptotic and anti-apoptotic markers investigated (not shown). These findings suggest the existence of an anti-erythroblast autoimmunity in bone marrow of early MDS patients. This could be related to the observed defective apoptosis, which in turn determines survival of auto-reactive marrow effectors. MDS Controls Medium PMA Medium PMA *denotes statistical significance versus controls. Values are mean±SE Anti-erythroblasts (pg/ml) 407±116* 487±146* 76±15 99±20 NF-kB (OD units) 294±50* 358±83 59±20 67±21 Caspase-3 (OD units) 132±24 145±26 209±53 186±46

Description: Background: the use of high sensitive FLAER in the last 10 years improved the detection of very small PNH clones (50% on granulocytes), we observed a significant worsening of cytopenia and raise of LDH along with clone size increase. Likewise, lower IPSS risk patients more frequently displayed a greater clone size. As regards therapy, PNH+MDS showed significantly higher response rates to immunosuppressive therapies (ATG and CyA, 84% vs 44.7%, p=0.01) and to HSCT (71% vs 56.6%, p=0.09) compared to PNH-, and the cumulative probability of response to any treatment significantly improved along with clone size increase (from 52 to 100%, p=0.03). In addition, PNH+MDS showed lower rate of progression and AML evolution, and a longer OS [mean 11.9+0.7 years (10.5-13.3) vs 7.3+0.3 (6.6-7.9), p

Description: Abstract 3206 Background: Conventional therapy of warm autoimmune hemolytic anemia (WAIHA) include administration of corticosteroids and immunosuppressive agents, or splenectomy, whereas no effective treatment exists for cold hemagglutinin disease (CHD). A substantial proportion of patients with WAIHA do not respond to or relapse after corticosteroid therapy and may experience clinically relevant side effects. Favorable responses to rituximab at standard doses (375 mg/m2 weekly for 4–6 courses) have been reported in both WAIHA and CHD, idiopathic or secondary, as well as in other autoimmune diseases, such as rheumatoid arthritis and primary immune thrombocytopenia. Recently, low dose (LD)-rituximab (100 mg fixed dose weekly for 4 courses) has been proven effective in patients with autoimmune cytopenias, particularly immune thrombocytopenia. Aims: To evaluate the safety, activity and the duration of the response of LD-rituximab associated with standard oral prednisone (PDN) as first line therapy in newly diagnosed WAIHA and CHD, and as second line therapy in WAIHA relapsed after standard oral PDN. To correlate the clinical response to biological parameters (cytokine and anti-erythrocyte antibody production). Methods: In this single-arm prospective pilot study, LD-rituximab was administered at 100 mg fixed dose weekly on days +7, +14, +21, +28 along with standard oral PDN (1 mg/kg/die p.o. from day +1 to + 30, followed by quick tapering: 10 mg/week until 0.5/mg/kg/die, then 5 mg/week until stop). Complete and partial initial responses (iCR and iPR) were defined as Hb 〉/= 12 g/dL and 〉/= 10 g/dL at month +2 from the beginning of therapy, respectively; sustained response (SR) was defined as Hb 〉/= 10 g/dL at month +6 and +12, in the absence of any treatment. Mitogen stimulated cultures were performed to measure anti-RBC antibodies (at enrolment and month +1, +3, +6 and +12) and cytokines (at enrolment and month, +3, and +6) by ELISA. Results: Twenty-one patients (14 female, 7 male; median age 52 yrs, range 28–77) were enrolled after informed consent. The median follow-up was 11 months (range 2–25). An iCR and iPR were observed in 14 (67%) and 4 (19%) out of 21 patients, respectively; the median Hb level increased from 9.1 g/dL (range 4.4–12.3) at enrolment to 12.5 g/dL (range 9.1–15.3) at month +2. A SR at month +6 was observed in 13/14, and at month +12 in 11/11 evaluable patients. A total of 3 patients relapsed: one at month +16 (retreated with the same protocol with iPR at month +18), another at month +7 (retreated with a SR at month +13), ad the last at month +5 (splenectomy planned). No side effects or serious adverse events were observed. For 10 relapsed AIHA patients (with a follow-up post treatment of at least 12 months) laboratory data (Hb, LDH, reticulocytes) and steroid administration were available before LD-rituximab treatment: a lower cumulative dose (roughly 50%) of steroid was administered to patients during LD-rituximab study compared with previous therapy, without significant differences in the general trend of Hb, LDH, and reticulocytes. Biological studies showed that anti-RBC antibody production decreased until month +6 (from 556 ng/ml at enrolment to 125 ng/ml) and slightly increased at month +12 (307 ng/ml). Regarding cytokine production, at enrolment TNF-α, IFN-γ, IL-12 and TGF-β were lower in patients vs controls, and increased at month +6, without reaching normal values. IL-17 was 10-fold normal values at enrolment, and diminished during the follow-up without reaching normal values. These preliminary results seem to indicate that the addition of LD-rituximab to standard corticosteroid therapy is a feasible and active treatment in AIHA. Data on SR are intriguing, particularly regarding the possible steroid sparing effect of LD-rituximab. Cytokine and anti-erythrocyte antibody production offer new insights into the immunomodulating activity of the drug. Disclosures: No relevant conflicts of interest to declare.