Abstract: Introduction The protein microarrays are becoming the leading technology in proteomic research area. They enable to implement both features of proteins that can be altered in disease as quantitative proteomics (levels in biological samples) as well as functional proteomics (determination of their selective interactions with other biomolecules). Protein microarray techniques such as sandwich immunoassays, antigen capture immunoassay and direct immunoassays use labeling and antibodies. However, these labels can interfere with the analyte binding site and thus affect protein activity. Using antibodies requires a prior knowledge of the studied proteins which is in the case of a heterogeneous disease with little knowledge in its proteomic field a huge disadvantage. Surface plasmon resonance (SPR) is a label-free and direct method allowing quantification as well as monitoring of protein-protein interactions simultaneously and in real time. Myelodysplastic syndrome (MDS) is a heterogeneous group of hematological malignancies. It affects pluripotent hematopoietic stem cell and is manifested by variety of clinical symptoms according to predominant involvement of development lineage. The high risk of MDS to transform into acute myeloid leukemia makes it a suitable model for study of biological processes leading to leukemia development. In this work, we use SPR imaging for simultaneous screening of blood plasma of MDS patients followed by mass spectrometry (MS) for identification of interacting partners and analysis of protein network properties. Proteins, whose levels are either elevated during MDS disease or interaction with their receptors/ligands is a part of signaling pathway, were employed. Method SPR imaging system with polarization contrast and internal referencing was combined with dispersionless microfluidics for parallel screening of blood plasma samples. Proteins involved in pathogenesis of MDS and their physiological counterparts were immobilized under flow to create 6x6 sensing spots. Specifically, these include integrin αMβ2 (LFA1), intercellular adhesion molecule 1 (ICAM1), integrin α4β1 (VLA4), vascular cell adhesion protein 1 (VCAM1) and cytotoxic T-lymphocyte protein 4 (CTLA4). The sensor surface functionalization was optimized with respect to its ability to provide a low-fouling sensing surface with biologically active receptors. Plasma samples of controls and MDS patients were flowed along the functionalized surface and differences in individual interactions were evaluated. Selected interacting partners were further identified using 2D-HPLC/ESI-MS/MS. Identified proteins were analyzed by String Networks and Power Graph Analysis. Results and Conclusion Significant differences in the protein profiles among different MDS groups of patients as well as relative to control healthy donors were observed using SPR imaging; tens of interacting proteins were identified by mass spectrometry. Protein interaction networks were explored through clustering of proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. Identified proteins are involved in several processes; regulation of immune system, ubiquitinylation and protein degradation, cell signaling, hemostasis, protein synthesis, cell adhesion, metastasis, and inhibition of blood coagulation. Using of Power Graphs, a novel representation of (protein) networks, provided valuable insight into the existence of protein complexes, their internal organization, and their relationships. Interaction networks also indicated possible pathways involved in MDS pathogenesis (especially Src tyrosine kinases). The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Abstract: Background Double induction using two subsequent 7+3 regimens of cytarabine plus anthracycline is commonly performed in AML patients with an adequate performance status in order to maximize dose intensity upfront. However, for patients with a good early response at day 15 of first induction, there is no prospective randomized evidence on the necessity or value of a second induction cycle. Aims In order to answer the question if good responders of the first 7+3 induction could be spared a second induction cycle, we set up randomized-controlled SAL DaunoDouble trial. The study prospectively assesses the outcome of patients with a good early response with respect to the number of induction cycles (single versus double). We assumed non-inferiority of single induction in terms of complete remission (CR/CRi) rate, based on a margin of 7.5%. Here, we present the results of the planned interim analysis. Methods Patients (pts) 18-65 years with newly diagnosed AML, normal cardiac and organ function received a first induction cycle with seven days of cytarabine plus three days of daunorubicin ("7+3"). Response assessment in bone marrow was done on day 15 after the initiation of chemotherapy and confirmed by central review. A blast count & lt;5% was defined as good response. Pts with good response were randomized to receive a second induction cycle (arm D) or no second induction cycle (arm S). Primary endpoint was CR/CRi after completion of induction, secondary endpoints were RFS, and OS. Results Between 2014 and 2020, 624 evaluable pts were enrolled and received the first induction cycle with 7+3. A marrow blast clearance below 5% on day 15 was achieved in 298 pts (48%), providing eligibility for randomization. Of these patients, 150 were randomized into arm S and 148 into arm D, respectively. Median age was 52 years, 92% had de novo AML, NPM1 mutation was present in 53%, FLT3-ITD in 25% of pts. Favorable, intermediate and adverse risk (ELN 2017) were present in 56%, 34% and 10% of pts, respectively. CR/CRi rates at the end of induction were 86% after single induction and 85% after double induction. The CR/CRi rates in 224 pre-defined per-protocol pts were 88% versus 91%, resulting in a CR difference of 3% (95%-CI -0.047-0.111; p for non-inferiority test 0.145). After a median follow-up time of 24 months, RFS was slightly but not significantly lower after single induction with a 3-year RFS of 53% versus 64% (HR 1.4, p=0.125), whereas no differences were seen in 3-year OS, with a of rate of 74% versus 75% (HR 1.1, p=0.645) after single versus double induction. Conclusion The interim analysis results show that in good responders, the difference between CR rates after single versus double induction was even smaller than the predefined 7.5% margin, suggesting a trend for non-inferiority of single induction, although statistical significance was not reached. The trial continued recruitment. These findings suggest that in good responders, it may be safe to omit a second induction cycle if a second cycle poses a high risk. Figure. CR + CRi, RFS and OS after randomization to single versus double induction. Disclosures Alakel: Pfizer: Consultancy. Jost:Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; JAZZ: Other: travel support; Celgene: Other: travel support. Novak:Roche: Consultancy; Janssen: Other: Travel expenses; Takeda: Consultancy; Amgen: Consultancy, Other: Travel expenses; Pfizer: Consultancy; Novartis: Consultancy. Krause:Takeda: Honoraria; Celgene: Other: Travel Support; MSD: Honoraria; Pfizer: Honoraria; Siemens: Research Funding; Gilead: Other: Travel Support. Held:Roche: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; MSD: Consultancy; Acrotech: Research Funding; Spectrum: Research Funding; Amgen: Research Funding. Platzbecker:AbbVie: Consultancy, Honoraria; Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria. Thiede:AgenDix GmbH: Other: Co-owner and CEO. Müller-Tidow:Daiichi Sankyo: Research Funding; Pfizer: Research Funding, Speakers Bureau; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau.

Abstract: Introduction: Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoesis, associated with cytopenias and high risk of leukemic transformations with common morbidity. MDS are hematological malignancies of unclear etiology where oxidative/nitrative stress may contribute to the pathogenesis1. The posttranslational oxidative modifications of proteins and low molecular weight compounds are induced, revealing dysbalance of redox systems in vivo. Nitration of tyrosine either in free form or bound in proteins is important marker of nitric oxide synthase (NOS) activity shift in the presence of oxidative stress in favour of superoxide formation. The aim of this work was to assess whether 3-nitrotyrosine (3-NT) serum concentrations are enhanced also in MDS patients. Methods: Serum samples were obtained using blood of either MDS patients or healthy donors. All tested individuals agreed to the study at the time of blood collection. We proposed HPLC-MS/MS method to estimate 3-NT concentration in serum samples using QTRAP 4000 mass spectrometer (ABSciex, Prague, Czech Republic). Serum proteins were precipitated using ethanol, supernatants were evaporated, reconstituted in 0.1% HCOOH/2% methanol and injected onto HALO C18 microcolumn 100x0.5 mm (ABSciex, Prague, Czech Republic). Oxidative stress in MDS patients and controls was assessed by serum malondialdehyde concentrations measured by HPLC of 2-thiobarbituric acid MDA derivative using UV detection. Results: The sensitivity of method proposed for analysis of 3-NT in sera was sufficient for estimation of differences of 3-NT in patients and control samples. We have found enhanced concentrations of both MDA and 3-nitrotyrosine in serum of MDS patients as compared with healthy donors. Discussion: Enhanced MDA concentrations in MDS patients confirmed the presence of oxidative stress in MDS patients. The reactive oxygen species may oxidize tetrahydrobiopterin, important cofactor of NOS, resulting into nitric oxide synthase uncoupling with enhanced superoxide and consequently peroxynitrite production2. It is known that methylarginines, naturally occurring inhibitors of NOS, can profoundly increase superoxide generation from uncoupled NOS. Recently, we have found significantly enhanced concentration of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome3. The observed increased concentrations of 3-NT in MDS patients correspond with assumed enhanced peroxynitrite formation as compared with controls. 3-nitrotyrosine concentrations thus could serve as a new criterion of NOS changed activity in MDS patients. Literature: 1. Farquhar MJ, Bowen DT. Oxidative stress and the myelodysplastic syndromes. Int J Hematol. 2003;77:342-350. 2. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315-424. 3. Štikarová J, Suttnar J, Pimková K, Chrastinová-Mášová L, Čermák J, Dyr JE. Enhanced levels of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome. Journal of Hematology & Oncology. 2013;6:58. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction MicroRNAs (miRNAs) are involved in the control of hematopoiesis, their deregulation also appears to play role in the pathogenesis of several hematopoietic diseases. Multiple miRNAs have been reported to be abnormally expressed in hematologic cancers; moreover, specific miRNA expression profiles have been proposed as diagnostic and prognostic markers in various hematologic malignancies, including Myelodysplastic Syndromes (MDS). MDS are a heterogeneous family of clonal disorders of hematopoietic stem cells. They are characterized by ineffective hematopoiesis and frequent leukemic progression. It has been shown that the mature erythrocytes are rich in diverse miRNAs species, even though they lack ribosomal and other large-size RNAs. miRNAs are expressed during erythrocyte differentiation and have an important role in erythropoiesis and mRNA degradation. Methods Four miRNAs, i.e. miR-16, miR-181, miR-34a, and miR-125b were selected as a model set of potential MDS biomarkers and evaluated using a surface plasmon resonance imaging (SPRi) biosensor and real-time PCR. The sensor calibration curves were measured with miRNAs spiked in a complex erythrocyte lysate. The total RNA was extracted from erythrocyte lysate using the acid guanidinium-thiocyanate-phenol-chloroform method. The levels of selected miRNAs were measured in all samples by quantitative reverse transcription - real-time polymerase chain reaction. Results A high-capacity array SPRi system for rapid simultaneous detection of multiple miRNAs in erythrocyte lysate was developed and demonstrated at the Institute of Photonics and Electronics. The ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes-coated gold surfaces of the SPR sensor were shown to reduce the non-specific interaction between surface of the sensor and sample. A two-step miRNA detection assay for multiplexed miRNA detection in erythrocyte lysate was demonstrated in which hybridization of probe-functionalized pCBAA with target miRNA bound to biotinylated oligonucleotide probes was followed with capture of streptavidin-functionalized gold nanoparticles to biotinylated probes. In preliminary experiments, the array was shown to be capable of detecting multiple miRNAs spiked in erythrocyte lysate without the need for complex lysate sample pretreatment at concentrations as low as 0.5 pM in less than 45 minutes. Conclusions Using the newly developed high-capacity array SPRi system we have found significantly increased levels of miR-16 in erythrocyte lysate samples (at the concentration range of ~10-100 pM) compared to other miRNAs tested within this study. The results were confirmed by a reference real-time polymerase chain reaction method. In addition, our results indicate an over-expression of miR-16 in erythrocyte lysate samples of MDS patients with both Refractory Cytopenia with Multilineage Dysplasia (RCMD)and Refractory Anemia with Excess Blasts (RAEB) diagnosis. This clearly demonstrates the potential of SPRi array technology for clinical applications. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Primary mediastinal large B-cell lymphoma (PMBCL) is a rare subtype of diffuse large B-cell lymphoma with typical clinical, pathological and genetic features. No standard frontline protocol is widely accepted and role of adjuvant radiotherapy is not established yet. New intensive etoposide-based (DA-EPOCH; Dunleavy K, NEJM 2013) regimens with added rituximab show survival benefit but certain patients are still at risk of early treatment failure. As residual masses (CT) after therapy are frequent, assessment of tumor metabolic response is of utmost importance. Complete metabolic response (CMR) measured by positron emission tomography (PET) has high predictive value after intensive treatment (Martelli M, JCO 2014), but the role of interim scans is unclear. Aim: To analyze the prognostic impact of interim and final whole-body PET-CT fusion scans using visual and Deauville criteria in PMBCL patients receiving intensive etoposide-based regimens with rituximab. Patients: Thirty-four consecutive PMBCL patients were treated in a single center from 5/2005 to 6/2013. The median age at diagnosis was 32.5 (19-73) years; male-to-female ratio 0.62:1. Ann Arbor stages I through IV were observed in 1, 19, 3 and 11 patients, respectively. Large mediastinal mass (≥11cm) was present in 19 (56%) patients. Thirteen patients (38%) had concurrent extranodal disease but unaffected bone marrow. IPI and age-adjusted IPI scores were: low 16 (47%) and 8 (24%), low-intermediate 11 (32%) and 11 (32%), high-intermediate 5 (15%) and 12 (35%), and high 2 (6%) and 3 (9%) patients, respectively. All patients received rituximab; intensive etoposide-doxorubicin-based therapy was given to 30 (88%; sequential n=29, MegaCHOP-ESHAP n=1), intensified CHOP (PACEBO) to 1 and CHOP to 3 patients. Treatment was consolidated with ASCT (BEAM 200) in high-risk bulky cases with extranodal involvement (n=20, 59%). Involved-field radiotherapy was used in only 9 (26%) patients. Whole-body PET-CT scans were planned after 2nd chemotherapy cycle (interim; iPET-2) and treatment completion (final; fPET). The PET results were expressed as positive/negative (IHP; Juweid ME, JCO 2007) and 5-point Deauville scale (D1-5; Meignan M, Leuk Lymphoma 2014). Treatment response was assessed using revised criteria (Cheson BD, JCO 2007). Results: After treatment, 28 (82%) patients achieved complete remission (CR), one partial remission, three stable disease and two progressed. Seventeen CR patients (61%) had residual mass on CT (median longest diameter 40mm). After a median follow-up of 58.7 months, 8 (24%) patients relapsed or progressed and 6 (18%) of them died. Five-year overall survival (5-y OS) reached 81.2% (95% CI 0.68-0.95), 5-year progression survival (5-y PFS) was 75.5% (95% CI 0.61-0.90). There were 27 (79%) and 33 (97%) cases assessable with iPET-2 and fPET, respectively. Using visual criteria, iPET-2 was negative in 10/27 (37%) and fPET in 28/33 (85%) cases. With Deauville criteria, iPET-2 scores were D1-2 in 6 (22%), D3 in 4 (15%) and D4-5 in 17 (63%) cases, and fPET scores D1-2 in 19 (58%), D3 in 8 (24%) and D4-5 in 6 (18%). All visually negative iPET-2 were scored as D1-3. In fPET, there was only one discordant case (visually negative scored as D4). Visually negative (D1-3) iPET-2 was associated with superior 5-y OS (34% vs 100%, p=0.08) and 5-y PFS (65% vs 100%, p=0.049). Visually negative visual fPET was linked with superior 5-y OS (20% vs 96.0%, p 〈 0.01) and 5-y PFS (20% vs 88.6%); D1-3 with 5-y OS (33.3% vs 95.8%, p 〈 0.01) and 5y-PFS (33.3% vs 88.2%, p 〈 0.01). Negative (NPV) and positive (PPV) predictive values for PFS were 100% and 35.3% for iPET-2 and 89.3% (visual), 90.0% (D) and 80% (visual), 66.7% (D) for fPET, respectively. Residual PET(-) mass have no impact on OS (p=0.47) or PFS (p=0.85). Conclusion: Early CMR resulted in excellent survival and superior NPV (100%). However, two thirds of iPET-positive patients may achieve long remission (PPV is low). Final PET brings twice higher PPV but despite relatively high NPV, more than 10% of fPET-negative patients relapse later. Visual and Deauville scales are comparable; intermediate (D3) results should be considered negative. Thus, iPET-2 identifies good responders and may serve as an indicator for tailored therapy. Further studies including tumor metabolic volume analysis are needed for better early identification of poor responders. Acknowledgment: LF-2014-001 Figure 1 Figure 1. Disclosures Procházka: Takeda pharmaceuticals: Speakers Bureau; Roche: Honoraria, travel grants, travel grants Other.

Abstract: Background: Guadecitabine (G) is a next generation subcutaneous (SC) hypomethylating agent (HMA) resistant to degradation by cytidine deaminase which results in prolonged in vivo exposure to the active metabolite decitabine. We conducted a large global randomized phase 3 study of G vs Treatment Choice (TC) with azacitidine (AZA), decitabine (DEC), or low dose Ara-C (LDAC) in 815 TN AML patients unfit for IC (ASTRAL-1 study). The primary ITT results were previously presented (Fenaux et al, EHA abstract S879, 2019). Clinical guidelines for single agent HMAs recommend a minimum of 4 to 6 treatment cycles for maximum benefit. We describe here the results of the study based on number of treatment cycles administered. M ethods: TN-AML patients ineligible for IC due to age ≥ 75 y, or coexisting morbidities, or ECOG PS 2-3 were randomized 1:1 to either G (60 mg/m2/d SC for 5-days Q28 days) or a preselected TC of AZA, DEC, or LDAC at their standard dose/schedule. AML diagnosis and response status were assessed by an independent central pathologist blinded to randomization assignment. Complete response (CR) and overall survival (OS) were co-primary endpoints. We analyzed patients' characteristics, number of treatment cycles, reasons for treatment discontinuation, CR, and OS including analyses by number of cycles received including prospective subgroups, and OS analyses of responders and non-responders. Results: 815 patients were randomized to G (408) or TC (407). Preselected TCs prior to randomization were DEC (43%), AZA (42%), and LDAC (15%). Baseline variables were well balanced across the 2 treatment arms. For G vs TC respectively, age ≥75 y in 62% vs 62.4%, PS 2-3 in 50.5% vs 50.4% (including 10.8% vs 8.8% PS 3), and poor risk cytogenetics in 34.3% vs 34.6%. Most patients were assigned to an HMA at randomization (759, 93%) with only 56 patients (7%) randomized to receive LDAC. Both CR (19.4% for G and 17.4% for TC), and OS Hazard Ratio (0.97; 95% CI 0.83-1.14) were similar and not significantly different between G and TC. Many patients in both arms did not receive the recommended minimum of 4 cycles (42.4% vs 40.8% for G vs TC respectively), or 6 cycles (54.2% vs 53.8% for G vs TC). The proportions were well balanced between the 2 treatment arms. Characteristics of patients who received at least 4 or 6 cycles were also well balanced between the 2 treatment arms for age, PS 2-3, secondary AML, poor risk cytogenetics, BM blasts 〉 30%, and proliferative AML (total white cell count ≥20,000/uL). The primary reasons and proportions for treatment discontinuation were similar for the 2 treatments arms. For patients with 〈 4 and 〈 6 cycles respectively they are, in descending order, early deaths (16.7% and 20.7% of the overall ITT population), progression (7.6% and 11.7%), adverse events (5.8% and 6.9%), and patient decision (5.5% and 7.1%). In patients who received at least 4 cycles more patients achieved CR on G (33.6%) vs TC (28.6%), and median OS was longer on G (15.6 months for G vs 13 for TC, HR 0.78, 95% CI 0.64-0.96, log-rank p 0.02, Fig 1). Similarly, in patients who received at least 6 cycles, there were more CR on G (40.1%) vs TC (36.2%) and median OS was longer on G (19.5 months for G vs 15.0 for TC, HR 0.69, 95% CI 0.54-0.88, log-rank p 0.002, Fig 2). Subgroup analyses of OS in patients who received at least 4 or 6 cycles showed that survival benefit from G over TC was consistent in all prospective subgroups including against each of the 3 TCs (AZA, DEC, and LDAC). OS analyses in patients who received at least 4 or 6 cycles also favored G vs TC in both responders (CR, CRp, CRi, or PR) and non-responders with maximum benefit in patients who received at least 6 cycles (G vs TC OS HR 0.66, 95% CI 0.45-0.96, log-rank p 0.028 for responders, and HR of 0.73, 95% CI 0.53-1.00, log-rank p 0.048 for non-responders). Summary/Conclusions: In a large global 815-patient randomized study of G vs TC composed mainly of first generation HMAs, G was at least as effective as TC based on the primary ITT analysis of CR and the narrow 95% CI of OS HR (0.83-1.14). Analyses of patients by number of treatment cycles showed that those who received at least 4 or 6 cycles achieved longer OS in G vs TC with the largest benefit in those who received at least 6 cycles. The benefit was observed in all subgroups, and in both responders and non-responders. Treatment with single agent guadecitabine should continue as long as the patient can still benefit and for at least 6 cycles to gain the maximum survival benefit. Disclosures Roboz: Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding. Krauter:Pfizer: Honoraria. Robak:Takeda: Consultancy, Research Funding; UCB: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grant, Research Funding; Amgen: Consultancy, Other: Travel grant; Roche: Consultancy, Other: Travel grant, Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel grant, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Acerta: Research Funding; Morphosys AG: Research Funding. Kantarjian:Agios: Honoraria, Research Funding; Astex: Research Funding; Jazz Pharma: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Novartis: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Takeda: Honoraria; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Ariad: Research Funding. Novak:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel,Accommodations; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel,Accommodations. Jedrzejczak:Takeda: Consultancy; Amgen: Consultancy, Other: travel support for hematology meetings; Celgene: Other: travel support for hematology meetings; Novartis: Research Funding; Roche: Other: travel support for hematology meetings. Thomas:PFIZER: Honoraria; ABBVIE: Honoraria; DAICHI: Honoraria; INCYTE: Honoraria. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria. Brandwein:Jazz Pharma: Consultancy, Honoraria; Otsuka: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Novartis: Consultancy, Honoraria. Demeter:Angelini: Other: Advisory Board; Pfizer: Other: Advisory Board; Novartis: Other: Advisory Board; Bristol Myers Squibb: Other: Advisory Board; Amicus: Other: Advisory Board; Amgen: Other: Advisory Board; Roche: Other: Advisory Board. Griffiths:Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Genentech, Inc.: Research Funding; Appelis Pharmaceuticals: Other: PI on a clinical trial; New Link Genetics: Consultancy; Novartis Inc.: Consultancy; Novartis Inc.: Consultancy; Onconova Therapeutics: Other: PI on a clinical trial; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Abbvie, Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; Onconova Therapeutics: Other: PI on a clinical trial; Partner Therapeutics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial. Yee:Agensys, Astex, Hoffman La Roche, MedImmune, Merck, Millenium, Roche/Genentech: Research Funding; Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas, Celgene, Otsuka, Shire, Takeda: Membership on an entity's Board of Directors or advisory committees. Hao:Astex Pharmaceuticals, Inc.: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Fenaux:Celgene Corporation: Honoraria, Research Funding; Aprea: Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding.

Abstract: Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aα chain - fibrinopeptides A and the Bβ chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined two unrelated boys aged 4 (boy 1) and 14 (boy 2) for susp. dysfibrinogenemia. Routine coagulation tests revealed prolonged thrombin and reptilase time in both boys and also showed decreased functional fibrinogen levels in both kids (Tab. 1). Boy 1 presented with bleeding manifestation – easy bruising, epistaxis, and bleeding after tooth extraction. His 26-year-old mother presented with similar coagulation findings and with mild bleeding complications. Boy 2 was asymptomatic. Fibrin polymerization experiments carried out on plasma samples showed prolonged lag time and significantly reduced final turbidity in both cases. Measurement of kinetics of fibrinopeptide release showed a decreased amount of the released fibrinopeptide A in both patients. DNA sequencing of boy 1 revealed a point mutation in exon 2 of the FGA gene at the position 3456 G/A, which causes the substitution of Aα 16 Arg to His (fibrinogen Praha V). The boy was found to be heterozygous for the mutation as well as his mother. DNA analysis of boy 2 revealed the same point mutation in the FGAgene, causing the same substitution of Aα 16 Arg to His (fibrinogen Kralupy nad Vltavou). Conclusion In this study we report two cases of congenital defects in the fibrinogen Aα Arg16-Gly17 bond found during childhood. It has been described earlier that the replacement of Aα 16 arginine by histidine decelerates thrombin catalyzed fibrinopeptide A release. Although both kids presented with the same genetic defect and with similar coagulation results, they have different clinical manifestation of the disease. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Table 1: Routine coagulation test results Boy 1 Boy 1's mother Boy 2 Normal APTT 40.1 s 34.6 s 35.3 s 27.5 - 36.1 s Prothrombin time 17.5 s 14.4 s 16.9 s 11.7 - 15.1 s Thrombin time 40.9 s 36.2 s 44.8 s 17.6 - 21.6 s Reptilase time 58.3 s 51.6 s 55.7 s 16.0 - 20.0 s Fibrinogen (Clauss) 0.51 g/l 1.12 g/l 0.58 g/l 2.00 - 4.20 g/l Fibrinogen (Immuno) 2.47 g/l 2.68 g/l 2.31 g/l 2.00 - 4.20 g/l Age 4 26 14 - Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Guadecitabine (G) is a next generation subcutaneous hypomethylating agent (HMA) resistant to degradation by cytidine deaminase which results in prolonged in vivo exposure to the active metabolite decitabine. We conducted a large global randomized phase 3 study of G vs TC of azacitidine (AZA), decitabine (DEC), or low dose Ara-C (LDAC) in 815 TN AML patients unfit for IC (ASTRAL-1 study). The ITT results for the primary endpoints of Complete Response (CR), and Overall Survival (OS) were previously presented (Fenaux et al, EHA abstract S879, 2019). There is no consensus on definition of disease progression particularly with HMA treatment which may continue to benefit patients in the absence of objective response. EFS analysis based on end of treatment benefit (treatment discontinuation, or start of an alternative therapy, or death) regardless of progression may offer a simpler way of assessing HMA treatment benefit. We describe here the results of the study based on both PFS and EFS analyses and how they compare with OS analyses in the overall ITT population, and in subgroups of patients based on number of cycles administered. M ethods: TN-AML ineligible for IC due to age ≥ 75 y, or comorbidities, or ECOG PS 2-3 were randomized 1:1 to either G (60 mg/m2/d SC for 5-days Q28 days) or a preselected TC of AZA, DEC, or LDAC at their standard regimens. AML diagnosis, and response status by IWG 2003 criteria, were assessed by an independent central pathologist blinded to randomization assignment. CR and OS were co-primary endpoints. PFS was a secondary endpoint calculated from date of randomization to the earliest date of progression by investigators or central assessment, relapse after response, start of an alternative therapy, or death. Since progression date is sometimes difficult to ascertain under HMA treatment, an EFS analysis was conducted post hoc using the concept of time to treatment failure. EFS was therefore calculated from date of randomization to the earliest date of discontinuation of randomized treatment, start of an alternative therapy, or death. PFS, EFS, and OS data are presented for the overall ITT population, and for patients who received at least 4 cycles or 6 cycles, and patients who had an objective response. Results: 815 patients were randomized to G (408) or TC (407). Preselected TCs prior to randomization were DEC (43%), AZA (42%), and LDAC (15%). Baseline variables were well balanced across the 2 treatment arms. The majority of patients were randomized to receive an HMA: 759 patients (93%) with only 56 patients (7%) randomized to receive LDAC. In the primary ITT analysis, CR (19.4% for G and 17.4% for TC), and OS Hazard Ratio (0.97; 95% CI 0.83-1.14) were not significantly different between G and TC. An equal proportion of patients received at least 4 cycles (57.6% for G vs 59.2% for TC), or 6 cycles (45.8% for G vs 46.2% for TC) so there was no obvious bias in terms of adherence to treatment in the 2 study arms. Table shows OS, PFS, and EFS median survival, G/TC HR with 95% CI, and p values for the primary ITT population as well as for patients who received at least 4 cycles (N=476 patients), and those who received at least 6 cycles (N=375 patients). G/TC HR for all analyses favored guadecitabine (HR 〈 1). However only OS, and EFS seemed to significantly favor G in patients who received adequate treatment duration by number of cycles. EFS was also the only analysis to significantly favor G in the overall ITT population suggesting that it may be a better predictor of OS benefit in patients who went on to receive adequate treatment with at least 4 or 6 cycles (Table). In addition, EFS also significantly favored G in patients who achieved an objective response (CR, CRp, CRi, or PR): median EFS for G 17.4 vs 14.6 m for TC, HR 0.68, 95% CI 0.5-0.93, p 0.016. Summary/Conclusions: In a large global 815-patient randomized study of G vs TC (composed mainly of first generation HMAs), EFS analyses that do not rely on progression date which is sometimes difficult to define favored G over TC in the ITT population, and seemed to better predict OS benefit in patients who went on to receive at least 4 or 6 cycles. EFS calculated from date of randomization to the earliest date of randomized treatment discontinuation, start of alternative therapy, or death as conducted here could be a simple surrogate for cessation of treatment benefit particularly for patients treated with HMAs. Table Disclosures Fenaux: Celgene Corporation: Honoraria, Research Funding; Aprea: Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding. Roboz:Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding. Robak:Takeda: Consultancy, Research Funding; UCB: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grant, Research Funding; Roche: Consultancy, Other: Travel grant, Research Funding; Amgen: Consultancy, Other: Travel grant; Abbvie: Consultancy, Honoraria, Other: Travel grant, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Acerta: Research Funding; Morphosys AG: Research Funding. Kantarjian:Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; Novartis: Research Funding; Immunogen: Research Funding; Astex: Research Funding; Ariad: Research Funding; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Jazz Pharma: Research Funding; Amgen: Honoraria, Research Funding. Novak:Janssen: Other: Travel,Accommodations; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel,Accommodations; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Jedrzejczak:Roche: Other: travel support for hematology meetings (ASH, EBMT, EHA) ; Novartis: Research Funding; Takeda: Consultancy; Celgene: Other: travel support for hematology meetings (ASH, EBMT, EHA) ; Amgen: Consultancy, Other: travel support for hematology meetings (ASH, EBMT, EHA) . Thomas:ABBVIE: Honoraria; PFIZER: Honoraria; DAICHI: Honoraria; INCYTE: Honoraria. Miyazaki:Kyowa-Kirin: Honoraria; Dainippon-Sumitomo: Honoraria; Nippon-Shinyaku: Honoraria; Novartis: Honoraria; Chugai: Research Funding; Otsuka: Honoraria. Brandwein:Jazz Pharma: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Otsuka: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Research Funding. Demeter:Pfizer: Other: Advisory Board; Amgen: Other: Advisory Board; Bristol Myers Squibb: Other: Advisory Board; Novartis: Other: Advisory Board; Amicus: Other: Advisory Board; Angelini: Other: Advisory Board; Roche: Other: Advisory Board. Griffiths:Celgene, Inc: Consultancy, Research Funding; Genentech, Inc.: Research Funding; Genentech, Inc.: Research Funding; Abbvie, Inc.: Consultancy; New Link Genetics: Consultancy; Celgene, Inc: Consultancy, Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Boston Scientific: Consultancy; Partner Therapeutics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Boston Scientific: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Abbvie, Inc.: Consultancy, PI on a clinical trial; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; New Link Genetics: Consultancy; Onconova Therapeutics: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Novartis Inc.: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding. Yee:Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, Hoffman La Roche, MedImmune, Merck, Millenium, Roche/Genentech: Research Funding; Astellas, Celgene, Otsuka, Shire, Takeda: Membership on an entity's Board of Directors or advisory committees. Hao:Astex Pharmaceuticals, Inc.: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding.