ЦЕЛЬ ИССЛЕДОВАНИЯ Оценить возможность прогнозирования наличия транслокации t(12;21)(p13;q22)/ETV6::RUNX1 методом проточной цитометрии с использованием антигенов CD27 и CD44 у детей с острым лимфобластным лейкозом из B-линейных предшественников (ВП-ОЛЛ). МЕТОДЫ ИССЛЕДОВАНИЯ В исследование вошло 437 случаев ВП-ОЛЛ у детей в возрасте от 1 до 18 лет. Процентное количество CD27 и CD44 позитивных опухолевых клеток, а также средняя интенсивность флуоресценции (MFI) были сопоставлены с результатами молекулярно-генетических методов исследования. Оптимизация порогового уровня для определения позитивной экспрессии антигенов CD27 и CD44 была проведена с помощью ROC-анализа. РЕЗУЛЬТАТЫ Было доказано, что транслокация t(12;21)(p13;q22)/ETV6::RUNX1 ассоциирована с сочетанием позитивной экспрессии CD27 и сниженной, либо отсутствующей экспрессией CD44. Тем не менее, оптимальными пороговыми уровнями для определения позитивной экспрессии CD27 и CD44 в процентах оказались 80,7 и 86,1% соответственно. Использование отношения MFI CD27 к MFI CD44 показало наиболее высокий параметр диагностического отношения шансов — 953,1. ЗАКЛЮЧЕНИЕ Использование сочетания антигенов CD27 и CD44 позволило с высокой диагностической эффективностью предсказывать наличие t(12;21)(p13;q22)/ETV6::RUNX1 при ВП-ОЛЛ у детей методом проточной цитометрии.

The purpose of this work was evaluation of prognostic significance of 11q23/KMT2A rearrangements in infants (aged under 365 days) with B-cell precursor acute lymphoblastic leukemia (ALL) enrolled in Russian-Belarus multicenter trial MLLBaby. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Research Institute of Medical Cell Technologies (Ekaterinburg). Various 11q23/KMT2A rearrangements were revealed in 100 (72%) of 139 patients. Event-free survival (EFS) in the intermediate risk group of MLL-Baby trial was 35.1% (standard error (SE) 6.9%), in the high risk group – 38.3% (SE 7.1%) (p = 0.941). The most unfavorable prognosis had infants with translocation t(9;11)/KMT2A-MLLT3: EFS 18.8% (SE 9.8%), cumulative incidence of relapse (CIR) 75.0% (SE 9.7%). Intermediate results were obtained in patients with translocations t(4;11)/KMT2A-AFF1 and t(11;19)/KMT2A-MLLT1: EFS 36.9% (SE 7,2%) and 32,7% (SE 10.4%), respectively; CIR 46.3% (SE 7.8%) and 50.9% (SE 12.3%). The most favorable treatment outcome was achieved in infants carrying translocation t(10;11)(p12;q23)/KMT2A-MLLT10: EFS 83.3% (SE 15.2%), CIR 0,0%. In the multivariate analysis unfavorable outcome of KMT2A-rearranged infant ALL was associated with initial CNS involvement (p = 0.020), initial white blood cell count higher than 300 × 109 /L (p = 0.028), more than 5% blast cells on day 15 in bone marrow (p = 0.012) and presence of translocation t(11;19)/KMT2A-MLLT1 (p = 0.012).

Aim. To study characteristics of 11q23 involvement, age-specific differences in the incidence of these chromosomal markers in acute myeloid leukemias (AML) in children, and to determine their prognostic significance in patients treated according to the protocols applied in leading Russian pediatric hematological clinics. Methods. The chromosomal analysis of bone marrow and peripheral blood cells has been performed prior to initiation of treatment in 395 children with primary AML aged from 0 to 16 years. The patients were treated in pediatric hematological clinics of Moscow and Moscow Region and in Yekaterinburg District Children’s Hospital No. 1. Clinical outcomes of 300 followed-up pediatric patients treated with similar modern therapy protocols were analyzed to evaluate the prognostic impact of 11q23/MLL abnormalities. To determine the incidence of 11q23/MLL rearrangements in AML of different age groups, we examined not only children, but also adult patients (n = 212). Results. In AML, the frequency of changes in the 11q23 region exceeded 40 % in children aged from 0 to 2 years. The frequency decrease with age and in patients over 40 years it was only 2 %. Significant heterogeneity of changes in karyotypes with 11q23/MLL rearrangements was observed: both various translocations with different regions of other chromosomes, and 11q23 deletions were detected. In addition, a great variability of numerical and structural additional chromosomal abnormalities was observed. The 10-year relapse-free survival rates (30.4 ± 6.7 %) and overall survival rates (35.1 ± 7.0 %) in AML with changes in the 11q23 region (n = 61) were significantly lower than those in patients from the intermediate risk group (n = 103): 48.9 ± 5.8 % and 43.8 ± 7.5 %, respectively (p = 0.035). The data are close to those in the high-risk group (n = 44): 35.9 ± 8.1 % and 38.3 ± 7.6 %, respectively. The study failed to confirm the published data that t(9;11) is a more favorable prognostic factor, and that t(6;11) and t(10;11) are less favorable ones than all other 11q23 translocations. Our results did not confirm a negative prognostic effect of additional chromosome abnormalities associated with 11q23 rearrangements. Conclusion. Pediatric AML patients with 11q23 abnormalities should be included in a high-risk group if therapy is performed according protocols applied in leading hematological centers of Russia.

Aim. To study characteristics of 11q23 involvement, agespecific differences in the incidence of these chromosomal markers in acute myeloid leukemias (AML) in children, and to determine their prognostic significance in patients treated according to the protocols applied in leading Russian pediatric hematological clinics. Methods. The chromosomal analysis of bone marrow and peripheral blood cells has been performed prior to initiation of treatment in 395 children with primary AML aged from 0 to 16 years. The patients were treated in pediatric hematological clinics of Moscow and Moscow Region and in Yekaterinburg District Children's Hospital No. 1. Clinical outcomes of 300 followed-up pediatric patients treated with similar modern therapy protocols were analyzed to evaluate the prognostic impact of 11q23/MLL abnormalities. To determine the incidence of 11q23/MLL rearrangements in AML of different age groups, we examined not only children, but also adult patients (n = 212). Results. In AML, the frequency of changes in the 11q23 region exceeded 40 % in children aged from 0 to 2 years. The frequency decrease with age and in patients over 40 years it was only 2 %. Significant heterogeneity of changes in karyotypes with 11q23/MLL rearrangements was observed: both various translocations with different regions of other chromosomes, and 11q23 deletions were detected. In addition, a great variability of numerical and structural additional chromosomal abnormalities was observed. The 10-year relapse-free survival rates (30.4 ± 6.7 %) and overall survival rates (35.1 ± 7.0 %) in AML with changes in the 11q23 region (n = 61) were significantly lower than those in patients from the intermediate risk group (n = 103): 48.9 ± 5.8 % and 43.8 ± 7.5 %, respectively (p = 0.035). The data are close to those in the high-risk group (n = 44): 35.9 ± 8.1 % and 38.3 ± 7.6 %, respectively. The study failed to confirm the published data that t(9; 11) is a more favorable prognostic factor, and that t(6; 11) and t(10; 11) are less favorable ones than all other 11q23 translocations. Our results did not confirm a negative prognostic effect of additional chromosome abnormalities associated with 11q23 rearrangements. Conclusion. Pediatric AML patients with 11q23 abnormalities should be included in a high-risk group if therapy is performed according protocols applied in leading hematological centers of Russia.

We performed clinical and laboratory characterization of patients with rare translocation t(1;11)(p32;q23) leading to MLL-EPS15 fusion gene formation. Study cohort consisted of 33 primary acute leukemia (AL) cases including 6 newly diagnosed and 27 patients previously described in literature. Among study group patients t(1;11)(p32;q23) was found most frequently in infant AL cases (median age 8 months). In acute lymphoblastic leukemia (ALL) male/female ratio was 1:3, in acute myeloid leukemia (AML) it was 1:1. Additional cytogenetic aberrations in 38 % of patients were revealed. The most frequent breakpoint position in EPS15 gene was intron 1. Four different types of MLLEPS15 fusion gene transcripts were detected. Primers-probe-plasmid combination for MLL-EPS15 fusion gene transcript monitoring by realtime quantitative polymerase chain reaction (RQ-PCR) was developed and successfully applied. In 3 patients RQ-PCR was done on genomic DNA for absolute quantification of MLL-EPS15 fusion gene. High qualitative concordance rate (92 %) was noted between minimal residual disease data obtained in cDNA and genomic DNA for MLL-EPS15 fusion detection.

The mechanisms of chimeric gene formation with MLL gene involvement in infants aged under 1 year, suffering from acute leukemia, are described. Rearrangement of MLL gene in DNA was detected in the patients by long inverted polymerase chain reaction (LI-PCR). The study was carried out in 72 patients aged 1 day to 11 months, 52 of these with acute lymphoblastic leukemia (ALL), 19 with acute myeloid leukemia (AML), and 1 with acute undifferentiated leukemia. The most incident (53.8%) chimeric gene in ALL was MLL-AF4; other ones were more rare: MLL-MLLT7 (23.1%), MLL-MLLT3 (13.5%), MLL-EPS15 (7.7%), and MLL-AFF3 (1.9%). In AML the most incident (36.8%) chimeric gene was MLL-MLLT3; other chimeric genes were MLL-MLLT10 (26.3%), MLL-MLLT11 and MLL-MYO1F (10.5% each), MLL-AF4, MLL-SEPT6, and MLL-SEPT9 (5.3% each). The most frequent aberration zone in MLL gene DNA was intron 11(48.1% cases) in ALL and intron 9 (42.1%) in AML. Patients with ALL with the rupture site in intron 11 were the youngest (p = 0.025). No relationship between the location of rupture sites and patient's gender, initial leukemia level, and partner gene type was detected. The aberration zones in MLL partner genes most often involved one or two introns, except AF4 and MLLT10 genes, in which the aberration zones were longer. The most incident mechanism of chimeric gene formation with MLL participation was reciprocal translocation (73.6%), trans-splicing or insertions were significantly more rare (15.3 and 11.1%, respectively). Rare MLL partner genes were found by LI-PCR: AFF3, MYO1F, SEPT6, SEPT9, as well as atypical locations of ruptures in the presence of MLL-AF4: MLL gene intron 7 and AF4 gene intron 10, not detected by the standard reverse transcription PCR. Hence, we characterized in detail the structure of chimeric genes with MLL participation in a large group of infants aged under 1 year, suffering from acute leukemia.

MYCN gene amplification and 1p deletion in neuroblastoma patients are associated with poor prognosis and commonly used for patient's stratification into risk groups. MYCN copy number and 1p deletion status were analyzed with multiplex ligase-dependent probe amplification (MLPA), PCR and FISH. MYCN amplification was revealed in 21 patients (17.2%) simultaneously by MLPA and PCR. In 28 cases (23.0%) 2p gain was detected. 1p deletion was revealed in 28 patients (23.0%) while concordance between PCR and MLPA achieved 95.8%, PCR and FISH - 90.9%. Mean follow-up time achieved 42 months (ranged from 1 month to 13 years). Event-free survival and overall survival in MYCN-amplified patients as well as in patients with 1p deletion were significantly lower comparing with MYCN-negative patients or patients without 1p deletion.