[Biological microchip for establishing the structure of fusion transcripts involving MLL in children with acute leukemia].

MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.

[THE COMPARISON OF RESULTS OF DETECTION OF MINIMAL RESIDUAL DISEASE IN PERIPHERAL BLOOD AND MARROW IN CHILDREN OF THE FIRST YEAR OF LIFE WITH ACUTE LYMPHOBLASTIC LEUCOSIS].

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).

[Prognostic value of the determination of bone marrow lesion in patients with neuroblastoma based on the gene PHOX2B and TH expression].

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).

[Prognostic value of bone marrow lesions in children with neuroblastoma detected by flow cytometry].

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.

[Methods of detection and prognostic significance of genetic material increase of the short arm of chromosome 2 and a deletion of the short arm of chromosome 1 in patients with neuroblastoma].

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 TH, ELAVL4 and GD2 gene expression as diagnostic markers of bone marrow lesions in patients with neuroblastoma].

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.

Suppressed miR-128-3p combined with TERT overexpression predicts dismal outcomes for neuroblastoma.

BACKGROUND: Molecular and clinical diversity of neuroblastomas is notorious. The activating TERT rearrangements have been associated with dismal prognosis. Suppression of miR-128-3p may complement and enhance the adverse effects of TERT overexpression. OBJECTIVE: The study aimed at evaluation of prognostic significance of the miR-128-3p/TERT expression in patients with primary neuroblastoma. METHODS: RNA samples isolated from fresh-frozen tumor specimens (n= 103) were reverse transcribed for evaluation of miR-128-3p and TERT expression by qPCR. The normalized expression levels were tested for correlations with the event-free survival (EFS). ROC-analysis was used to establish threshold expression levels (TLs) for the possible best prediction of the outcomes. The median follow-up was 57 months. RESULTS: Both TERT overexpression and miR-128-3p downregulation were independently associated with superior rates of adverse events (p= 0.027, TL =-2.32 log10 and p= 0.080, TL =-1.33 log10, respectively). The MYCN single-copy patients were stratified into groups based on the character of alterations in expression of the studied transcripts. Five-year EFS in the groups of patients with elevated TERT/normal miR-128-3p expression and normal TERT/reduced miR-128-3p expression were 0.74 ± 0.08 and 0.60 ± 0.16, respectively. The patients with elevated TERT/reduced miR-128-3p expression had the worst outcomes, with 5-year EFS of 0.40 ± 0.16 compared with 0.91 ± 0.06 for the patients with unaltered levels of both transcripts (p< 0.001). Cumulative incidence of relapse/progression for the groups constituted 0.23 ± 0.08, 0.40 ± 0.16, 0.60 ± 0.16 and 0.09 ± 0.06, respectively. Moreover, the loss of miR-128-3p was qualified as independent adverse predictor which outperformed the conventional clinical and genetic risk factors in the multivariate Cox regression model of EFS. CONCLUSIONS: Combined expression levels of miR-128-3p and TERT represent a novel prognostic biomarker for neuroblastoma.

[The limited possibility of using a simplified approach to detect minimal residual disease by the flow cytometry technique in children with precursor B-lineage acute lymphoblastic leukemia].

Minimal residue disease (MRD) is a state in which the tumor cells remain in the patient in the amounts unrecognizable with the standard cytological techniques. Flow cytometry is one of the basic methods for evaluation of MRD in precursor B-lineage acute lymphoblastic leukemia (PBLALL). The so-called simplified three-color analysis using the combination of CD19/CD10/CD34 antibodies has been proposed to detect MRD in the midcourse of induction therapy. Four-to-nine-color is presently used to identify MRD. One hundred and thirty-four bone marrow samples taken at different stages of therapy in 55 children with PBLALL were examined to estimate the possibility of using the flow cytometry technique using the 3-color simplified approach to determining MRD. The results of the simplified and standard approaches were compared in the samples stained with 6-8 monoclonal antibodies in the combinations that always included CD19, Cd10 and CD34. The comparison revealed that MRD had been incorrectly identified by the simplified method in 8.0, 17.6, and 75.8% of the patients on therapy days 15, 36, and 85, respectively. In addition, the content of residual tumor cells with respect to the threshold values more frequently proposed to stratify patients was found to be incorrectly calculated in some true positive samples. Thus, when the simplified approach was applied using the results of MRD detection to stratify the patients into risk groups, 16.0, 27.4, and 81.8% of the samples would yield incorrect information on therapy days 15, 36, and 85, respectively. Thus, the simplified approach to identifying MRD is most applicable on day 15 of therapy; however, there may be mistakes in this point of observation. This method used on day 36 more frequently yields incorrect results and is inapplicable on day 85.

[Minimal residual disease monitoring by flow cytometry in children with acute lymphoblastic leukemia].

The cells that have avoided the action of antitumor drugs may be retained after remission achievement during induction therapy and consolidation. A combination of these cells is given the name minimal residual disease (MRD). Multicolor flow cytometry has recently attracted considerable interest as the most promising method for measuring the content of residual tumor blasts. This technique is based on the detection of the so-called leukemia-associated immunophenotype (LAIP), i.e., a tumor-specific combination of the expression of membrane and cytoplasmic markers. Flow cytometry may be successfully used to monitor MRD in 90-95% cases of acute lymphoblastic leukemia (ALL) and in 80-85% of patients with acute myelocytic leukemia. The sensitivity of flow cytometry, which is real for routine flow techniques, is a possibility of identifying one cell among 10(4)-10(5) cells. Multicolor flow cytometry (that involves the simultaneous analysis of the expression of a few markers) is the most reasonable tool for MRD monitoring. The monoclonal antibody panels recommended by different groups of investigators for MRD monitoring in B-lineage ALL include antibodies to the pan-B-cell antigen CD19, markers of different stages of differentiation of B-lineage precursors of CD10, CD34, and CD20 and leukemia-associated markers different for each panel, such as CD22, CD38, CD58, CD45, TdT, CD13, CD33. The hyperexpression of CD10, CD34, CD19, TdT, the decreased expression of CD38, CD45, CD22, CD19, the simultaneous expression of markers of different stages of differentiation of B lymphocytes, such as CD10 and CD20, and the lymphoblast coexpression of myeloid markers of CD13, CD33, CDS15 are the most frequently described immunophenotype aberrations in B-lineage ALL. The selection of combinations of markers for MRD monitoring in children with T-ALL is based on the simultaneous expression of combinations of the antigens characteristic for early stages of differentiation of normal T lymphocytes, namely TdT and cytoplasmic CD3. Some authors consider the use of CD99 versus TdT to be most appropriate. There is recent evidence that MRD-positive patients have a higher cumulative risk for recurrences as compared with those without residual blasts. Moreover, the longer the tumor cells are retained during therapy, the worse the prognosis is. Thus, for choice of the adequate intensity of antitumor therapy, it is necessary to qualitatively and quantitatively assess MRD by multicolor flow cytometry at different stages of therapy.

[Genetic polymorphism of glutathione-S-transferase M1 and T1 in children with acute lymphoblastic leukemia].

Children with acute lymphoblastic leukemia (ALL) and deletions of glutathione-S-transferase M1 (GSTMI) and glutathione-S-transferase T1 (GSTT1) have better event-free survival and lower rates of relapses than those with GSTM1 and/or GSTT1. It is concluded that deletions of GSTM1 and/or GSTT1, double-null genotype are closely associated with the good prognosis of childhood ALL treated according to the ALL-BFM 90 and ALL-MB 91 protocols.