Rituximab and reduced-intensity chemotherapy in children and adolescents with mature B-cell lymphoma: interim results for 231 patients enrolled in the second Russian-Belorussian multicentre study B-NHL-2010M.

The value of adding rituximab to chemotherapy in children with aggressive B-cell non-Hodgkin lymphoma (B-NHL) is still insufficiently studied. We enrolled 231 patients [mean age 9 years old (range 2-17); male:female ratio 3·4:1] with Burkitt (BL, 179 patients, 76·7%), diffuse large B-cell (32 patients, 14%), primary mediastinal B-cell (14 patients, 6%), and other (6 patients, 2·6%) B-cell lymphomas in a prospective study of immuno-chemotherapy. Stages were I-II in 32% and III-IV in 68% of the patients. Four doses of 375 mg/m2 rituximab were added to the Berlin-Frankfurt-Munster-NHL-90-like chemotherapy, with methotrexate being reduced or omitted in the first 2 induction blocks. The complete remission rate was 100% in limited-stage and 91·4% in advanced-stage patients. Five advanced-stage patients (2·2%) died in induction and 1 patient with stage 2 B-NHL died in remission; 11 patients in the high-risk group progressed on therapy (3 non-BL are alive after salvage) and 5 relapsed. Sixteen patients (9·7%) with advanced stage disease proceeded to transplant. With a median follow-up of 46 months, 98·5 ± 1% of patients with limited disease and 88·1 ± 2% (88·1% in Risk Group 3; 82·6% in Risk Group 4) in advanced stages are alive. This study confirmed that combined immunochemotherapy for B-lymphomas is highly effective in children, despite reducing the intensity of the induction blocks.

Therapy of advanced-stage mature B-cell lymphoma and leukemia in children and adolescents with rituximab and reduced intensity induction chemotherapy (B-NHL 2004M protocol): the results of a multicenter study.

Pediatric mature B-cell non-Hodgkin lymphomas (B-NHLs) are highly aggressive malignant tumors that are curable with chemotherapy (ChT). High-dose methotrexate (MTX) is considered indispensable for successful treatment, but this therapy frequently induces severe mucositis and infectious complications, especially in induction, which can cause treatment failure. A prospective multicenter trial of combined immunochemotherapy for advanced-stage B-NHL with rituximab and the modified NHL-BFM-90 protocol was conducted. The major differences from the original protocol were a decrease in the dose of MTX from 5000 to 1000 mg/m/24 h in the first 2 ChT blocks and the addition of rituximab at 375 mg/m to each of the first 4 blocks of ChT. Eighty-three newly diagnosed patients with a median age of 8.84 years with Burkitt lymphoma/leukemia and diffuse large B-cell lymphomas stage III to IV were included. Four patients died during induction ChT due to tumor lysis syndrome and infection. Two additional patients died subsequently due to tumor resistance. Complete remission was achieved in 77 (92.8%) patients; 2 patients relapsed at 1 and 3 months, and 2 developed secondary malignancies at 1 and 6.5 years, respectively, after the completion of therapy. The overall survival probability was 82%±8% with a median follow-up of 65.2 months. Combined therapy with rituximab and intensive ChT with a reduced MTX dose of 1 g/m in the 2 induction courses was feasible and produced high cure rates in patients with pediatric advanced-stage mature B-NHL.

Additional flow cytometric studies for differential diagnosis between Burkitt lymphoma/leukemia and B-cell precursor acute lymphoblastic leukemia.

The differentiation between Burkitt lymphoma/leukemia (BL) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is sometimes complicated. Laboratory findings that favor BL (e.g., surface expression of µ heavy chain and/or one of the light chains of immunoglobulin, FAB L3 morphology of blasts, MYC gene rearrangements) are not always present simultaneously. Our previous work demonstrated that BL differed from Ig(+) BCP-ALL by expression of Ig and other surface markers. In the current study, we have evaluated additional flow cytometric markers for reliable differentiation between BL and BCP-ALL. Among three studied surface antigens (CD44, CD38, CD58), only CD58 demonstrated significantly higher expression in BL as compared to BCP-ALL. Moreover, BL cases were associated with an increased level of Ki-67 and a higher percentage of cells in the S-phase of cell cycle. These two features reflect an aggressive proliferative potential of BL. Thus, when BL is suspected and results of surface Ig evaluation are controversial, the flow cytometric analysis of CD58, Ki-67 and cell cycle could assist in the differential diagnosis.

TPMT genetic variations in populations of the Russian Federation.

BACKGROUND: Polymorphisms that reduce the activity of thiopurine S-methyltransferase (TPMT) cause adverse reactions to conventional doses of thiopurines, routinely used for antileukemic and immunosuppressive treatment. There are more than 20 variant alleles of TPMT that cause decreased enzymatic activity. We studied the most common variant alleles of TPMT and their frequency distribution in a large cohort of multiracial residents in the Russian Federation and compared their frequencies in children with and without malignancy to determine whether TPMT gene abnormality is associated with hematologic malignancy. PROCEDURE: The TPMT biochip was used to detect 6 TPMT single nucleotide polymorphisms (SNPs) corresponding to 7 TPMT-deficiency alleles (TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8). We analyzed allele frequencies in the whole cohort, the childhood cancer group, and the non-cancer group. We also characterized disease features and outcome according to the presence of TPMT SNPs in children with acute lymphoblastic leukemia (ALL). RESULTS: Fifty-five (5.5%) study participants overall had heterozygous TPMT genotypes (1 variant and 1 wild-type allele): TPMT*1/*3A (n = 45; 4.5%), TPMT*1/*3C (n = 8; 0.8%), and TPMT*1/*2 (n = 2; 0.2%). TPMT SNPs were more frequent in children with hematologic malignancy than in other participants (7.5% vs. 4.0%, P = 0.02). We found no significant association between TPMT SNPs and ALL treatment outcome (median follow-up, 31.3 months). CONCLUSIONS: TPMT*3A is the most prevalent variant allele in the Russian Federation. The estimated frequency of variant alleles in the study cohort (5.5%) was similar to that observed in the White populations in the U.S. and Eastern Europe.

Heterogeneity of childhood acute leukemia with mature B-cell immunophenotype.

BACKGROUND: Flow cytometry (FCM) plays a crucial role in the differential diagnosis of Burkitt lymphoma/leukemia (BL) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The presence of surface IgM (sIgM) alone or with light chain restriction indicates a mature blast phenotype (BIV by EGIL) and is usually observed in BL. However, sIgM expression could also be detected in transitional BCP-ALL cases. These similarities in immunophenotype and ambiguous correspondence with other laboratory findings may challenge the correct BL diagnostics. METHODS: We retrospectively reviewed the available data from immunophenotypic, morphological, cytogenetic, and molecular genetic studies of 146 children (85 boys and 61 girls) with a median age of 10 years (range 0-18 years) who were diagnosed with BL and BCP-ALL. The blasts' immunophenotype was studied by multicolor FCM. The conventional cytogenetic analysis included G-banded karyotyping and fluorescence in situ hybridization (FISH). RESULTS: In 54 children classified as BIV-ALL according to the EGIL, it was demonstrated that sIgM in a minority of cases can be associated with various types of BCP-ALL. Analysis of the antigen expression profile of 105 patients with verified BL (n = 21) and BCP-ALL (n = 84) showed significant differences in BL and the sIgM(+) vs BCP-ALL immunophenotype. Thus, even in cases of ambiguous sIgM expression, these two diseases could be reliably discriminated by complex immunophenotyping. Moreover, 10 patients (7 boys and 3 girls) with BL leukemic cells did not express sIgM, and they were diagnosed with BL on the basis of other laboratory and clinical signs. CONCLUSIONS: In conclusion, our study shows that BIV subtype is heterogeneous group of leukemia including not only the BL, but also BCP-ALL. In ambiguous cases, only a combination of multiple immunophenotypic, cytomorphologic, and genetic diagnostic technologies can allow the precise discrimination of BL and BCP-ALL and selection of the appropriate treatment scheme.

Bodyweight-adjusted rivaroxaban for children with venous thromboembolism (EINSTEIN-Jr): results from three multicentre, single-arm, phase 2 studies.

BACKGROUND: Rivaroxaban has been shown to be efficacious for treatment of venous thromboembolism in adults, and has a reduced risk of bleeding compared with standard anticoagulants. We aimed to develop paediatric rivaroxaban regimens for the treatment of venous thromboembolism in children and adolescents. METHODS: In this phase 2 programme, we did three studies to evaluate rivaroxaban treatment in children younger than 6 months, aged 6 months to 5 years, and aged 6-17 years. Our studies used a multicentre, single-arm design at 54 sites in Australia, Europe, Israel, Japan, and north America. We included children with objectively confirmed venous thromboembolism previously treated with low-molecular weight heparin, fondaparinux, or a vitamin K antagonist for at least 2 months or, in children who had catheter-related venous thromboembolism for at least 6 weeks. We administered rivaroxaban orally in a bodyweight-adjusted 20 mg-equivalent dose, based on physiologically-based pharmacokinetic modelling predictions and EINSTEIN-Jr phase 1 data in young adults, in either a once-daily (tablets; for those aged 6-17 years), twice-daily (in suspension; for those aged 6 months to 11 years), or three times-daily (in suspension; for those younger than 6 months) dosing regimen for 30 days (or 7 days for those younger than 6 months). The primary aim was to define rivaroxaban treatment regimens that match the target adult exposure range. The principal safety outcome was major bleeding and clinically relevant non-major bleeding. Analyses were per-protocol. The predefined efficacy outcomes were symptomatic recurrent venous thromboembolism, asymptomatic deterioration on repeat imaging at the end of the study treatment period. These trials are registered at ClinicalTrials.gov, numbers NCT02564718, NCT02309411, and NCT02234843. FINDINGS: Between Feb 11, 2013, and Dec 20, 2017, we enrolled 93 children (ten children younger than 6 months; 15 children aged 6 months to 1 year; 25 children aged 2-5 years; 32 children aged 6-11 years; and 11 children aged 12-17 years) into our study. 89 (96%) children completed study treatment (30 days of treatment, or 7 days in those younger than 6 months), and 93 (100%) children received at least one dose of study treatment and were evaluable for the primary endpoints. None of the children had a major bleed, and four (4%, 95% CI 1·2-10·6) of these children had a clinically relevant non-major bleed (three children aged 12-17 years with menorrhagia and one child aged 6-11 years with gingival bleeding). We found no symptomatic recurrent venous thromboembolism in any patients (0%, 0·0-3·9). 24 (32%) of 75 patients with repeat imaging had their thrombotic burden resolved, 43 (57%) patients improved, and eight (11%) patients were unchanged. No patient deteriorated. We confirmed therapeutic rivaroxaban exposures with once-daily dosing in children with bodyweights of at least 30 kg and with twice-daily dosing in children with bodyweights of at least 20 kg and less than 30 kg. Children with low bodyweights (<20 kg, particularly <12 kg) showed low exposures so, for future studies, rivaroxaban dosages were revised for these weight categories, to match the target adult exposure range. 61 (66%) of 93 children had adverse events during the study. Pyrexia was the most common adverse event (ten [11%] events), and anaemia and neutropenia or febrile neutropenia were the most frequent grade 3 or worse events (four [4%] events each). No children died or were discontinued from rivaroxaban because of adverse events. INTERPRETATION: Treatment with bodyweight-adjusted rivaroxaban appears to be safe in children. The treatment regimens that we confirmed in children with bodyweights of at least 20 kg and the revised treatment regimens that we predicted in those with bodyweights less than 20 kg will be evaluated in the EINSTEIN-Jr phase 3 trial in children with acute venous thromboembolism. FUNDING: Bayer AG, Janssen Research and Development.

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique.

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent.