Preventive Use of Azacitidine in Patients with Acute Myeloid Leukemia after Haploidentical Allo-BMT

Background. Haploidentical bone marrow transplantation (BMT) can be a reliable alternative if a fully matched donor is not available. The main challenges after BMT are a relapse of major disease, graft-versus-host disease (GVHD), and infections. Azacitidine possesses antileukemic effect together with immunomodulating properties and being administered soon after BMT can significantly improve the outcome. Aim. To study azacitidine effect on the outcome of haploidentical BMT in patients with acute myeloid leukemia (AML) in the early post-transplantation period. Materials & Methods. The trial included 18 AML patients who received haploidentical BMT at VA Almazov National Medical Research Center. In all patients MRD-negative remission was achieved on the 30th day after BMT. Azacitidine therapy was initiated not earlier than 2 months after BMT with a complete engraftment of transplant and no GVHD. Azacitidine 100 mg/day was administered on D1–D5 every 28 days within a year after BMT. When a molecular relapse was detected, donor lymphocytes were additionally infused during every other cycle of therapy. Results. Eleven patients received preventive azacitidine treatment, 7 patients were included in control group. Median onset of azacitidine treatment after haploidentical BMT was 4 months (range 2–10 months), median number of azacitidine courses was 3.5 (range 1–9). During azacitidine treatment acute GVHD was identified in 5 (45.4 %) patients. In 4 of them an exacerbation of earlier GVHD was detected (3 with cutaneous form and 1 with intestinal form), and only in 1 patient de novo acute intestinal GVHD was discovered. Conclusion. Azacitidine treatment of AML patients after haploidentical allo-BMT is safe and well tolerated. Preventive azacitidine treatment after haploidentical BMT improves overall survival of AML patients

Manufacturing of CD19 Specific CAR T-Cells and Evaluation of their Functional Activity in Vitro

Background. The most promising variant of adoptive immunotherapy of the B-line oncohematological diseases includes the use of cells with the chimeric antigen receptor (CAR T-cells), that showed extraordinary results in clinical studies. Aim. To manufacture CAR T-cells for the clinical use and to study their cytotoxicity in vitro. Methods. Human T-lymphocytes were transduced by the lentiviral vector containing anti-CD19-CAR, RIAD, and GFP genes. The T-cell transduction efficacy was assessed on the basis of GFP protein signal by flow cytometry. Propidium iodide was used to analyse the cell viability. Cytotoxic activity of the manufactured CAR T-cells was studied in the presence of the target cells being directly co-cultivated. Analysis of the number and viability of CAR T-cells and cytokine expression was performed by flow cytometry. Results. The viability of the transduced T-cells and GFP expression reached 91.87 % and 50.87 % respectively. When cultured in the presence of IL-2 and recombinant CD19 (the target antigen), the amount of CAR-T after 120 h of the process was 1.4 times larger compared with the period of 48 h. In the cytotoxic test of co-cultivation CAR-T with the K562-CD19+ cells the percentage of CAR-T increased to 57 % and 84.5 % after 48 h and 120 h of exposure respectively. When cultured with the K562 cells (test line not expressing CD19) the number of CAR T-cells decreased to 36.2 % within 48 h while the number of K562 cells increased to 58.3 %. The viability of target cells in the experimental and control groups was 3.5 % and 36.74 % respectively. Comparison of IL-6 level in the control and experimental groups revealed that the differences are insignificant, as opposed to the level of other cytokines (IFN-γ, IL-2, TNF) which proved to be different in both groups. Conclusion. The present work resulted in the production of anti-CD19 CAR T-cells with adequate viability. The in vitro model demonstrated their cytotoxicity. Manufacturing of CAR T-cells for clinical use is the first step of the development of adoptive immunotherapy in the Russian Federation

Plerixafor in Patients with Decreased Mobilizing Ability of Autologous Hematopoietic Stem Cells

