Treatment of patients with high-risk neuroblastoma (NB) is a complex challenge, and it is based on response to certain elements of therapy. The development and introduction of new treatment approaches, such as GD2-targeted immunotherapy (IT), leads to improved survival in this cohort of patients. The aim of the study was to retrospectively assess the effectiveness of therapy in patients with high-risk NB before the introduction of IT into clinical practice. We retrospectively analyzed the data of 151 NB patients stratified into a high-risk group who had received treatment in accordance with the modified NB2004 protocol of the German Society for Pediatric Oncology and Hematology (GPOH) at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology from 01.2012 to 12.2017. This study was approved by the Independent Ethics Committee and the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. All the study subjects (or their legal representatives) signed a voluntary informed consent form indicating their agreement to treatment and use of their data for research purposes. Overall survival (OS), event-free survival (EFS), and risk factors were analyzed in the patients with high-risk NB including those who had completed multimodal therapy with autologous hematopoietic stem cell transplantation and post-consolidation therapy with isotretinoin and had achieved a satisfactory response to induction therapy (complete response (CR), very good partial response (VGPR), partial response (PR)) (population of special interest). The main unfavorable prognostic clinical and molecular genetic factors affecting survival in the high-risk NB patients were older age, MYCN gene amplification, and stage 4 of the disease. The use of the modified GPOH NB2004 protocol resulted in a satisfactory response (CR/VGPR/PR) to the induction therapy in most patients: 124/151 (82.1 %). Surgery (other than primary tumor biopsy) led to improved survival, with no statistical difference between macroscopic radical surgery and macroscopic residual tumor. At the same time, radiation therapy (RT), as the second element of local control, had a significant impact on EFS in the group of the patients with stage 4 disease: the 3-year EFS was 39.4 % (95 % confidence interval (CI) 23.1–55.4) in the patients with RT versus 25.7 % (95 % CI 17.5–34.7) in the patients without RT (p = 0.0295). The introduction of a new high-dose TreoMel chemotherapy regimen did not result in worse survival rates but led to a decrease in transplant-related toxicity. The 5-year OS and 5-year EFS were 49.4 % (95 % CI 40.9–57.3 %) and 33.3 % (95 % CI 25.9–40.9) respectively for all the study subjects, and 81.6 % (95 % CI 70.3–88.9) and 55.1 % (95 % CI 43.1–65.5) respectively for the patients from the population of special interest. The analysis of the results of therapy in the high-risk NB patients who had received treatment at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, yielded results comparable to those of the original GPOH NB2004 protocol. The patients with CR/VGPR/PR to the induction therapy who had completed the protocol treatment with autologous hematopoietic stem cell transplantation and isotretinoin post-consolidation therapy demonstrated higher 5-year EFS rates. However, there remains a need to develop more effective treatment regimens for high-risk NB.

Neuroblastoma (NB) is a malignant neoplasm of the sympathetic nervous system of embryonic origin, consisting of undifferentiated neuroectodermal cells of the neural crest.In the structure of the incidence of malignant neoplasms in patients under one year of age, NB is the most common tumor. At the same time, mortality of this disease ranks third, behind leukemias and tumors of the central nervous system, and amounts to 13% in the structure of child mortalityfrom malignant tumors in developed countries. The stratification of patients to the risk groups and the subsequent determination of treatment tactics depends on several prognostic factors, including genetic aberrations identified in tumor cells. Moreover, processes such as spontaneous regression and transformation into benign tumors are due to the genetic characteristics of NB. Thus, the study of genetic disorders underlying the pathogenesis of NB is necessary for adequate subdivision of patients into risk groups and developing of new methods of treatment.

Nowadays much attention is paid to non-invasive methods of cancer diagnostics and monitoring. One of the promising methods is the sequencing of platelet RNA (tumor-educated platelets), in which, as it was previously established, the mRNA repertoire changes in various oncological diseases. Thus, platelets can contain information about the molecular genetic characteristics of tumor. This review summarizes the current understanding of the mechanisms of interaction between tumor cells and platelets, and also discusses the possibilities of using platelet transcriptome analysis methods for diagnosing and assessing the dynamics of the tumor state, in particular, in response to therapy, as well as the current limitations for a wide range of introduction of this method into clinical practice.

Активное внедрение высокопроизводительного секвенирования в клиническую практику требует общего подхода к интерпретации обнаруженных генетических вариантов, в частности, вариантов с соматическим статусом. В 2017 году Ассоциация молекулярной патологии США (AMP), Американская коллегия медицинской генетики и геномики (ACMG), Американское общество клинической онкологии (ASCO) и Коллегия американских патологов (CAP) опубликовали руководство по интерпретации соматических генетических вариантов и выдаче заключений по результатам высокопроизводительного секвенирования опухолевой ДНК. Данный обзор посвящен специфике применения руководства AMP/ACMG/ASCO/CAP для интерпретации результатов генетических исследований детских солидных опухолей. В статье приводятся критерии, на которых основана классификация соматических генетических вариантов, обсуждаются проблемы оценки клинической значимости генетических находок и приводятся примеры классификации генетических вариантов, выявленных в различных типах детских солидных опухолей. Active clinical implementation of high-throughput DNA sequencing requires a common approach to the interpretation of detected genetic variants, including variants with somatic status. In 2017, the United States Association of Molecular Pathology (AMP), the American College of Medical Genetics and Genomics (ACMG), the American Society of Clinical Oncology (ASCO), and the College of American Pathologists (CAP) published the guidelines for interpreting and reporting the somatic genetic variants in cancer identified using high-throughput sequencing analysis. This review focuses on the specific application of the AMP/ACMG/ASCO/CAP guidelines in the field of genetic research on paediatric solid tumors. In particular, the review provides the criteria for classification of somatic genetic variants, discusses the problems of evaluating the clinical significance of genetic findings in paediatric tumors, and provides examples of classification of genetic variants specific for certain types of childhood solid malignancies.

The main pathogenetic mechanism of the development of pediatric low grade gliomas (pLGGs) is genetic aberrations in BRAF gene. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. We analyzed the clinical and molecular characteristics of 69 patients with LGGs. Molecular genetic testing for BRAF V600E mutation was performed by allele-specific real-time PCR and Sanger sequencing. BRAF V600E mutation was detected in 15 (21.7%) patients with LGG. The majority of BRAF-mutated cases of LGGs had the midline location: OPG – 7, subcortical ganglia – 1, brainstem – 2. The 2-year PFS was much worse in patients with BRAF V600E compared to patients without this mutation – 30% and 66.2%, respectively. The median time to progression for patients with BRAF V600E mutation was 9.5 months compared to 3.1 years for patients without indicated substitution. 5 patients with BRAF V600E-mutated LGGs who experienced progression after the conventional treatment, received targeted therapy (BRAF-inhibitor-3, BRAF + MEK inhibitors – 2) with good response (complete response – 2, partial response – 3). BRAF V600E mutation contributes to poor outcome in patients with LGGs Targeted therapy could be effective in this cohort of patients.

Correspondence: Ludmila I. Papusha Cand. of Sci. (Med.), Senior Physician, Head of the Department of Optimization of CNS Tumor Therapy, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Healthcare of Russian Federation Address: 1 Samory Mashela St., Moscow 117997, Russia E-mail: ludmila.mur@mail.ru DOI: 10.24287/1726-1708-2020-19-4-58-65

This article describes the use of allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of a patient with metastatic alveolar rhabdomyosarcoma. The reported case is unusual in terms of its presentation, diagnosis, and choice of treatment. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. In the last decades, the survival rates of patients with metastatic malignant neoplasms, including those with soft-tissue sarcomas, have not improved significantly. The prognosis for these patients remains extremely poor despite the intensification of chemotherapy and the application of radiation therapy. The use of autologous HSCT has not brought about any positive changes in treatment outcomes. Experimental approaches to treatment, including immunotherapeutic techniques, aimed at improving the prognosis for very high-risk patients are currently under development. We chose to report this case because of the unconventional treatment approach that had helped to achieve long-term control over the disease and to prevent systemic progression. It demonstrates that allogeneic HSCT can be regarded as one of the promising options for treatment intensification mainly due to the “graftversus-tumour” effect. The patients' parents gave their consent to the use of their child's data, including photographs, for research purposes and in publications.

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.

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.

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 objective of the present study was to elucidate the etiopathological significance of various Streptococcus pneumoniae serotypes in the children presenting with ENT infections and carrying nasopharyngeal pathogens. The incidence of the latter condition was 19.5% in the children free from S. pneumoniae infection in comparison with 20.9% and 30.7% in those having diagnosis of otitis media and rhinosinusitis respectively. Fifty five (88.8%) of the 62 isolated streptococcal strains were grouped into types with the use of multiplex PCR. Twelve serotypes were identified in the patients presenting with rhinosinusitis with the predominance of 6A/6B and 3 (40.5%) compared with seven isolated from the carriers of nasopharyngeal pathogens. In this group, type 3 also prevailed (26.5%) whereas other serotypes occurred less frequently: 23F (13,4%), indivisible totality of 8, 9V, 9A, 1F, 11A, 211B, 11C, 11D, 12F, 15A, and 33F (13.4%), 20 (6.7%), 19A (6.7%), 14 (6.7%), 6A,6B (6.7%). The serotypes of S. pneumoniae isolated from the patients with rhinosinusitis were found to show 55.3% identity with those present in the composition of the conjugated 7-valent pneumococcal vaccines, 63.2% identity with the 10-valent vaccine, 81.6% identity with the 11p-valnet vaccine, and 84.2% identity with the 13-valent vaccine.

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.

This article presents results of the multiplex PCR investigation of the serotypes distribution of S. pneumoniae strains circulating in Ekaterinburg and the Sverdlovsk region. This study was performed in children with invasive, noninvasive pneumococcal infections and carriers. 118 strains of pneumococci typed out of 129 ones (91.5%) referred to the 15 serotypes: 6A, 6B (20,8%); 23F (13,9%); 19F (11,5%); 8, 9V, 9A, 11F, 11A, 11B, 11C, 11D, 12F, 15A, 33F (11,5%); 3 (10%) 2, 15F, 17F, 22F, 23B (3,9%); 18B, 18C (3.9%), 19A (3,2%); 7F, 19B, 19C, 23A (3,2%); 5,10A (1.6%), 20 (1.6%), 14 (1, 6%); 9L, 9N, 15B, 15C (1,6%); 18F (1,6%); 18A (1.6%). Coincidence rate of serotypes S. pneumoniae, isolated from children with chronic infectious and inflammatory diseases of the lung with serotypes included into the content of the conjugate vaccines is: 7-valent 69.3%, 10-valent 98.2%, 11 and 13-valent 100%.

A total of 132 bone marrow specimens from 35 patients with B-acute lymphoblastic leukemia were studied. The minimal residual disease (MRD) was evaluated combined by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), and real time PCR. Qualitative compatibility of the results of flow cytometry and RT-PCR was 93.94%. Quantitative data of flow cytometry and real time PCR differed significantly (r(2) = 0.291). The time course of MRD evaluated by flow cytometry and real time PCR was similar. Simultaneous detection of MRD by flow cytometry and of the chimeric transcript by PCR can be an alternative to evaluation of heavy chain clone restructuring in immunoglobulins and T-cell receptors.