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Heat shock proteins (HSPs) attract the attention of scientists and clinicians due to their potential role as diagnostic and prognostic factors in a variety of cancers. HSP90 is one of the most important and well-known family members, necessary for maintaining intracellular homeostasis. In the extracellular space, it is responsible for the transmission of alarm signals to the immune system. Numerous reports have indicated that the level of intra - and extracellular HSP90 can correlate either with a poorer prognosis or with a better outcome, depending on the type of cancer. Still, little is known why the level of this chaperone is increased in some tumors and decreased in others, reflecting dual role of protein in cell death processes. Currently, there is no database reporting levels of serum HSP90 in children with acute lymphoblastic leukemia (ALL). As such, using enzyme-linked immunosorbent assay (ELISA) method, we aimed to determine this parameter in a group of 21 patients with newly diagnosed ALL. We found decreased protein serum levels in patients at disease presentation and after induction block of chemotherapy in comparison to healthy controls. Furthermore, we observed a negative correlation between HSP90 serum levels and one of the earliest prognostic factors of the treatment response - peripheral blood lymphoblasts on the 8th day of treatment. Our results indicate that HSP90 serum may play an important role in leukemogenesis and could be used as a marker to predict treatment failure in children with ALL.

HSPs demonstrate a strong association with gamma-delta (γδ) T cells. Most of the studies regarding interactions between the parameters were conducted in the 1990s. Despite promising results, the concept of targeting γδ T cells by HSPs seems to be a forgotten direction due to potent non-peptidic phosphoantigens rather than HSPs have been found to be the essential stimulatory components for human γδ cells. Currently, with greater knowledge of lymphocyte diversity, and more accurate diagnostic methods, we decided to study the correlation once again in the neoplastic condition. Twenty-one children with newly diagnosed acute lymphoblastic leukaemia (ALL) were enrolled on the study. Serum HSP90 concentrations were evaluated by an enzyme-linked immunosorbent assay (ELISA), subsets of γδ T cells (CD3+ γδ, CD3+ γδ HLA/DR+, CD4+ γδ and CD8+ γδ) by flow cytometry. We have shown statistically relevant correlations between serum HSP90 and CD3+ HLA/DR+ γδ T cells in paediatric ALL at diagnosis (R = 0.53, p &lt, 0.05), but not after induction chemotherapy. We also have demonstrated decreased levels of both serum HSP90 and CD3+ HLA/DR+ γδ T cells before treatment, which may indirectly indicate dose-dependent unknown interaction between the parameters. The results of our study may be a good introduction to research on the association between HSPs and CD3+ HLA/DR+ γδ T cells, which could be an interesting direction for the development of anti-cancer strategies, not just for childhood ALL.

Background/aim The risk factors, clinical features and non-hematological toxicity profiles during chemotherapy in acute lymphoblastic leukemia (ALL) patients treated in pediatric hematology centres were analysed. Materials and methods A total of 902/1872 children were reported as having grade 3 or 4 toxicity. Results Among the analysed toxicities, infection and gastrointestinal and liver toxicities were the most common. The median follow-up was 6.8 years. Overall survival and event-free survival rates for the analysed group were lower than those reported for the group without grade ≥3 toxicity. In univariate analysis, we identified the number of toxic episodes, the risk group and remission status that had a significant impact on the outcome. Multivariate analysis demonstrated the risk group and the number of toxic episodes ≥3 to be statistically significant for the results. Conclusion The toxic profiles investigated in our report should be used in future efforts to decrease the burden of side effects during chemotherapy.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children despite recent advances showing improved responses to chemotherapy treatment. Gamma-delta (γδ) T cells are a recent topic of growing interest in the field of adoptive immunotherapy. These cells have also been proven to be an optimal predictor of a favorable outcome in numerous malignancies. We evaluated subgroups of γδ T cells in the peripheral blood of 19 children with newly diagnosed ALL at the time of diagnosis and following chemotherapy induction. Due to the fact that serum HSP90 serves as a ligand for γδ T cells, we also checked the correlation between HSP90 and γδ T cells. As a result, we found, that the number of CD8+ γδ T cells in peripheral blood at disease presentation was almost three times higher in the intermediate risk group compared to patients in high risk group. Furthermore, we observed higher percentages of the subset in younger patients at diagnosis and after induction, but not in healthy controls. We also noticed negative correlations between CD8+ γδ T cells and minimal residual disease (MRD) before chemotherapy and after induction. We showed a positive association between activated CD3+ γδ T cell and serum HSP90 at presentation. In conclusion, our data suggests that CD8+ γδ T cells may be an early marker of favorable prognosis in childhood ALL while serum HSP90 may act as an agent activating CD3+ γδ T effector cells.

The aim of the study was to retrospectively compare the effectiveness of the ALL IC-BFM 2002 and ALL IC-BFM 2009 protocols and the distribution of risk groups by the two protocols after minimal residual disease (MRD) measurement as well as its impact on survival. We reviewed the medical records of 3248 patients aged 1–18 years with newly diagnosed ALL who were treated in 14 hemato-oncological centers between 2002 and 2018 in Poland. The overall survival (OS) of 1872 children with ALL treated with the ALL IC 2002 protocol was 84% after 3 years, whereas the OS of 1376 children with ALL treated with the ALL IC 2009 protocol was 87% (P P = 0.006). Our study shows that the ALL IC-BFM 2009 protocol improved the results of children with ALL compared to the ALL IC-BFM 2002 protocol in Poland. This analysis confirms that MRD marrow assessment on day 15 of treatment by FCM-MRD is an important predictive factor.

Objective The aim of this study was to analyse the clinical characteristics and outcome of children diagnosed with Ph+ ALL. Material and methods A total of 2591 newly diagnosed ALL children were treated in Poland between the years 2005 and 2017. Of those, 44 were diagnosed with Ph(+) ALL. The patients were treated according to protocols: ALL IC-BFM 2002 and 2009 (26 patients), EsPhALL (12 patients), initially ALL IC-BFM and then EsPhALL (6 patients). Results The median of follow-up in the observed group was 3 years. Overall survival (OS) and event-free survival (EFS) of Ph+ ALL group were 0.73 and 0.64. OS and EFS of patients after HSCT were 0.78 and 0.66, while without HSCT were 0.6 and 0.6, P = 0.27 and 0.63. OS was 0.8 for patients treated with chemotherapy plus imatinib and 0.61 for chemotherapy alone, P = 0.22, while EFS was 0.66 (imatinib therapy) and 0. 61 (without imatinib), P = 0.41. Conclusion Our study suggests that adding imatinib to intensive chemotherapy seems to improve outcome. However, this study was limited by a small number of patients and a variety of chemotherapy regimens with or without imatinib.

The aim of this study was to evaluate the risk factors of relapse and treatment-related deaths in acute lymphoblastic leukemia (ALL) in children residing in Poland. A total of 1872 patients with newly diagnosed ALL, treated according to the ALL IC-BFM 2002 protocol in 14 Polish pediatric hematology centers from 2002 to 2012 were included in the study. Three-hundred eighty-four patients experienced treatment failure. The last follow-up was 31 December, 2016. Univariate analysis identified factors in each risk group that were significantly different between children whose treatment failed and those who remained in the first remission. Multivariate analysis demonstrated that only the age of 10 years or over at primary diagnosis in the high-risk group was an adverse prognostic factor. To facilitate the analysis, patients were divided into three groups: relapsed children who survived; relapsed children who died; children without relapse who died due to toxicity. Our analysis showed that age older than 10 years is a particular risk factor for the failure of first-line of treatment, both in terms of relapse and treatment-related mortality.

Regulatory T-cells (Tregs) are a very important subtype of lymphocytes when it comes to self-control in the human immunological system. Tregs are decisive not only in the protection against destruction of own tissues by autoimmune immunocompetent cells but also in the immunological answer to developing cancers. On the other hand, Tregs could be responsible for the progression of acute and chronic leukemias. In our study, we review publications available in the PUMED database concerning acute leukemia, with a particular emphasis on child’s leukemias. The percentage of regulatory T-lymphocytes in peripheral blood and bone marrow was elevated compared to those in healthy individuals and correlated with progressive disease. Regulatory T-cells taken from children diagnosed with leukemia showed a higher suppressive capability, which was confirmed by detecting elevated levels of secreted IL-10 and TGF-beta. The possibility of pharmacological intervention in the self-control of the immunological system is now under extensive investigation in many human cancers. Presumably, Treg cells could be a vital part of targeted therapies. Routine Treg determination could be used to assess the severity of disease and prognosis in children with acute lymphoblastic leukemia. This proposition results from the fact that in some studies, higher percentage of Treg cells in peripheral blood was demonstrated. However, observations confirming these facts are scarce; thus, extrapolating them to the population of children with hematological malignancies needs to be verified in additional studies.

CD4+CD25highCD127low/−FoxP3+ regulatory T cells (Tregs) are currently under extensive investigation in childhood acute lymphoblastic leukemia (ALL) and in other human cancers. Usually, Treg cells maintain the immune cell homeostasis. This small subset of T cells has been, in fact, considered to be involved in the pathogenesis of autoimmune diseases and progression of acute and chronic leukemias. However, whether Treg dysregulation in CLL and ALL plays a key role or it rather represents a simple epiphenomenon is still a matter of debate. Treg cells have been proposed as a prognostic indicator of the clinical course of the disease and might also be used for targeted immune therapy. Our study revealed statistically higher percentage of Treg cells in the bone marrow than in peripheral blood in the group of 42 children with acute lymphoblastic leukemia. By analyzing Treg subpopulations, it was shown that only memory Tregs in contact with leukemic antigens showed statistically significant differences. We noticed a low negative correlation between Treg cells in the bone marrow and the percentage of blasts (R=−0.36) as well as a moderate correlation between Treg cells in the bone marrow and Hb level (R=+0.41) in peripheral blood before therapy. The number of peripheral blood blasts on day 8th correlates negatively (R=−0.36) with Tregs. Furthermore, statistical analysis revealed low negative correlation between the number of Tregs in the bone marrow and the minimal residual disease measured on day 15th, the percentage of blasts in the bone marrow and leukocytosis after 15 days of chemotherapy. These results indicate the influence of Tregs on the final therapeutic effect.

Background Chronic myeloid leukemia (CML) constitutes only 2-3% of all leukemias in pediatric patients. Philapelphia chromosome and BCR-ABL fusion are genetic hallmarks of CML, and their presence is crucial for targeted molecular therapy with tyrosine kinase inhibitors (TKIs), which replaced hematopoietic stem cell transplantation (HSCT) as a standard first-line therapy. The disease in pediatric population is rare, and despite molecular and clinical similarities to CML in adults, different approach is needed, due to the long lifetime expectancy and distinct developmental characteristics of affected children. Objectives The objective of this study is to evaluate treatment with imatinib in Polish pediatric patients with CML. Material and methods We analyzed the results of treatment with imatinib in 57 pediatric patients (June 2006 - January 2016) from 14 Polish pediatric hematology and oncology centers. Results In the study group, 40 patients continued imatinib (median follow-up: 23.4 months), while in 17 the treatment was terminated (median follow-up: 15.1 months) due to therapy failure. In the latter group, 13 patients underwent HSCT, while 4 switched to second-generation TKIs. The 5-year overall survival rate (OS) in the study group was 96%, and the 5-year event-free survival (EFS) was 81%. Conclusions Our results confirm that the introduction of TKI therapy has revolutionized the treatment of CML in the pediatric population by replacing the previous method of treatment with HSCT and allowing a high percentage of OS and EFS.

Background. Hematopoietic stem cell transplantation (HSCT) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including malignancies, bone marrow failure syndromes, disorders of the immune system, and metabolic disorders. Objective. Analysis of results of hematopoietic stem cell transplantations performed over a period of 12 years in a single pediatric center. Patients and methods. All transplants performed between 2003 and 2015 in the Department of Pediatric Hematology and Oncology in Bydgoszcz were included in this analysis. The results of therapy with stem cell transplantation were analyzed in three time periods: 2004-2007, 2008-2011 and 2012-2015. Results. A total number of transplants was 318, including 132 auto-HSCT and 186 allo-HSCT. Among allogeneic transplants, 68 were done from matched-sibling donor and 118 from alternative donor. The mean survival for all patients estimated by Kaplan-Meier method was 8.1 years. Probability of overall survival (pOS) after all transplants was 0.64±0.03. pOS after allo-HSCT was 0.62±0.04, and 0.67±0.05 after auto-HSCT. Overall survival for patients transplanted in the second (2008-2011) and third (2012-2015) time period was comparable both for auto- and allo-HSCT. However, it was significantly higher than for patients transplanted in the first period of time (2004-2007) for all patients, and for those undergoing auto-HSCT. In allo-HSCT patients, in spite of increase of over 20% in pOS (43% vs 66% vs 64% in respective time periods), the difference was not statistically significant. Conclusion. Presented results of HSCT obtained in our center are comparable with those from other international registries and centers. Wstęp. Transplantacja komórek krwiotwórczych (HSCT) jest ważną metodą terapeutyczną w wielu wrodzonych i nabytych chorobach, w tym nowotworowych, zespołach niewydolności szpiku oraz zaburzeniach immunologicznych i metabolicznych. Celem pracy jest analiza wyników HSCT w pojedynczym ośrodku pediatrycznym w okresie 12 lat. Pacjenci i metodyka. Analizie poddano wyniki przeszczepień wykonanych w latach 2003-2015 w Klinice Pediatrii, Hematologii i Onkologii w Bydgoszczy. Transplantacje analizowano w trzech przedziałach czasowych: 2004-2007, 2008-2011 oraz 2012-2015. Wyniki. Wykonano 318 HSCT, w tym 186 alloge-nicznych (68 zgodnych rodzinnych i 118 od dawców alternatywnych) oraz 132 autologiczne. Średnie przeżycie po HSCT, wyznaczone metodą Kaplana-Meiera wyniosło 8,1 lat. Całkowite prawdopodobieństwo przeżycia (pOS) wyniosło 0,64±0,03; pOS po allo-HSCT wynosi 0,62±0,04, a po auto-HSCT 0,67±0,05. Nie wykazano znamiennych różnic w pOS zarówno po allo-HSCT, jak i po auto-HSCT pomiędzy drugim (2008-2011) i trzecim (2012-2015) analizowanym okresie. Jednakże, pOS było wyższe w dru-gim i trzecim okresie w stosunku do okresu pierwszego (2004-2007), zarówno dla wszystkich pacjentów, jak i u pacjentów po auto-HSCT. W grupie pacjentów allo-HSCT, uzyskano wzrost pOS o ponad 20% (43% vs 66% vs 64% w kolejnych przedziałach czasu; ns). Wnioski. Wyniki HSCT uzyskiwane aktualnie w na-szym ośrodku są porównywalne z wynikami podawanymi w międzynarodowych rejestrach.

Background Currently, there are three major maturational stages of CD19 antigen expressing B-cell precursors (hematogones). In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), the malignant counterpart of hematogones, the leukemic blasts share common phenotypic features. The aim of the study was to enumerate the actual differences between the leukemic blasts in the CD10+ and CD10− subgroups of BCP-ALL and hematogones by assessing the expression of the antigens: TdT, CD34, CD45, CD10, CD38, CD20 and CD22. Methods To enable quantitative assessment of antigen expression on the different cell types, an objective scale of antigen expression was developed, the basis of which was direct fluorescence measurement using multicolor flow cytometry. Results All cases of CD10+ BCP-ALL clustered with type 1 hematogones. Among CD10−- BCP-ALL subgroup, 54.5%, 27.3% and 18.2% of cases clustered with type 1, 2 and 3 hematogones, respectively. In contrast to the CD10− blasts, the CD10+ blasts exhibited significantly higher levels of TdT, CD22, CD34 and CD20 expression. Conversely, CD10− blasts showed significantly higher expression of CD45 than CD10+ blasts, and a higher rate of CD45 antigen overexpression than CD10+ blasts (54.5% vs. 14.9% of cases, respectively). Conclusions Multiparameter flow cytometry combined with the use of absolute antigen expression scale based on direct fluorescence measurement, has enabled a clear distinction between blasts in BCP-ALL cases and their normal counterparts. This novel and previously undescribed method has allowed the comparative analysis of antigen expression between leukemic blasts and different types of their normal counterparts. © 2014 International Clinical Cytometry Society

Individualization of the topotecan dosing can reduce inter-patient variability, toxicity, and at the same time increases chemotherapy efficacy. Topotecan dosing based on simultaneous drug monitoring and pharmacokinetic analysis can yield more accurate and precise estimation of the topotecan systemic exposure than that attainable with the fixed dosing approach. Therefore, a combined approach could provide a tool assisting the clinicians in individualization of the topotecan dosing. The aim of the study was to estimate the topotecan exposure in pediatric patients with acute myeloid leukemia (AML) based on the plasma concentration-time data and using the pharmacokinetic analysis. The primary goal was achieve the correct estimation of the target plasma area against the topotecan concentration-time curve (AUC) in a 5 day course of cladribine followed by monitored topotecan in pediatric patients with recurrent/refractory AML. A sensitive and selective reversed-phase liquid chromatographic-mass spectrometry (LC-MS) assay was developed to quantify total topotecan in the human plasma samples. This method, with its lower quantification limit of 1 ng/ml, was validated over a linear range of 1-150 ng/ml. Under the proposed approach, the topotecan dosing was selected so as to achieve the final AUC value of 140±20 ng/ml h. The presented analytical and pharmacokinetic data demonstrate that the proposed approach can be a practical, useful, efficient, and accurate tool for individualizing the topotecan dosing in children with AML.

Background: Diabetic ketoacidosis (DKA) development in children with new-onset type 1 diabetes (T1DM) is often the main consequence of delayed diagnosis. The aim of the study was to estimate the frequency of difficulties in T1DM diagnosis and to investigate if and how the demographic factors (gender, patient's age at presentation, family history of T1DM, level of maternal education, place of residence, and health service unit the patient called at) have any influence on diagnostic delays. Subjects and methods: Retrospective analysis of 474 children (243 boys—51.27% and 231 girls −48.73%) with new-onset T1DM aged below 17 yr and living in the Pomeranian region of Poland was carried out. The delay in diagnosis was recognized if the patient was not diagnosed on the first visit because of omission, wrong interpretation of main diabetic symptoms, exclusive treatment of additional signs, or concomitant diseases. Results: Difficulties in diagnosing T1DM were found in 67 cases (14.13%) and they are the main cause of DKA development in these children (p = 0.00). Among the examined demographic factors, mainly the patient's age at presentation has a significant influence on diagnostic delays (p = 0.01), especially in children below 2 yr (p = 0.00). Most frequently family doctors were responsible for wrong preliminary diagnosis. Conclusions: Difficulties in diagnosing T1DM are a significant cause of DKA development in children with new-onset disease. Patient's age at presentation is the main risk factor of delayed diagnosis, especially in children below 2 yr. The increase in awareness among pediatricians concerning the possibility of T1DM development in children is needed.