Background & Aims. Autologous hematopoietic stem cell transplantation (autoHSCT) is an effective treatment for patients with malignant lymphoproliferative disorders, multiple myelomas and solid tumors sensitive to chemotherapy. Harvesting of hematopoietic stem cells (HSC) prior autoHSCT may be ineffective in up to 40 % of cases, if aggravating factors are present. One of methods to overcome the reduced mobilization ability is to include a CXCR4-inhibitor (plerixafor) to the mobilization strategies. The aim was to evaluate the efficacy and safety of different autologous HSC mobilization regimens containing plerixafor. Methods. 63 patients with solid and hematological malignancies were included into the study. 2 mobilization regimens were used: filgrastim + plerixafor (n = 47) and pegfilgrastim + plerixafor (n = 16). Filgrastim was prescribed at a dose 5 mg/kg twice a day subcutaneously on days 1–4; on day 4, at 12.00 am, plerixafor was prescribed at a dose of 0.24 mg/kg subcutaneously; on day 5, filgrastim 5 mg/kg was administered subcutaneously, and then a cytapheresis session was performed at 10.00 am. Pegfilgrastim was administered subcutaneously at a dose of 6 mg on day 1; on day 4, plerixafor was administered subcutaneously at a dose of 0.24 mg/kg at 06.00 am; then, 11 hours later, cytapheresis was performed. The cytapheresis was performed at a level of CD34+ cells ³ 20 ´ 106/mL. Results. In 73.7 % of cases (n = 42), patients had an advanced stage disease and underwent more than one chemotherapy line prior to mobilization of autologous HSC. After mobilization with G-CSF (filgrastim or pegfilgrastim), the CD34+ cell count in peripheral blood was 0–17 ´ 106/mL (median 9.8 ´ 106/mL). Further injection of plerixafor increased the CD34+ cell count to 2–89 ´ 106/mL (median 31.6 ´ 106/mL) (p = 0.0001). In 85.7 % of cases (n = 54), the sufficient amount of CD34+ cells (³ 2 ´ 106/kg; median 5.1 ´ 106/kg) was harvested for transplantation. The effectiveness of mobilization in two groups was comparable 90.2 % for the filgrastim + plerixafor regimen and 68.7 % for pegfilgrastim + plerixafor (p = 0.08). The use of the filgrastim + plerixafor combination in patients with low baseline CD34+ cell counts increased the number of hematopoietic stem cells up to 6.6–63 ´ 106/mL (median 27.1 ´ 106/mL), thus allowing to harvest a good quality graft in 83.3 % of cases (p = 0.0001). When the level of CD34+ cell counts was in the «grey zone», successful graft harvesting was performed in 90 % of cases: 1.74–4.6 ´ 106/kg; median 3.1 ´ 106/kg (p = 0.0001). Complications associated with plerixafor were observed in 2 cases: diarrhea (n = 1) and hypocalcaemia (n = 1). Conclusion. In patients who are poor mobilizers, the use of plerixafor-containing regimens increased the chance of successful graft harvesting with good tolerability

Molecular Monitoring of RUNX1-RUNX1T1 Transcript Level in Acute Myeloblastic Leukemias on Treatment

Background. The current approach to treatment of acute myeloblastic leukemia (AML) includes the achievement of maximum tumor reduction and, therefore, eradication of a leukemic clone. The goal of the therapy is to achieve undetectable levels of the target gene, except an isolated molecular rearrangement of RUNX1-RUNX1T1. Aim. To estimate the dynamics of the RUNX1-RUNX1T1 level and relevant clinical manifestations during the monitoring of various stages of the program therapy and after its completion. Methods. The article presents a description of 10 cases of AML with isolated RUNX1-RUNX1T1 expression (n = 4) and the expression in combination with different molecular and cytogenetic abnormalities (n = 6). In addition, a long-term monitoring of the gene expression by quantitative determination of RUNX1-RUNX1T1 using a real-time PCR was presented. Results. The incidence of relapses in a group with a decreased RUNX1-RUNX1T1 expression level of >2 log is 75 % as compared to patients with a less significant reduction of the transcript level (with the relapse incidence equal to 0 %) (p = 0.05). The increase of the RUNX1-RUNX1T1 level against the background of bone marrow remission by more than 1 log coincided with a bone marrow relapse within 5–18 weeks. In addition, long-term persistence of a certain transcript level after the completion of a program therapy without relapse is possible. Conclusion. The study analyzed possible molecular background of different clinical outcomes of long-term persistence of the RUNX1-RUNX1T1 transcript that might lead to an individualized approach to AML patients

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.Journal ArticleResearch Support, N.I.H. IntramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe