ALBERT

Description: Introduction Refractory anemia with ring sideroblasts (RARS), as a form of acquired primary sideroblastic anemia, represents about 11% of myelodysplastic syndromes (MDS) and is classified as a low risk MDS. It´s defined by the WHO as a pure dyserythropoietic disorder with presence of 〉15% ringed sideroblasts in the bone marrow. Abnormal expression of several genes of heme synthesis in this MDS subgroup and excessive accumulation of iron especially in mitochondria, the main place of reactive oxygen species (ROS) formation, contributes to the elevated oxidative stress. Oxidative stress is also known as one of the factors involved in the pathogenesis of MDS. High iron concentrations catalyse a Fenton reaction, where a hydroxyl radical is produced from hydrogen peroxide and causes an increase in ROS which may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. The aim of this study was to find a useful method for detection and identification of oxidatively modified proteins in plasma unique for RARS patients. Methods Carbonylated protein levels were determined spectrophotometrically using dinitrophenylhydrazine (DNPH) derivatization. Oxidatively modified proteins of plasma samples were derivatized with biotin hydrazide. The dialyzed biotin hydrazide labeled samples and negative controls were mixed with monomeric avidin resin. Captured carbonylated proteins were digested by trypsin and then identified by MS/MS mass spectrometry coupled to a nano-LC system. Mascot (Matrix Science, London, UK) was used for database searching (Swiss-Prot). Two unique peptides (with a higher Mascot score than the minimum for identification, P

Description: 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.

Description: Introduction Acute myeloid leukemia (AML) represents an aggressive bone marrow malignancy with diverse genetic abnormalities. The tumor cells, to support their own growth and proliferation, may alter metabolism 1) to accelerated glycolysis to provide energy/biosynthetic precursors and 2) to produce intermediates by active TCA cycle for synthesis of essential biomolecules [1]. Mutations of isocitrate dehydrogenase (IDH1/2) as frequent mutations (affecting approximately 20% of AML patients) cause the reduction of α-ketoglutarate to D-2-hydroxyglutarate instead of oxidative decarboxylation of isocitrate to α-ketoglutarate. IDH2 mutations occur almost exclusively in hematopoietic tumors. Whereas IDH2R140 mutation is frequently accompanied by normal cytogenetics and NPM1 mutations, IDH2R172 is frequently the only mutation detected in AML. In addition, IDH2R172 confers a poor prognosis in patients with AML [2, 3]. The aim of this study was to characterize and uncover the differences in metabolism of AML patients with or without IDH2 mutations in diagnosis (the initial stage), in remission (after chemotherapy treatment), and before transplantation (after conditioning). Methods Serum metabolomics profiles were generated with samples obtained at diagnosis, in remission, and before transplantation from patients (n=20) treated in the Institute of Hematology and Blood Transfusion, Prague, Czech Republic. Patients were assigned in IDH2WT (n=7) and IDH2R140/ IDH2R172 (n=7/6) groups. Targeted metabolomic profiling of 19 metabolites related mainly to TCA cycle and glycolysis was performed by LC-MS/MS. Results Using non-parametric randomized block analysis of variance (Friedman test) we found significant differences in levels of 13 metabolites among treatment periods for all AML patients. Moreover, when comparing each treatment periods for all patients, we found significantly decreasing levels of 12 metabolites between the initial stage and the period before transplantation. If we considered the division of patients into groups according to IDH2 mutations, we found significantly different levels of 4 metabolites among treatment periods for samples of patients with IDH2R140,namely 2 metabolites of TCA cycle: isocitrate, succinate; and 3-hydroxybutyrate and urate. We obtained significantly different levels in the same number of other metabolites for IDH2WT. In samples of patients with IDH2R172 we found significantly different levels of 8 metabolites among treatment periods, namely 5 metabolites of TCA cycle: citrate, 2-hydroxyglutarate, succinate, fumarate, malate; 2 metabolites of glycolysis: 3-phosphoglycerate, phosphoenolpyruvate; and pyroglutamate of glutathione metabolism. Furthermore, using Nemenyi post-hoc test we ascertained significantly decreasing levels of named metabolites in IDH2R140 and IDH2R172 samples before transplantation with respect to the initial stages of AML (diagnosis). Conclusion Overall, this study identified significant changes in several metabolites of TCA cycle and glycolysis between initial and final treatment periods and also between remission and period before transplantation in AML patients. With respect to IDH2 mutations we found more significant changes in metabolites specifically in TCA cycle for IDH2R172 patients relative to IDH2R140. The results of our preliminary targeted metabolomic profiling of AML patients with IDH2 mutations are corresponding with the already described differences in morphological and genetic patterns in the patients and, moreover, support the classification of IDH2R172 as separate AML subtype. Metabolomic profiling thus seems to be a new valuable tool providing additional important information. Acknowledgment This work was supported by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports), by the project of the Ministry of Health, Czech Republic, 00023736, by Grant from the Academy of Sciences, Czech Republic, P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001. [1] DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. Cell Metab. 2008;7(1):11-20. [2] Rakheja D, Konoplev S, Medeiros LJ, Chen W. Hum Pathol. 2012;43(10):1541-51. [3] Meggendorfer M, Cappelli LV, Walter W, Haferlach C, Kern W, Falini B, Haferlach T. Leukemia. 2018;32(5):1249-1253. Disclosures No relevant conflicts of interest to declare.

Description: 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 (Aa, Bb, and g) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aa chain - fibrinopeptides A and the Bb 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 four unrelated children suspected dysfibrinogenemia. The first patient was a 5-yr old boy with prolonged thrombin time and low functional fibrinogen level. He presented with easy bruising and epistaxis. Genetic analysis revealed a heterozygous substitution Aalpha R16H. The second patient was a 13-yr old girl with prolonged thrombin and reptilase times and low functional fibrinogen level. She presented with menorrhagia. Genetic analysis revealed a heterozygous substitution Aalpha G17V. The third patient was a 12-yr old asymptomatic boy. Genetic analysis revealed a heterozygous substitution Aalpha R16H. Kinetics of fibrinopeptide release was impaired in all these cases. The fourth patient was a 3-yr old girl with low functional fibrinogen level. She presented with easy bruising. Genetic analysis revealed a heterozygous substitution Aalpha K448N. All patients presented with impaired fibrin polymerization. Conclusion All patients were found to be heterozygous for point mutations in FGA gene. Three mutations were found in the site of fibrinopeptide cleavage and one in the alphaC-domain. Mutations had different clinical manifestations from asymptomatic to bleeding. Mutations Aalpha Arg16His and Aalpha Gly17Val are among the most common fibrinogen mutations and both decelerate fibrinopeptide A cleavage from mutant fibrinogens. We describe here a novel previously unreported mutation Aalpha Lys448Asn affecting fibrin formation. 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. Disclosures No relevant conflicts of interest to declare.

Description: Background Cardiovascular diseases are linked with oxidative stress which is the source of reactive oxidative and nitrative species, contributors of post-translational modification. Fibrinogen due to its high concentration in blood is considered as one of the most sought of targets of oxidative stress substances. Post-translational modifications of fibrinogen might influence its physiological function, thus affect hemostasis in the terms of fibrin nets forming and architecture or interaction with platelets. The aim of this study was to observe influence of in vivo fibrinogen modifications on formation of fibrin net and to identify amino acid residues prone to changes related to oxidative stress. Methods Plasma samples were collected from patients of The Military University Hospital Prague in the agreement with ethical committees of participating institutions and with informed consents from all subjects. Samples were divided into 4 groups: patients with acute coronary syndrome (A), patients with stroke (B), patients with thrombus localized in carotid vein (C) and control group (patients without coronary atherosclerosis; D). Fibrin net architecture was studied by scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). For identification of modified amino acids residue mass spectroscopy was used (Triple TOF 6600, Sciex). Molecular dynamics simulations of hydrated protein were performed in Gromacs software with Gromos force fields. Crystal structure 3GHG was used as a reference structure to which post-translational modifications were introduced manually in Yasara View. Results We found extensive both qualitative and quantitative changes in the structure of fibrinogen molecule in all groups of patients. Oxidative stress level differed among patient groups and between the control group. Different oxidative changes caused by in vivo modifications of fibrinogen affected quite distinctly the architecture of fibrin net. Modified amino acids were detected in all three fibrinogen chains. In gamma chain the localisation of modified amino acid residues correlated with the part of fibrinogen important for fibrin polymerisation. The impact of the most pronounced post-translational modifications on the secondary structure of fibrinogen was described by molecular dynamics simulations. Conclusions The results show that the degree of impairment of fibrinogen functions in the cardiovascular diseases is related to the level of oxidative stress. Characterization of oxidative fibrinogen modification and its precise meaning to the function of fibrinogen in hemostasis appears to be extremely helpful to better understanding of thrombotic/bleeding complications linked with various cardiovascular diseases. Acknowledgments This work was supported by the Ministry of Health, Czech Republic, no. 00023736, by the Academy of Sciences, Czech Republic no. P205/12/G118 and NV18-08-00149, by ERDF OPPK CZ.2.16/3.1.00/24001 and by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports). Disclosures No relevant conflicts of interest to declare.

Description: Omega-3 polyunsaturated fatty acids (ω-3PUFAs) are introduced into parenteral nutrition (PN) as hepatoprotective but may be susceptible to the lipid peroxidation while olive oil (OO) is declared more peroxidation resistant. We aimed to estimate how the lipid composition of PN mixture affects plasma and erythrocyte lipidome and the propensity of oxidative stress. A cross-sectional comparative study was performed in a cohort of adult patients who were long-term parenterally administered ω-3 PUFAs without (FO/–, n = 9) or with (FO/OO, n = 13) olive oil and healthy age- and sex-matched controls, (n = 30). Lipoperoxidation assessed as plasma and erythrocyte malondialdehyde content was increased in both FO/– and FO/OO groups but protein oxidative stress (protein carbonyls in plasma) and low redox status (GSH/GSSG in erythrocytes) was detected only in the FO/– subcohort. The lipidome of all subjects receiving ω-3 PUFAs was enriched with lipid species containing ω-3 PUFAs (FO/–˃FO/OO). Common characteristic of all PN-dependent patients was high content of fatty acyl-esters of hydroxy-fatty acids (FAHFAs) in plasma while acylcarnitines and ceramides were enriched in erythrocytes. Plasma and erythrocyte concentrations of plasmanyls and plasmalogens (endogenous antioxidants) were decreased in both patient groups with a significantly more pronounced effect in FO/–. We confirmed the protective effect of OO in PN mixtures containing ω-3 PUFAs.

Description: Atherosclerosis is a leading cause of major vascular events, myocardial infarction, and ischemic stroke. Tryptophan (TRP) catabolism was recognized as an important player in inflammation and immune response having together with oxidative stress (OS) significant effects on each phase of atherosclerosis. The aim of the study is to analyze the relationship of plasma levels of TRP metabolites, inflammation, and OS in patients with neurovascular diseases (acute ischemic stroke (AIS), significant carotid artery stenosis (SCAS)) and in healthy controls. Blood samples were collected from 43 patients (25 with SCAS, 18 with AIS) and from 25 healthy controls. The concentrations of twelve TRP metabolites, riboflavin, neopterin (NEO, marker of inflammation), and malondialdehyde (MDA, marker of OS) were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Concentrations of seven TRP metabolites (TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), anthranilic acid (AA), melatonin (MEL), tryptamine (TA)), NEO, and MDA were significantly different in the studied groups. Significantly lower concentrations of TRP, KYN, 3-HAA, MEL, TA, and higher MDA concentrations were found in AIS compared to SCAS patients. MDA concentration was higher in both AIS and SCAS group (p 〈 0.001, p = 0.004, respectively) compared to controls, NEO concentration was enhanced (p 〈 0.003) in AIS. MDA did not directly correlate with TRP metabolites in the study groups, except for 1) a negative correlation with kynurenine acid and 2) the activity of kynurenine aminotransferase in AIS patients (r = −0.552, p = 0.018; r = −0.504, p = 0.033, respectively). In summary, TRP metabolism is clearly more deregulated in AIS compared to SCAS patients; the effect of TRP metabolites on OS should be further elucidated.

Description: Introduction Myelodysplastic syndromes are clonal hematopoetic stem cell disorders characterized by refractory cytopenias with displasia as a result of ineffective hematopoiesis. Approximately 30 percent of MDS cases progress to acute myeloid leukemia. Excess blasts are the strongest predictors for poor outcome and are associated with disease progression. Because of its heterogeneity and lack of molecular and protein markers that effectively monitor disease progression, clinical management of MDS patients is challenging. The concentrations of several proteins were found elevated during MDS disease. Their interaction with receptors/ligands is a part of signaling pathways that may play a crucial role in pathogenesis of the disease. Serum levels of vascular cell adhesion protein 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) were found higher in MDS patients in comparison with control group and were related to MDS severity. Alpha-2-HS-glycoprotein (FETUA) was analyzed in plasma of MDS patients. Leucine-rich alpha-2-glycoprotein (LRG) was found increased in RAEB-I and RAEB-II patients by proteomic studies. In this work we developed a protein chip for study of molecular interactions in plasma of MDS patients using surface plasmon resonance (SPR) imaging. Interacting proteins were analyzed and identified using LC-MS/MS. Methods Patients with diagnosis of refractory anemia and refractory anemia with ringed sideroblasts (RA/RARS, n=13), refractory cytopenia with multilineage dysplasia (RCMD, n=16), and refractory anemia with excess blasts (RAEB, n=15) as a subgroup of MDS, and MDS patients who progress to acute myeloid leukemia (AML, n=8) were chosen for this study. The high-resolution SPR imaging (SPRi) system with polarization contrast and internal referencing combined with dispersionless microfluidics was used to study protein interactions. Passive mixing structures were employed to further improve the sensing performance of this SPRi biosensor. The respective biomarkers (ICAM1, VCAM1, FETUA and LRG) were covalently attached to the functionalized sensor surface and interaction with their counterparts in MDS plasma samples were monitored. Interacting proteins were removed from the chip surface by diluted NaOH, treated by trypsin, analyzed by nanoLC-MS/MS (TripleTOF 5600) and identified using ProteinPilot database. Results and Conclusion Significant differences were observed between analyzed MDS subtypes for VCAM1, FETUA, and LRG (ANOVA: P = 0,00755, P = 0,0306 and P 〈 0.001 respectively). Using post hoc Tukey contrasts tests we ascertained that sensor responses were significantly higher in group RCMD and AML with respect to the healthy donors for immobilized VCAM1 and in group RA/RARS, RCMD, RAEB and AML with respect to the healthy donors for LRG. Moreover sensor responses were significantly higher in group AML with respect to the RA, RCMD or RAEB for immobilized LRG. Based on our results LRG seems to be a suitable marker of MDS progression. The identified proteins presented in MDS plasma samples unlike in healthy donor plasma are clusterin, protein S100-A8, serum amyloid A-1 protein, annexin A2, and zinc-alpha-2-glycoprotein among others. S100-A8 and clusterin have been connected with MDS or AML previously; it could be tested as a new protein marker of MDS by SPRi. The results show that SPR biosensors combined with nanoLC-MS/MS are a promising tool for improvement of the diagnosis in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Myelodysplastic syndromes (MDS) are a heterogenous group of oncohematologic diseases, characterized by bone marrow hypercellularity, dysplasia of myeloid lineage cells, peripheral cytopenias, and an increased risk of evolution to acute myeloid leukemia (AML). Pathophysiology of MDS is not fully understood, however it has been described that oxidative stress plays an important role in initiation and disease progression. Oxidative stress is defined as an imbalance between prooxidative and antioxidative processes that prefers the production of reactive oxygen species (ROS) over an antioxidant defence. The association between MDS and oxidative stress was discussed in several studies, where functional mitochondrial abnormalities in MDS patients have been demonstrated as one of the possible sources of oxidative stress. Another mechanism could be associated with mitochondrial dysfunction via iron overloading, mitochondrial DNA mutation, systemic inflammation, and bone marrow stromal defects. Protein carbonylation is ROS-mediated protein oxidation and it is defined as an irreversible post-translational oxidative modification. The detection of protein carbonyl groups is a specific useful marker of oxidative stress. The aim of this study was to determine the levels of carbonylated proteins in plasma of MDS patients resp. in plasma of individual subtype of MDS patients and evaluate changes of carbonylated plasma proteome in MDS patients compared with healthy controls. Methods: The content of protein carbonyls was measured after derivatization with dinitrophenylhydrazine by monitoring absorbance between 250 and 500nm. Carbonylated proteins of plasma samples were purified after reaction with biotin hydrazide. The negative controls were processed using the exact same procedure, except biotin hydrazide labeling. The dialyzed biotin hydrazide labeled samples and the negative controls were isolated using monomeric avidin resin. All the samples were prepared in doublets. One part of samples was used for the on bead tryptic digestion and the second part of samples was separated by SDS-PAGE after eluation with Laemmli buffer. The obtained carbonylated proteins were identified by mass spectrometry (LC-MS/MS). Results: We have examined 32 patients with different MDS subtype (10 patients RAEB-1,2; 12 RCMD; 10 RARS) and 20 healthy controls. Diagnoses of different subgroups of MDS were established according to the WHO classification criteria. The median age of patients group was 64,5 and presented 17 males (57 %); the median age of healthy controls group was 43 and presented 10 males (50 %). We have found significantly (P

Description: Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by a high risk of evolution into acute myeloid leukemia (AML). Different processes are involved in its pathogenesis, such as (epi)genetic alterations and immunological dysfunctions. Till now, in plasma several protein markers for MDS were found. One of them is a misfolded receptor protein VEGFR-1 for which we have created a detection assay based on the surface plasmon resonance (SPR) (Pimkova K, Suttnar J, et al., Anal Bioanal Chem. 2012, 402, 381-387). An elevated level of circulating 70-kDa heat shock proteins (Hsp70) has been detected in the blood of patients with myeloid leukemia. The Hsp70 are ATP-dependent molecular chaperones which interacts with unfolded/misfolded and aggregated proteins, thus providing cytoprotective role against various cellular stresses. Hsp70 released from cancer cells can carry peptide antigens including ones specific for a tumour and contribute to the development of anti-cancer immune response. The goal of this study is to identify proteins in HSP70 complexes using SPR biosensor followed by mass spectroscopy (MS) in the blood plasma of patients from MDS subgroups. Methods A six-channel spectroscopic SPR biosensor for detection of protein comlexes in diluted blood plasma samples was used. Preincubated ATP-Hsp70 complex was immobilized on the sensor surface via covalent attachment by dispersionless microfluidics. The biosensor utilizes Hsp70 rate-limiting step of ATPase cycle, ADP/ATP exchange, which allows in an ATP-controlled manner binding of protein substrates and their elution with minimal non specificity. Eluted proteins were subsequently analyzed and identified by using LC-MS/MS (TripleTOF 5600). Patients of MDS (n=10) and MDS patients who progress to AML (n=5) were chosen. Results and Conclusion Using preincubated ATP-Hsp70 complex significant differences in SPR responses of MDS patients in comparison to healthy donors as well as differences among MDS patients were observed. The responses represented hundreds of proteins identified by mass spectrometry. Protein interaction networks were visualized and analyzed by bioinformatics tools which cluster 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. The identified proteins are known to be involved in several processes including ubiquitinylation, protein degradation, cell signaling, stress response, protein synthesis, and also regulation of the immune system. We used Power Graphs, a novel representation of (protein) networks which provided a valuable insight into the presence of protein complexes, their internal organization and relationships. Interaction networks also indicated possible proteins involved in immunomodulatory functions of MDS. 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.