ALBERT

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: Introduction and Objectives: MV in thrombocytopenic pts could be complicated by life threatening tracheobronchial bleeding. The systemic treatment with tranexamic acid or rFVIIa increases the risk of thromboembolic events. Presented in airspace tissue factor (TF) reacts as a cofactor in augmenting the activity of FVIIa 1000-fold. Topical treatment of the inhalation with rFVIIa has not been studied yet. The aim of the study was to evaluate hemostatic effect of rFVIIa inhalation for treatment of the tracheobronchial bleeding in MV thrombocytopenic pts. Materials and Methods: The results of rFVIIa inhalation were investigated in 5 acute leukemia pts (2 males, 3 female). All pts were mechanically ventilated through tracheostomy tube and had life threatening tracheobronchial bleeding. Before intervention all pts had low platelet count 50-120х109/l (median 65 х109/l), lowered Quick prothrombin (55-64%, median 58%), prolonged APTT (55-57s). Results. All pts received rFVIIa as an inhalation over five to ten minutes. For inhalation rFVIIa (Koagil VII, Generium, Russia, Vial of 2.4 mg) was diluted in 10 ml of isotonic saline. If, after 20 min, there is no response to rFVIIa second dose of rFVIIa (2.4 mg) as an inhalation was administrated. Bleeding was stopped in 3/5 pts, in 2 pts the intensity of bleeding decreased. There were no changes in APTT and Quick prothrombin registered. Median plasma FVII activity before inhalation was 82%, after inhalation - 73%. There were no changes on the TEG (figure 1). Thus, despite local hemostatic effect we did not registered systemic effects of rFIIa after inhalation Conclusion. Topical treatment of the tracheobronchial bleeding with rFVIIa inhalation provided hemostatic effect. There were not be systemic adverse effects in as much as the alveolocapillary membrane does not allow the transmembranous passage of rFVIIa. Disclosures No relevant conflicts of interest to declare.

Description: Acute myelogenous leukemia (AML) is the most sensitive to natural killer (NK)-cell reactivity among blood malignancies treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Function of NK cells is regulated by their inhibitory and activating surface receptors, including killer-cell immunoglobulin-like receptors (KIRs). KIRs with specificity to different HLA class I-encoded ligands seem to play a major role in allorecognition: HLA-C allotypes with asparagine at position 80 (HLA-C1ligands) are recognized by KIR2DL2/3, HLA-C allotypes with lysine at position 80 (HLA-C2 ligands) are recognized by KIR2DL1and KIR2DS1, and HLA-B allotypes with a polymorphic sequence motif at position 77-83 (Bw4 ligands) are recognized by KIR3DL1.There are numerous but sometimes contradictory data about the role of KIR genes and genes of their HLA ligands in outcomes after HLA-identical related and HLA-matched unrelated HCST in patients with AML. The goal of our prospective study was to evaluate the influence of KIR genes and HLA class Iligands on overall survival (OS) and event-free survival (EFS) after allo-HSCT in adult patients with AML. 35 patients with AML (median age 36 years, range 19-60) treated with allogeneic HSCT in our transplant center from HLA-identical related (n=19) and HLA-matched (10/10) unrelated (n=16) donors were included in the study. Median follow-up was 18 months (range, 5-56). The pre-transplantation risk category included standard or high risk. Patients in complete remission 1 (CR1) were categorized as standard risk (n=22), whereas patients in 〉 CR1 were considered as high risk (n=13). In case of HLA-identical related HSCT donor KIR genotyping was performed simultaneously with HLA-typing. In case of HLA-matched unrelated HSCT donor KIR genotyping was performed in time of confirmatory HLA-testing or in day of HSCT. KIR genotyping was done using a PCR-SSP kit (Olerup, Sweden). Overall survival (OS) and event-free survival (EFS) were calculated by Kaplan-Meier method, and compared with log-rank test. OS was defined as survival without lethal event from any cause, EFS was defined as survival in complete remission without lethal event from any cause. The 3-year estimated OS and EFS in all patients were 66 and 43%, respectively. 10 patients died (29%), relapse (hematological and/or molecular) was diagnosed in 18 patients (51%). The pre-transplantation risk category was the main factor affecting OS and EFS after HSCT. The probability of the 3-year OS and EFS for standard-risk patients was 86 and 57%, respectively. Nobody of our patients with high risk lived more than18 months. Conditioning regimen, graft source and donor/patient gender combination had no significant effect on OS and EFS. The patients with unrelated HLA-matched donors had better (not significantly) EFS in comparison with recipients of related HLA-identical grafts (p=0.12). The influence of KIR and HLA- ligand genes on EFS after allo-HSCT was investigated in patients with standard risk. We did not find the immunogenetic factors (presence of B- haplotypes in donors, donor KIR B content, presence of centromeric or telomeric B- motifs in donors, presence of missing HLA ligand for any inhibitory KIR of donor), which significantly affected EFS, but we found two obvious tendencies. The patients with HLA-C1/C1 homozygosity had a tendency to improved EFS compared with patients having either HLA-C1/C2 or HLA-C2/C2 ligands (p=0.09). There was a tendency to better EFS in HLA-C1/x recipients of KIR2DS1 -positive allografts (p=0.09). Our data suggest that KIRs and their HLA class I ligands play role in the graft versus leukemia effect in AML. It seems that HLA-C1 homozygosity improves EFS in patients after allo-HSCT, and KIR2DS 1-positive donors are preferable for HLA-C1/x patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2696 Background: Follicular lymphoma (FL) has considerable clinical heterogeneity. There is a need for easily quantifiable prognostic biomarkers. There is no consensus on the prognostic significance of microvessel density. Aim: Characterize the blood and lymphatic vessel density of tumor tissue basing on immunohistochemical study (IHC) of sections from paraffin blocks of lymph nodes biopsies in two groups of patients with different clinical course. Patients and Methods: The study included 59 patients with follicular lymphoma: 39 women (67%) and 20 men (33%). The mean and median age was 53 years (range: 27–83 years). 49 patients were observed in the National Research Center for Hematology (Moscow) from April 2001 to May 2011 and 10 patients – in Cancer Research Center (Moscow) at the same period of time. The first group (1) included 28 patients followed during 2 years or more without therapy; with remission of the underlying disease; with relapse in five or more years from the start of their treatment (late relapse). The second group (2) included 31 patients who died due to tumor progression in the first 1–2 years from time of putting this diagnosis; with resistance to the passage of the tumor; with relapsed FL in the first year from the start of treatment (early relapse). Patients in both groups received the same initial treatment. Vascularization of tumor tissue was assessed by IHC on sections from paraffin blocks of tumor biopsies of lymph nodes from the visualization of blood and lymphatic vessels using antibodies CD34 and D2–40. CD34 is an endothelial marker of blood vessels. D2–40 is a novel monoclonal antibody to an Mr 40,000 O-linked sialoglycoprotein that reacts with a fixation-resistant epitope on lymphatic endothelium. Morphometric analysis was performed using light microscopy and a digital camera Leica (vol. × 40). The pictures are processed by a computer program “VideoTesT-Morphology 5.2” in order to estimate the specific area of vessels in relation to the tumor tissue (%) during visual inspection of the researcher. IHC-specimen evaluation was carried out using a table of random numbers. Results: 5-year OS rate in group 1 was significantly higher than in group 2 (83±7% vs 28±13%; ð=0,03) (Figure 1). Evaluation of the specific area of blood and lymph vessels was conducted in two comparable groups. The specific area of blood vessels in group 2 was significantly higher than in group 1: 0,04 (95% CI, 0,03–0,05) vs 0,02 (95% CI, 0,01–0,03) (Figure 2). The specific area of the lymph vessels in group 2 was significantly higher than in group 1: 0,06 (95% CI, 0,04–0,07) vs 0,03 (95% CI, 0,01–0,04) (Figure 3). Conclusion: The microvascular density can be estimated using the markers CD34 and D2–40. Our results demonstrate an association between prognosis and angiogenic sprouting and revealed higher angiogenic activity in the poor prognostic group. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2572 Adult ALL regardless recent advantages in adolescents and young adults is still considered to be a therapeutical problem. So called “a pediatric-like approach” applied in adult ALL is reported to be more reasonable even in the older adults (up to 55 y). RALL has initiated a prospective multicenter trial for adult Ph-neg ALL based on: 1) evaluation of b/m blast clearance after 7 days of Prednisolone (PRD) prephase and its substitution by Dexamethasone (DEXA) if b/m blast count was 〉25%; 2) continuous 2,5 years treatment schedule with prolonged L-asparaginase (Σ=590.000 IU); 3) evaluation of the impact of the late intensification (2 courses of HD of methotrexate and ARA-C) on MRD clearance. The study is registered on the ClinicalTrials.gov public site; NCT01193933. From Nov, 2008, till June, 2012, 24 centers enrolled 173 pts: median age 28 y (15–55), 71f/101m, 112 (64,7%) = B-lin, 54 (31,2%) = T-lin, 1 pt -undifferentiated AL (0,6%), 6 - uknown phenotype (3,5%), median LDH=848 ME (72–13061), median L=13,5 (0,6–556*109/l). Cytogenetics was evaluable in 58,3% of pts (n=101) and 46,5% of them (n=47) had normal karyotype (NK). Initial risk group was evaluated in 154 pts among whom 46 patients (29,9%) were in the standard risk (SR) group (WBC 100 for T-Lin, EGIL BI, T-I-II-IV; LDH 〉 2N, No late CR, t(4;11)-positive). 19/173 pts (11%) were not qualified. The analysis was performed in June, 2012. +8 day b/m blast count was reported in 149 pts and b/m blasts less than 25% were detected in 36,2% of pts. The portion of PRD non-responders was statistically different in SR and HR groups: 24/45 (53,3%) and 68/101 (67,3%) (p=0,01), confirming the initial risk groups identification. Induction results were obtained in 150 pts, and CR rate was identical in both risk groups (SR=86,9%; HR=84,1%) with total 12 induction deaths (8,0%) and 6 resistant leukemias (4,0%). With a median follow-up of 12 mo (1–36 mo) death in CR was reported in 9/132 (6,8%) pts. OS at 36 mo was 58,6%, DFS-68,3%. MRD analysis for clonal IgH and TCR rearrangements was carried out in 25 pts. And as in our previous studies (ASH 2006, abstr 2294) the clearance was slow with only 41,6% pts negative for MRD at day +133 (4mo) of the protocol. Two late intensification courses (day +157 = 5 mo) increased MRD negativity only up to 50%. Such slow MRD clearance did not correspond to higher relapse rate so far. Age, WBC, immunophenotype, LDH, risk group, +8 day b/m blast count, time to CR, time without treatment (8days), L-asparaginase cessation did not influence survival. OS and DFS differed in pts with NK vs all other abnormalities: 87,4% vs 57,9% (p=0,002) and 88,9% vs 66,5%, respectively (p=0,02). So, our data demonstrated that ALL-2009 protocol provided 58% 3-years overall survival and 68,3% DFS. In adult Ph-neg ALL normal karyotype predicts better survival. The MRD clearance is very slow while on this protocol. Disclosures: No relevant conflicts of interest to declare.

Description: ALK-negative anaplastic large T-cell lymphoma (ALK-negative ALTCL) is a rare aggressive disease, constituting 12% of all T-cell lymphomas of adults. When using CHOP/CHOEP chemotherapy overall and event-free survival is less than 50%. Factors of poor prognosis include IPI (3-5), CNS and skin involvement as well as molecular predictors. The aim of current study was to detect rearrangements (R) of DUSP22 gene, t(6;7)(p25.3;q32.3), inv(3)(q26q28), involving TP63 gene using fluorescence in situ hybridization and ТР63 mutation by Sanger sequencing. 23 patients with ALK-negative ALTCL were involved in current study: 14 males, 9 females. Advanced stage of disease was diagnosed in 70 % of patients. All patients were treated with high-dose chemotherapy. In 20% of patients were revealed DUSP22-R, 13% - TP63 abnormalities, 67% of patients were triple-negative (without ALK-R, DUSP22-R, TP63-R). There weren't detected any mutations in DNA-binding domain of TP63 gene. Comparing data of molecular studies and clinical data it was revealed that presence or absence of above-mentioned gene rearrangements did not affect disease stage: III stage was diagnosed in 30% of cases with DUSP22-R and in 33% of triple-negative cases, IV stage in 100% cases with TP63-R, in 67% with DUSP22-R, in 40% with triple-negative variant. Majority of patients had IPI (3-5) in all three cytogenetic groups. In 100% of cases with DUSP22-Rwere revealed more than 2 extranodal site of involvement. Overall (OS) and progression-free survival (PFS) depending on molecular profile were 100% and 100%, respectively, in case with DUSP22-R. In case of TP63-R OS and PFS were 8% and 10%, respectively, in triple-negative cases - 60% and 39%, respectively. Thus, detection of DUSP22, TP63 gene rearrangements has great practical value in ALK-negative ALTCL. Statistical significance should be confirmed by further study. Disclosures No relevant conflicts of interest to declare.

