Immunogenicity, efficacy and safety of Nuwiq® (human-cl rhFVIII) in previously untreated patients with severe haemophilia A-Interim results from the NuProtect Study.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a fourth generation recombinant FVIII, produced in a human cell line, without chemical modification or protein fusion. No inhibitors developed in studies with Nuwiq® in 201 previously treated patients with haemophilia A (HA). The immunogenicity, efficacy and safety of Nuwiq® in previously untreated patients (PUPs) with severe HA are being assessed in the ongoing NuProtect study. METHODS: The study, conducted across 38 centres worldwide, is evaluating 110 true PUPs of all ages and ethnicities enrolled for study up to 100 exposure days (EDs) or 5 years maximum. The primary objective is to assess the immunogenicity of Nuwiq® (inhibitor activity ≥0.6 BU) using the Nijmegen-modified Bethesda assay at a central laboratory. RESULTS: Data for 66 PUPs with ≥20 EDs from a preplanned interim analysis were analysed. High-titre (HT) inhibitors developed in 8 of 66 patients after a median of 11.5 EDs (range 6-24). Five patients developed low-titre inhibitors (4 transient). The cumulative incidence (95% confidence interval) was 12.8% (4.5%, 21.2%) for HT inhibitors and 20.8% (10.7%, 31.0%) for all inhibitors. During inhibitor-free periods, median annualized bleeding rates during prophylaxis were 0 for spontaneous bleeds and 2.40 for all bleeds. Efficacy was rated as "excellent" or "good" in treating 91.8% of bleeds. Efficacy of surgical prophylaxis was "excellent" or "good" for 8 (89%) procedures and "moderate" for 1 (11%). No tolerability concerns were evident. CONCLUSION: These interim data show a cumulative incidence of 12.8% for HT inhibitors and convincing efficacy and tolerability in PUPs treated with Nuwiq® .

Recent thymic emigrants, T regulatory cells, and BAFF level in children with X-linked agammaglobulinaemia in association with chronic respiratory disease.

BACKGROUND: X-linked agammaglobulinaemia (XLA) is a genetic disorder affecting B cell maturation, which is characterised by a low number of B cells, agammaglobulinaemia and increased susceptibility to a variety of bacterial infections. This study was performed to assess T cell subpopulations in a group of children with XLA in association with chronic respiratory disease (CRD). METHODS: Numbers of T cell subpopulations (CD3+, CD4+, CD8+, CD3+DR+, naïve, memory, recent thymic emigrants (RTE), regulatory T cells, follicular T helpers) were measured by eight-colour flow cytometry in 22 XLA patients and 50 controls. BAFF level was measured by ELISA. RESULTS: XLA patients with CRD had a significantly lower percentage of RTE numbers and Tregs, while significantly higher absolute counts of lymphocytes, CD3+, CD8+, CD3+DR+ and CD4+CD45RO+ T cells were detected as compared with healthy controls. In patients with XLA without CRD, the number of follicular T helper cells was altered significantly (percentage and absolute), as compared with healthy controls. Additionally, they had significantly higher counts (percentage and absolute) of CD4+CD45RA+ cells and lower percentage of CD4+CD45RO+ cells in comparison with healthy controls. CONCLUSIONS: Our study affords new information concerning CRD and T cell subsets that differentiate or are maintained in the absence of B cells in children with XLA. T cell's homeostasis depends on the presence of chronic respiratory disease that may be caused by the delay in diagnosis.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

[Age-related characteristics of the efficacy of different glucocorticosteroids in the therapy of acute lymphoblastic leukemia].

AIM: To determine predictors for decision-making on a differential approach to choosing glucocorticosteroids (GCS) for children and adolescents with acute lymphoblastic leukemia (ALL). SUBJECTS AND METHODS: The analysis covered 1064 primary patients aged to 1 to 18 years with ALL who had been registered at the clinics of Russia and Belorussia in April 2002 to November 2006. Before induction therapy, the patients were randomized into a dexamethasone (DEXA) 6 mg/m2 group (n=539) and a methylprednisolone (MePRED) 60 mg/m2 one (n=525). RESULTS: The entire group showed no statistically significant differences in survival rates between the patients receiving DEXA or MePRED. However, an analysis of age groups revealed the benefits of DEXA in children younger than 14 years (the event-free survival (EFS) was 76±2 and 71±2%, respectively (p=0.048); the overall survival (OS) was 81±2 and 77±2%, respectively (p=0.046); therapy-induced mortality was 6.4% (DEXA) andl 1.1% (MePRED) (p=0.01 4); the rate of isolated extramedullary relapses was 1.5% (DEXA) and 4.4% (MePRED) (p=0.009). At the same time, EFS and OS in 14-to-18-year-old adolescents were statistically significantly higher than in those who used MePRED (EFS, 65±6 and 52±6%, respectively (p=0.087); OS, 72±6 and 61±6%, respectively; (p=0.l 7). CONCLUSION: The findings suggest that it is possible that the choice of a GCS for ALL therapy must be also based on a patient's age. There is a need for further studies of this matter in prospective randomized multicenter trials in children and adolescents.

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

[Mesenchymal stem cells as perspective method of fibrosis/cirrhosis treatment].

UNLABELLED: THE PURPOSE OF THE REVIEW: Discuss the background and available results of transplantation of bone marrow stem cells as a promising treatment of hepatic fibrosis. KEY PROVISIONS: Actuality of the problem of chronic hepatitis is present due to their progressive course with the formation of liver cirrhosis and a high level of mortality. Conservative treatment of patients with decompensated process requires a liver transplantation that is compounded by the high percentage of graft reinfection in infectious hepatology. It is necessary to study the use of bone marrow-derived stem cells transplantation, because MSCs have certain therapeutic potential, low immunogenicity and the capacity for directional migration. In experimental models MSCs mechanisms of action are shown to limit the progression of liver fibrosis and stimulation of regeneration processes. In clinical studies good tolerability and relative safety of administration of autologous MSCs have reported as well as the positive effects on liver synthetic function, a decrease in the severity of cirrhosis on class Child-Pugh and MELD, reduction in overall mortality are shown. The results of our own prospective pilot study using autologous MSCs from bone marrow in patients with HCV-associated liver cirrhosis are described. CONCLUSION: MSCs can exert multiple synergistic effects on the hepatic stellate cells, reduce inflammation in the liver tissue remodeling processes and fibrogenesis. For objective evidence of the clinical benefits of the method, evaluation of long-term efficacy and safety of MSCs, as well as developing rational strategies, further clinical studies are required.

Drug resistance associated properties of blasts subpopulations with different CD34 expression in childhood acute lymphoblastic leukemia (ALL).

AIM: The objective of this study was to investigate expression of drug resistance associated genes in CD34+ and CD34- leukemic subpopulations in childhood acute lymphoblastic leukemia (ALL). METHODS: ALL samples with heterogeneous CD34 expression were separated into CD34-positive and CD34-negative subpopulations and mRNA levels of MDR1, LRP, BCRP and BCL-2 genes were compared. RESULTS: BCL-2 gene expression levels did not differ significantly between CD34+ vs CD34- subpopulations in most analyzed ALL cases. Oppositely, MDR1 gene had >two-fold differences in expression levels between subpopulations in the majority of ALL cases. In T-lineage ALL CD34- fractions had increased level of BCRP and LRP genes in comparison with CD34+ ones whereas in most of B-lineage ALL expression of these genes did not differ. CONCLUSION: It was not found the unique pattern of resistance related genes expression in CD34+ vs CD34- subpopulations. However, in majority of studied pediatric ALL cases with CD34 heterogeneous expression one of subpopulations (positive or negative) could have an advantage for survival through elevated expression of drug resistance related genes.

[Efficiency of the ALL-MB-2002 protocol in children with acute lymphoblastic leukemia].

AIM: To evaluate the efficiency of the original ALL-MB-2002 protocol within the multicenter study of treatment of acute lymphoblastic leukemia (ALL) in children. SUBJECTS AND METHODS: A total of 1873 primary patients with ALL aged 1 to 18 years, of whom 1544 patients were enrolled in this study, were notified at 36 clinics of Russia and Belarus from April 15, 2002, to January 1, 2008. RESULTS: With the median observation of 4.12 years, 7-year event-free survival (EFS) was 73 +/- 13%; overall survival (OS) 78 +/- 2%; relapse-free survival 82 +/- 1%. The rates of EFS and OS were equal and amounted to 76 +/- 2 and 80 +/- 2% in the standard-risk group (SRG) and intermediate-risk group (ImRG), respectively. In the high-risk group (HRG) patients, EFS and OS were as high as 30 +/- 6 and 37 +/- 6%, respectively. The frequency of relapses with central nervous system lesion was as much as 4.7% in all the patients, 6-year cumulative risk for isolated neurorecurrences being 2.5% in the SRG patients. Adolescents, patients with the baseline leukocytosis (more than 100 x 10(9)/l), and those with a splenic size of over 4 cm or more from the costal arch margin had substantially worse survival rates. A poor early response to therapy (on induction days 8 and 15) was also associated with its lower efficiency. CONCLUSION: Despite a considerable rise in the number of centers and a slight increase in the intensity of therapy, the results of the new ALL-MB-2002 protocol are as minimum equivalents obtained in the use of the previous ALL-MB-91 protocol. A significant improvement in the overall results of therapy and a reduction in the cumulative risk for isolated neurorecurrences were noted in the ImRG patients.

[Use of LAK-cells and systemic chemotherapy with hyperthermia in the management of chemo-resistant tumors].

Tentative results of LAK-cell and whole-body hyperthermia (WBH) were evaluated in 19 children with advanced chemorefractory tumors. LAK-cells were obtained by extracorporeal incubation of peripheral blood lymphocytes: a germ-cell rhabdomyosarcoma was detected in 4, Askin's tumor--2--2, renal cell carcinoma--2 and miscellaneous--7. Autologous LAK-cells were infused twice: on completion of WBH as body temperature fell to as low as (+) 40 deg. C and on day after WBH. The latter was well tolerated. Complete or partial response to thermochemobiotherapy was reported in 8 patients. Overall 5-year survival was 43% (median follow-up--12.6 months).

[Treatment results of neuroblastoma in children in the Republic of Belarus].

Our investigation was concerned with effectiveness of infantile neuroblastoma treatment in Belarus and dependence of prognosis on extent of radical surgery. The study involved 115 patients with morphologically confirmed diagnosis of primary tumor who were treated at the Center (1997-2007). Ten-year overall and relapse-free survival rates for favorable prognosis, stage I, II, III, were 1.00 and 0.94 +/- 0.04, respectively. They were significantly higher than those for intermediate (0.70 +/- 0.09 and 0.61 +/- 0.09) or high risk (0.32 +/- 0.08 and 0.27 +/- 0.08), respectively, (p < or = 0.01). The results for radical local surgery were better: 5-year relapse-free survival--0.82 +/- 0.09 as compared with subtotal excision (0.62 +/- 0.12) and biopsy (0.5 +/- 0.25) among patients older than 12 months. Autologous stem cell transplantation (ASCT) was followed by significant improvement: 6-year overall and relapse-free survival (stage IV) (0.5 +/- 0.12 and 0.38 +/- 0.12, respectively) as compared those without ASCT (0.12 +/- 0.08 and 0.1 +/- 0.08), respectively, (p < or = 0.01). The main cause of death after ASCT was relapse. Poor results following post-ASCT treatment of stage IV tumor should be improved by more effective detection and removal of tumor cell harvest.

[Role of three-color flow cytofluorometry in the algorithm for detection of minimal residual disease in children with acute lymphoblastic and acute myeloblastic leukemia].

Three-color flow cytofluorometry (FCF) was used to identify minimal residual disease in 124 patients with acute lymphoblastic leukemia (ALL) treated in accordance with the ALL-myeloblastic leukemia (MBL)-2002/2008 protocol and 36 patients with MBL treated under the MBL-MM-2000 protocol. It was shown that approximately a fifth of children with ALL, 3-color FCF could not monitor minimal residual disease, which required the use of 4- or more-color FCF and parallel determination in these MBL samples by molecular genetic methods. An algorithm of study of MBL by FCF, by additionally using molecular genetic methods, is presented.

[Effectiveness of intensive chemotherapy in the treatment of medulloblastoma/primitive neuroectodermal tumor in children].

Standard and high-risk groups of 77 children with neuroectodermal medulloblastoma were given sandwich chemotherapy. The former group was treated with high-dose chemotherapy complemented with autotransplantation of bone marrow and peripheral stem cells. The treatment proved effective: 7-year recurrence-free survival (0.66 +/- 0.05) (overall survival--0.67 +/- 0.05; recurrence-free--0.62 +/- 0.06). Sandwich chemotherapy administered in standard risk group was followed by 7-year recurrence-free survival (0.84 +/- 0.08). High-dose chemotherapy complemented with autotransplantation of bone marrow and peripheral stem cells in conjunction with high-dose chemotherapy resulted in 6-year recurrence-free survival: 0.77 +/- 0.08 in patients after high-dose chemotherapy and 0.46 +/- 0.10--without it.

[Long-term results of combined treatment of acute promyelocytic leukemia in children and adolescents with gene therapy].

AIM: To analyse the results of treatment of children and adolescents with acute promyelocytic leukemia (APL) including polychemotherapy and ATRA (protocols APL 93, 98 and 2003). MATERIAL AND METHODS: The course of the disease, modification of the treatment protocols with reduction of anthracyclines and ATRA doses, results of molecular monitoring of PML/RARalpha transcript have been analysed for 107 APL patients. RESULTS: For prognosis of the disease important are initial characteristics of the patient and the time of the tumor regress assessed by molecular methods--establishment of molecular remission and molecular recurrence. CONCLUSIONS: In APL it is necessary to conduct molecular monitoring especially in patients at high risk and with poor prognosis in a decrease of treatment intensity for toxicity relief. Detection of molecular recurrence is indication for treatment. To raise efficacy of APL recurrence therapy it is necessary to design a special updated protocol.

Study of expression and functional activity of P-GP membrane glycoprotein in children with acute leukemia.

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp.

Fluorescent probing of the ligand-binding ability of blood plasma in the acute-phase response.

The acute-phase response alters the composition of carrier proteins in plasma, which may affect the blood deposition and transport of biomediators and drugs. The effect of the acute-phase response on the ligand binding ability of plasma was studied in leukemic children with and without systemic inflammation (sepsis and septic shock). To target different transport proteins, differentially charged fluorescent dyes were used: anionic ANS (8-anilinonaphthalene-1-sulfonate), uncharged Nile red, and cationic Quinaldine red. Human serum albumin was a principal carrier for ANS and competed for Nile red binding with lipoproteins. The synchro-scan fluorescence spectra of Nile red in plasma distinguished two species of the dye bound to serum albumin and to low-density and/or very low-density lipoproteins. The binding of Quinaldine red did not correlate with albumin and lipoprotein levels, and was probably determined by alpha(1)-acid glycoprotein. Compared with the control group, leukemia increased Quinaldine red binding by 65% and did not significantly affect the binding of other probes. Sepsis and septic shock did not change the binding of Quinaldine red, but progressively decreased ANS binding, finally by about 33%, and shifted Nile red distribution from serum albumin toward lipoproteins. These changes reflected a modified composition of the three principal transport proteins in plasma in the acute-phase response. Simple and rapid fluorescent tests developed in this study can be used to evaluate the acute-phase response and to optimize drug administration protocols in clinical practice.