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).

The purpose of the current work was the estimation of prognostic significance of cytogenetic and molecular markers, assessed by multiplex ligation-dependent probe amplification (MLPA) in 142 cases of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Good-risk genetic (GEN-GR) group consisted of 114 patients carrying either ETV6-RUNX1 or high hyperdiploidy together with normal copy-number status for all 8 genes (IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B and PAR1) or isolated deletions affecting ETV6/ PAX5/BTG1 and ETV6 deletions with a single additional deletion of BTG1/PAX5/CDKN2A/2B. All other patients (n = 28) were classified to genetic poor risk (GEN-PR) group. GEN-PR features were older age (p = 0.015), stratification to high-risk group of ALL-MB 2008 protocol (p = 0.001), higher initial WBC (p = 0.008), M3 marrow status on day 15 (p = 0.002) and lack of remission on day 36 (p = 0.039). GEN-PR patients had statistically significant lower event-free survival (EFS) (0.59 ± 0.11 vs 0.88 ± 0.03; p = 0.0008), overall survival (OS) (0.63 ± 0.15 vs 0.93 ± 0.02; p = 0.0050) and higher cumulative incidence of relapse (CIR) (0.38 ± 0.12 и 0.06 ± 0.02; p < 0.0001) in comparison to GEN-GR patients. Genetic risk group stratification retained its negative prognostic value in multivariate analysis affecting EFS (hazard ratio (HR) – 2.659; 95 % CI 1.047–6.755; p = 0.040) and CIR (HR – 3.864; 95 % CI 1.226–12.183; p = 0.021), nut did not influenced to OS (HR – 1.479; 95 % CI 0.356–6.139; p = 0.590). There was no prognostic significance of genetic risk group classifier in the “B-other ALL” group. Majority of unfavorable events (9 out of 10) and relapse (8 out of 9) in GEN-PR patients were revealed in case of IKZF1 deletion co-occurrence. Moreover all 15 patients carrying IKZF1 deletions were stratified to GEN-PR group. So when we added IKZF1 deletion as extra variable in the multivariate analysis genetic risk group classification lost its prognostic significance on EFS (HR – 0.696; 95 % CI 0.086–5.636; p = 0,735), and CIR (HR – 0.511; 95 % CI 0.053–4.924; p = 0.561), while IKZF1 deletion remained its prognostic value both to risk of unfavorable event (HR – 4.292; 95 % CI 1.521–12.911; p = 0.006) and risk of relapse (HR – 9.163; 95 % CI 3.131–26.815; p < 0.001). Thus, combination of cytogenetic risk group and MLPA markers did not bring any advantage over detection of isolated IKZF1 deletion for the estimation of prognosis in pediatric BCP-ALL.

This article represents results of work of Center of pediatric oncology and hematology of Regional pediatric clinical hospital № 1 of Yekaterinburg on Hematopoietic stem cell transplantation (HSCT) during the period 2006–2016. One hundred seventeen HSCT performed during this time including 70 autologous, 33 allogenic, 14 haploidentical at patients with solid tumors (n = 55), acute leukemias (n = 27), lymphomas (n = 9), non-malignant diseases (n = 11). Results of treatment depending on type of disease and HSCT were shown. It was indicated that presence of minimal residual disease (MRD) before HSCT in case of leukemia and neuroblastoma was indicated as negative prognosis. Possible way of treatment of persistent MRD after HSCT with the help of blinatumomab and donor lymphocytes infusion showed.

The occurrence of minimal residual disease is an important prognostic factor under acute lymphoblastic leucosis in children and adults. In overwhelming majority of research studies bone marrow is used to detect minimal residual disease. The comparative characteristic of detection of minimal residual disease in peripheral blood and bone marrow was carried out. The prognostic role of occurrence of minimal residual disease in peripheral blood and bone marrow under therapy according protocol MLL-Baby was evaluated. The analysis embraced 142 pair samples from 53 patients with acute lymphoblastic leucosis and various displacements of gene MLL younger than 365 days. The minimal residual disease was detected by force of identification of chimeric transcripts using polymerase chain reaction in real-time mode in 7 sequential points of observation established by protocol of therapy. The comparability of results of qualitative detection of minimal residual disease in bone marrow and peripheral blood amounted to 84.5%. At that, in all 22 (15.5%) discordant samples minimal residual disease was detected only in bone marrow. Despite of high level of comparability of results of detection of minimal residual disease in peripheral blood and bone marrow the occurrence of minimal residual disease in peripheral blood at various stages of therapy demonstrated no independent prognostic significance. The established differences had no relationship with sensitivity of method determined by value of absolute expression of gene ABL. Most likely, these differences reflected real distribution of tumor cells. The results of study demonstrated that application of peripheral blood instead of bone marrow for monitoring of minimal residual disease under acute lymphoblastic leucosis in children of first year of life is inappropriate. At the same time, retention of minimal residual disease in TH4 in bone marrow was an independent and prognostic unfavorable factor under therapy of acute lymphoblastic leucosis of children of first year of life according protocol MLL-Baby (OO=7.326, confidence interval 2.378-22.565).

191 consecutive unselected children with acute lymphoblastic leukemia aged from 1 to 16 years were enrolled in the study. Bone marrow samples were obtained at the time of initial diagnostics as well as at days 15 (n = 188), 36 (n = 191), and 85 (n = 187) of remission induction. Minimal residual disease (MRD) was assessed by 6–10-color flow cytometry. Flow cytometry data at day 15 allowed distinguishing three patients groups with significantly different outcome (p ˂ 0.0001): 35.64 % patients with MRD < 0.1 % represented 5-year event-free survival (EFS) of 100 %; 48.40 % cases with 0.1 % ≤ MRD< 10 % had EFS 84.6 ± 4.2 %; 15.96 % patients with very high MRD (≥ 10 %) belonged to group with poor outcome (EFS 56.7 ± 9.0 %). At the end of remission induction (day 36) 36 children (18.85 %) with MRD higher than 0.1 % had significantly worse outcome compared to remaining ones (EFS 49.4 ± 9.0 and 93.5 ± 2.1 % respectively; p ˂ 0.0001). From a clinical standpoint it is relevant to evaluate both low-risk and high-risk criteria. Multivariate analysis showed that day 15 MRD data is better for low-risk patients definition while end-induction MRD is the strongest unfavorable prognostic factor.

Bone marrow (BM) involvement in neuroblastoma patients is commonly detected by cytomorphology and associated with poor outcome. Molecular techniques, flow cytometry and immunocytochemistry were offered to detect low number of tumor cells in BM due to high value of analytical sensitivity, while prognostic significance of results, obtained with these methods is unclear. PHOX2B and/or TH genes expression was selected as molecular marker of BM involvement. It was determined in 411 BM samples obtained from 75 neuroblastoma patients. 263 BM samples were taken at the time of primary diagnosis, 80 during treatment and 68 before autologous stem cells (ASC) apheresis. Prognostic significance of BM involvement was defined using 5-year (in some groups 4-year) overall (OS), event free (EFS) and progression free (PFS) survival. 24 patients (32.0%) were positive for PHOX2B and/or TH expression in the BM at the time of primary diagnosis. They had decreased survival rates: EFS achieved 0.49+/-0.12, OS - 0.57+/-0.12, PFS - 0.54+/-0.12, comparing with 0.75+/-0.07, 0.80+/-0.07 and 0.77+/-0.07, respectively, in patients with negative BM, p=0.014, p=0.029 and p=0.033. The trend to decreased OS and PFS was detected in case of minimal residual disease presence at the end of the induction chemotherapy (OS and PFS both are 0.22+/-0.19 vs. 0.70+/-0.18 and 0.43+/-0.22, correspondingly, p=0.121, p=0.130). Detection of PHOX2B and/or TH genes expression in the BM before ASC harvesting led to significant decreasing of EFS and OS (0.00 vs. 0.59+/-0.14 and 0.75+/-0.13, respectively, p=0.021 and p=0.016).

The purpose of the study was to evaluate the prognostic value of the detection of tumor cells in the bone marrow (BM) in children with neuroblastoma (NB) by flow cytometry. The detection of tumor cells was performed in BM of 51 patients with NB (24 boys and 27 girls) aged from 6 days to 15 years (median--1 year 3 months). Flow cytometry allowed determining NB cells in BM in a much larger number of cases than cytomorphology (49.0% and 29.4% of patients, respectively). Patients, in whom NB cells were not detected in BM by flow cytometry, had significantly better event-free and overall survival rates as well as progression free survival (83.5%, 87.7% and 86,8%, respectively) compared with those in whom immunophenotyping revealed the tumor cells (28.0%, 35.87% and 34,3%, respectively). The prognostic value of the detection of BM lesion by flow cytometry was also confirmed in selected groups of patients with other criteria of stratification. Therefore the detection of tumor cells in BM by flow cytometry could potentially be considered in conjunction with other factors in choosing treatment strategy in patients with NB.

117 cases of infant acute lymphoblastic leukemia without Down syndrome (aged from 1 to 365 days) were included in the current study.Rearrangements of 11q23 (MLL) were revealed in 74 (63.2 %) patients. Among this group the most common rearrangement was t(4;11) q21;q23)/MLL-AF4 detected in 63.5 % cases, less frequently was found t(11;19)(q23;p13)/MLL-MLLT1 (in 18.9 % cases), t(10;11) p12;q23)/MLL-MLLT10 and t(1;11)(p32;q23)/ML L-EPS15 (each one in 6.8 %), t(9;11)(p22;q23)/MLL-MLLT3 in 2.7 %. Children under 6 months of age had significantly higher incidence of 11q23 (ML L) rearrangements in comparison with infants olde r than 6 months (84.0 % vs. 47.8 %, p < 0.001). P atients with translocations 11q23 (ML L) more frequently had BI-A LL and less frequently BII-ALL than children without these rearrangements (p < 0.001 f or both). Fusion gene transcript w as sequenced in 26 ML Lrearranged cases. Depending on breakpoint position within ML L and partner genes we detected 7 differ ent types of ML L-AF4 fusion gene transcript, 3 types of MLL-MLLT1, 2 types of MLL-EPS15. The most common fusion site within MLL gene was exon 11, detected in 14 (53.8 %) patients.

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.

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.

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.

The bone marrow (BM) TH, ELAVL4 and GD2 genes expression was evaluated in 331 samples from 57 different stage neuroblastoma (NB) patients, 26 BM samples from patients without NB and samples from 2 NB cell lines (IMR-32, Kelly) by real-time PCR. BM samples were considered NB-positive if PHOX2B expression was found or tumor cells were detected in BM smears. TH expression was not revealed in normal BM and was significantly lower in NB-negative samples. Expression of PHOX2B, TH and GD2 remained stable throughout NB treatment, while ELAVL4 expression was down-modulated. ROC-analysis revealed similar initial and follow-up values of TH and PHOX2B in NB patients' bone marrow making it possible to be used for disease detection and monitoring. The test prediction value was 0.994 and 0.952, respectively. The additional test for TH didn't increase the test effectiveness in comparison with PHOX2B test. ELAVL4 and GD2 assessment didn't add diagnostic value for BM involvement monitoring in NB patients.

The study was carried out in 39 patients with acute lymphoblastic leukemia aged 1 day to 11 months, treated by the MLL-Baby protocol. The minimal residual disease (MRD) was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR) of MLL fusion gene transcripts (FGt). Specimens without FGt detected by both RT-PCR and qPCR with sensitivity of at least 1 x 10(-4) were considered MRD-negative. Bone marrow specimens were collected at the time of diagnosis, on days 15 (time point 1 - TP1) and 36 (TP2) of remission induction and after each course of all-trans retinoic acid administration (TP3-TP9). Multivariate analysis showed that two most significant parameters essential for relapse prediction in this group of patients were MRD higher than 0.1% at TP3: hazard ratio 4.250; 95% confidence interval (Cl) (1.159-15.585; p = 0.029) and MRD-positivity atTP4: hazard ratio 3.771; 95% CI (1.033-13.674; p = 0.044), both parameters have similar prognostic value. Later TPs, such as TP5, did not bring any extra advantages.