Description: CONTEXT: Many hematological and non-hematological diseases can be hidden under the mask of isolated thrombocytopenia. The choice of therapeutic tactics is determinated by correct diagnosis. OBJECTIVE: to define the frequency of occurrence of primary immune thrombocytopenia (idiopathic thrombocytopenic purpura-ITP) in the group of patients with isolated thrombocytopenia. Materials and methods: We analysed clinical and laboratory data of 301 patients who applied to the outpatient department of National Research Center for hematology, Russian Federation with thrombocytopenia of unspecified origin. The first group is 183 patients who applied for the first time. The second group is 118 patients with long history of ITP. All patients were examined according to the extended differential diagnostic protocol used in isolated thrombocytopenia and based on international and National clinical recommendations for the diagnosis and treatment of ITP in adults. Results: Median age of patients in both groups was 36 years, male/female ratio in group 1 was 1:2, in group 2 - 1:4. In group 1, the count of platelets in the blood was more than 50*109/l in 87% of cases, while in the second group, in most cases (94%), there was a decrease in the count of platelets

Description: The stromal microenvironment regulating hematopoiesis in patients with hematologic malignancies undergoes significant alterations. The changes in the concentration of colony-forming fibroblast units (CFU-F) in the bone marrow (BM) and disruption in the functioning of multipotent mesenchymal stromal cells (MSCs) are shown in many studies for patients with acute leukemia. Often it is not possible to distinguish the cause of changes in stromal progenitor cells after treatment: interaction with tumor cells or the effects of therapy. Most of the patients with diffuse large B-cell lymphoma (DLBCL) do not have BM involvement. It was assumed that the properties of MSCs in these patients were not changed, and so this could be an attractive model for investigation the effect of antitumor drugs on human BM stromal microenvironment. The aim of the study was to compare the properties of MSCs in patients with DLBCL in the onset of the disease and a month after the end of therapy. Methods The study included 20 patients with DLBCL (11 male, 9 female) aged 42-60 years in the onset of the disease and a month after the end of treatment. 3-5 ml of BM were collected during diagnostic punctures after informed consent. MSCs and CFU-F were cultured by standard methods. The total MSCs production, the doubling-population level per day, the concentration of CFU-F, the relative gene expression level (REL) in MSC by real-time PCR and the mean fluorescence intensity (MFI) by flow cytometry were analyzed. The control group included 31 donors of the corresponding age. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Results The total cell production for 4 passages in primary patients' MSCs was significantly higher than in donors (11.4 ± 2 x 106 per flask versus 6.9 ± 1.1, p = 0.04). It remained elevated after therapy (10.2 ± 1.5). At the same time, the MSCs population-doubling level per day was significantly decreased in patients in comparison with donors (0.6 ± 0.03 vs. 0.4 ± 0.04, p

Description: Abstract 4874 Background Primary bone lymphoma (PBL) is a rare extranodal type of tumor. A diagnosis of diffuse large B-cell lymphoma (DLBCL) is made in 90% of all cases. The treatment standard for PBL is ÑÍÎ−D chemotherapy (CT) combined with radiation therapy. In the group of PBL patients with a local (IE) stage and without any unfavourable prognostic factors (elevated lactate dehydrogenase (LDH) concentrations, Â-symptoms, tumor size ≥10 cm) treated with standard chemotherapy and radiation, the relapse-free 5-year survival rate is as high as 80% to 100%. In advanced disease stages (stage II, IVE and any unfavourable prognostic factor) this treatment strategy leads to a remission in just 20% of patients (Ramadan KM, Shenkier T et al. Ann Oncol 18:129–135, 2007). Therefore, the results achieved with the ÑÍÎ−D regimen and radiation therapy in the treatment of adult patients with advanced PBL are unsatisfactory. Further research is thus needed to boost treatment efficacy for patients with advanced PBL. One of the ways to overcome the chemotherapy resistance of the tumor is primary intensification of CT. For instance, according to Children's Cancer Group data, high-dose CT was effective in children suffering from advanced PBL (Lones MA, Perkins SL et al. Clin Oncol 20:2293–2301, 2002). Intensive CT has not been used previously in adult patients with PBL. Purpose To evaluate the efficacy of intensive CT according to the mNHL-BFM-90 program in adult patients with advanced-stage PBL. Materials and methods The study enrolled all patients (22 subjects in total) with advanced-stage PBL monitored by the Research Centre of Haematology in the period from 2007 to 2012; 13 were male and 9 female, aged from 16 to 69 years (median age, 39 years). According to the WHO classification, all patients were diagnosed with DLBCL. The following evaluations were performed for disease staging: physical examination and medical history, complete blood count and blood chemistry, chest and abdominal computed tomography, whole-body positron emission tomography using 18F-fluorodeoxyglucose, magnetic resonance imaging and computed tomography of affected bones. Tumor staging was done according to the Ann-Arbor classification. Local IÅ stage (involvement of 1 bone) was verified in 6 cases (27%), stage IIÅ (involvement of regional lymph nodes) in 3 subjects (13%), and stage IVÅ (multiple bone involvement) in 13 patients (60%). Patients with the IIIÅ stage (distant nodal disease) were not recruited in this study. The main symptoms of the disease included pain and swelling. LDH elevation was observed in 13 cases (60%), Â-symptoms were found in 15 patients (68%), and large-size tumors were seen in 20 subjects (90%). The most common tumor sites were the skull bones, vertebrae, and femur. The average ECOG score for global condition assessment was 2 at the time of CT initiation. All patients received treatment according to the mNHL-BFM-90 intensive CT program (Reiter A, Schrappe M. et al. Blood 94(10): 3294–3306, 1999). This program was modified: adriamycin was administered on the 3rd day of course ÀÀ at a dose of 50 mg/m2. Methotrexate was administered on the 1rd day of course C. The dose of methotrexate was reduced to 1.0 g/m2 and the administration time shortened to 12 hours. Leucovorin was administered in 6 hours after the end of the methotrexate infusion until the serum methotrexate concentration fell below 0.1 μmol/L. The toxicity of this treatment was assessed using the NCIC Common Toxicity Criteria. Results Complete remissions were achieved in 20 patients (90%; 95th% CI, 70% to 95%). The mean follow-up period was 23 months (range, 1 to 68 months). Relapses were observed in two patients for 6 and 40 months. Grades 3, 4 haematological toxicity was observed in all study subjects. Non-haematological toxicities were not life-threatening (grades 1, 2). Blood component replacement therapy and administration of leukopoiesis stimulators were required in all patients. No deaths related to mNHL-BFM-90 treatment occurred. No patient was withdrawn from the treatment program. No second malignancies were observed. Conclusion The obtained results indicate a high chemosensitivity of PBL. The mNHL-BFM-90 program is the therapy of choice for patients with advanced PBL. Adult patients tolerate the intensive CT program well with complex accompanying therapy being administered. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction and data: Social and demographic personal data are important to be included in analysis and interpretation of results of any population studies in oncology. But it looks like social status is underestimated as cofounder for survival of patients with chronic myeloid leukemia (CML). The aim of this study was to check the prognostic value of social parameters like marital status and education level in comparison with standard risk factors. Russian CML Registry include more than 10 thousand patients (pts) data. In the analysis 8326 CML pts in chronic phase (CP) with first line TKI therapy were included: 91% of pts were treated by Imatinib and 9% by other TKIs. Mean age was 49years, 4607 f/ 3705 m. Me of follow-up-4.24years. Overall survival(OS) was estimated starting the diagnosis date, event was death from any reason, date of last contact was censored for alive pts. Survival analysis was performed by SAS procedures. Results:Firstly, we perform one-way survival analysis and estimate OS depending upon the 3 parameters Sokal Score, Marital Status and Education. There are high significant dependences OS on all three factors as shown on picture 1 (OS estimates depending on values of: 1A) Sokal score (low, intermediate, high), 1B) Marital Status (married, single, widowed/divorced), 1C) Education (low/secondary, higher). Marital Status and Education are correlated with Sokal Score (corresponding Pearson's coefficients are 0.19 and 0.12, p