ALL IC-BFM Research Articles

Outcome of Childhood and Adolescents and Young Adults ALL Treated with ALL IC-BFM 2009 Protocol–A Single-Center Experience in India

Outcome of Childhood and Adolescents and Young Adults ALL Treated with ALL IC-BFM 2009 Protocol–A Single-Center Experience in India

ETV6::RUNX1 Acute Lymphoblastic Leukemia: how much therapy is needed for cure?

Recent trials show 5-year survival rates >95% for ETV6::RUNX1 Acute Lymphoblastic Leukemia (ALL). Since treatment has many side effects, an overview of cumulative drug doses and intensities between eight international trials is presented to characterize therapy needed for cure. A meta-analysis was performed as a comprehensive summary of survival outcomes at 5 and 10 years. For drug dose comparison in non-high risk trial arms, risk group distribution was applied to split the trials into two groups: trial group A with ~70% (range: 63.5–75%) of patients in low risk (LR) (CCLSG ALL2004, CoALL 07-03, NOPHO ALL2008, UKALL2003) and trial group B with ~45% (range: 38.7–52.7%) in LR (AIEOP-BFM ALL 2000, ALL-IC BFM ALL 2002, DCOG ALL10, JACLS ALL-02). Meta-analysis did not show evidence of heterogeneity between studies in trial group A LR and medium risk (MR) despite differences in treatment intensity. Statistical heterogeneity was present in trial group B LR and MR. Trials using higher cumulative dose and intensity of asparaginase and pulses of glucocorticoids and vincristine showed better 5-year event-free survival but similar overall survival. Based on similar outcomes between trials despite differences in therapy intensity, future trials should investigate, to what extent de-escalation is feasible for ETV6::RUNX1 ALL.

PAX5 Alterations in a Consecutive Childhood B-Cell Acute Lymphoblastic Leukemia Cohort Treated Using the ALL IC-BFM 2009 Protocol.

In this study, we aimed to identify patients within our B-ALL cohort with altered PAX5. Our objective was to use a comprehensive analysis approach to characterize the types of genetic changes, determine their origin (somatic/germline), and analyze the clinical outcomes associated with them. A consecutive cohort of 99 patients with B-ALL treated at the Children's Hospital of the UMC Ljubljana according to the ALL IC-BFM 2009 protocol was included in our study. We used RNA sequencing data for gene expression analysis, fusion gene detection and single nucleotide variant identification, multiplex-ligation dependent probe amplification for copy number variation assessment, and Sanger sequencing for germline variant detection. PAX5 was impacted in 33.3% of our patients, with the genetic alterations ranging from CNVs and rearrangements to SNVs. The most common were CNVs, which were found in more than a third of patients, followed by point mutations in 5.2%, and gene rearrangements in 4.1%. We identified eight patients with a PAX5-associated genetic subtype that were previously classified as "B-other", and they showed intermediate outcomes. We showed higher minimal residual disease values at the end of induction and poorer event-free survival in hyperdiploid cases carrying duplications in PAX5 compared to other hyperdiploid cases. We also report an interesting case of a patient with PAX5::FKBP15 and a pathogenic variant in PTPN11 who underwent an early relapse with a monocytic switch. In conclusion, this study provides valuable insights into the presence, frequency, and prognostic significance of diverse PAX5 alterations in B-ALL patients, highlighting the complexity of genetic factors and their impact on patient outcomes.

Measurable Residual Disease Results & Survival with Augmented Early Intensification Therapy in Intermediate-Risk & High-Risk Group Pediatric Acute Lymphoblastic Leukemia Patients Treated with ALL IC-BFM 2009 Protocol: A Single-Center Study from India

Introduction Measurable residual disease (MRD) has proven to be one of the most effective disease monitoring responses in ALL. Multiple treatment groups have demonstrated End-of-Induction (day 33) flow MRD of <0.01% has excellent disease-free survival (DFS). Patients with end-of-consolidation MRD of <0.01% has an overall DFS of 75 % as per COG AALL1131 including the very high-risk B-ALL genotypes. We conducted a study to assess the efficacy & toxicity of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol for intermediate-risk & high-risk group pediatric ALL patients. Objectives To evaluate efficacy of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 in terms of achievement of day 78 MRD negative status. To determine the event-free survival in intermediate-risk & high-risk group patients. To appraise the adverse events (AEs) of augmented phase IB Induction. Methodology The study enrolled treatment-naïve ALL patients of 1-25 years' age between April 2021 to February 2023 at AIIMS Rishikesh, India. Patients were risk stratified into Standard (SR), Intermediate (IR) & High risk (HR) groups as per ALL IC BFM 2009 protocol. All patients received Augmented phase IB protocol. The regimen comprised of cyclophosphamide (2 doses), four blocks of cytarabine & 6-mercaptopurine, augmented with vincristine (4 doses), and L-Asparaginase (12 doses). Day 78 bone marrow flowcytometric MRD was performed in all patients irrespective of day 33 marrow morphology & MRD. The study defined complete remission as BM blasts <5% with no extramedullary disease and MRD negativity as <0.01%. The AEs was monitored as per NCI-CTCAE v5. As per the study protocol, patients of B-ALL with day 33 MRD >0.01%, ph-positive ALL, hypodiploidy and T-ALL with day 78 MRD > 0.01% was considered as HR & treated with High-risk blocks consolidation followed by transplant or delayed intensification & maintenance. Results A total of 75 patients enrolled in the study. The M: F ratio was 1.6:1. The median age was 8 years (range 1-25 yrs). B & T-ALL constituted 80% & 20% respectively. ph-positivity accounted 22% of B-ALL. The median Lansky performance was 40 & ECOG was 3 among the participants. The median hb, total white counts, and platelets were 5.9 g/dl, 28 x10 9/L and 30 x10 9/L respectively. At diagnosis, 28% of patients had hyperleukocytosis, 15% with bacterial sepsis, 8% with invasive fungal infections, and 8% with CNS leukemia. At baseline, the distribution of patients in SR, IR & HR was 15%, 68% & 17% respectively. The final risk strata at end-of-induction were 8%, 49% & 43% in SR, IR & HR groups. CNS leukemia accounted for 8% of all patients. Of the total evaluable patients, 86% achieved day 8 good steroid response (absolute blasts count <1000/cu.mm), 73% & 38% achieved day 15 marrow M1 response (<5%blasts) and MRD < 0.1%, 97% & 77% achieved day 33 M1 marrow response and day 33 MRD <0.01% respectively. Among the study participants who were MRD positive on day 15, three-fourths (75%) of them accomplished MRD negativity at day 33. Of the 23% patients with day 33 MRD positivity, 80% achieved day 78 MRD negativity of <0.01% with Augmented phase IB with 3 participants being persistent MRD positive. The median follow-up was 24 months and the overall survival was 74%. The mean survival time was 16.7 months (12.7-20.6) for HR & 21.2 months (18.3-24.1) for IR patients. The subgroup analysis demonstrated an overall survival of 80% for SR, 84% for IR & 64% for HR groups. Nevertheless, the proportion of patients in the SR group (8%) was lower compared to the others. The commonest hematological adverse events were hypofibrinogenemia (22%), followed by febrile neutropenia (19%), anemia (19%), and thrombocytopenia (18%) respectively. The commonest non-hematological AEs were peripheral neuropathy (5%) and typhlitis (5%). Conclusion In our single-center experience, the use of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol in intermediate-risk & high-risk pediatric ALL patients was associated with achievement of day 78 MRD negative status in 80% patients who had day 33 MRD-positivity without significant treatment-related toxicity, and encouraging event-free survival in this patient population.

Tooth Abnormalities and Their Age-Dependent Occurrence in Leukemia Survivors.

The multidrug nature of anticancer treatment and different treatment protocols used in the studies are likely to be a major limitation in establishing real risk factors determining the occurrence of dental abnormalities. The authors aimed to establish a relationship between the duration and the dose of chemotherapy and the number of tooth adverse effects in the group receiving the same treatment. Of the 40 anticancer therapy recipients who attended the outpatient dental clinic, 7 leukemia survivors receiving the treatment according to the ALL IC-BFM 2002 protocol were selected. The study group consisted of four females and three males aged 92 to 207 months at the time of dental examination and 29 to 91 months at leukemia diagnosis. As a result of the clinical and radiological examination, dental abnormalities such as agenesis, tooth size reduction, root abnormalities, and taurodontia were identified, and the medical records of all survivors were reviewed in terms of drugs administered, their doses, and treatment schedules. No correlation was observed between the treatment duration of an intensive therapy, the entire therapy, and the number of tooth abnormalities. No relationship was also found between the number of dental abnormalities and the cumulative dose of vincristine, L-asparaginase, methotrexate, cyclophosphamide, cytarabine, and 6-mercaptopurine. The age at the onset of antineoplastic therapy is likely to be the strongest risk factor for toxic injury during tooth development.

Electroencephlographic predictors of epileptiphorm activity in children with lymphoid tumors under therapy

Objective. To identify electroencephalographic predictors of symptomatic seizure in children and adolescent with lymphoid tumors during chemotherapy.Material and methods. EEG results of a study in 20 children aged 7 to 15 years with lymphoid tumors during chemotherapy under the В-NHLBFM-90/95 и ALL IC-BFM 2002 program are presented. Depending on the presence of clinical manifestations, patients were divided into groups: 1st group (n = 10 without epileptiform signs), 2nd group (n = 10 with epileptiform signs).Results. In patients of group 1, who did not have clinical manifestations (seizures), the EEG recorded a signifiantly better, compared with patients of group 2, spectral dencity in band alpha rhythm and sensorimotor rhythm (p < 0.001) and signifiantly less delta activity and beta frequencies (p < 0.001). It was found that, despite the absence of epileptiform signs in the EEG, these patients had fewer EEG correlates of the ‘spike-wave’ complexes, and signifiantly less power levels of delta and beta-2 frequency bands (p < 0.001).Conclusions. The use of comparative EEG mapping made it possible to identify predictors of epileptiform activity in children and adolescents with lymphoid tumors during chemotherapy.

Evaluation of MRD-status in post-induction period in pediatric patients with acute lymphoblastic leukemia.

The need to study the significance of minimal residual disease (MRD) at the induction therapy in patients with acute lymphoblastic leukemia (ALL) is beyond doubt. This has been confirmed by many years of work by many research groups. The role of MRD in the late stages of treatment and the impact of these values on patients survival requires research and discussion. To evaluate the influence of MRD-status in post-induction period on survival in patients with acute lymphoblastic leukemia. From 2010 to 2022, 135 patients with primary B-ALL enrolled in ALL-IC BFM 2009 protocol. Median age was 5.4 year (range 1-17). Male was 62 (49,5%) and female 73 (54,1%). The diagnosis was based on WHO 2016 criteria. Stratification on prognostic risk groups was carried out according to protocol criteria. Prednisone response evaluated at day 8 of treatment. The 15th, 33th, and 78th (as post-induction) day response was assessed by bone marrow cytology and level of MRD by flow cytometry. 5y-overall survival (OS) for patients with MRD-negative status on day 15 was 94,4±5,4% and 87,0±3,4% for MRD-positive (p=0,5). On day 33 patients with MRD-negative status achieved 5y-OS in 86,7±5,8% and 89,6±3,5% for MRD-positive (p=0,6).5y-OS for patients with MRD-negative status on day 78 was 90.8±4.0%, MRD-positive - 90,4+6.5%. DFS for MRD-negative status was 88.5±4.5%, for MRD-positive - 66.3±11.8% (p=0,1). EFS for MRD-negative patients was 87.2±4,6% and for MRD-positive 66.3±11.8% (p=0,09). We have found a tendency between MRD status on day 78 and the frequency of relapses in patients. At the moment, there are no reliable data on the effect of post-induction MRD status on survival. The assessment of MRD in the post-induction period has prognostic prospects and requires further study.

Dermatological toxicity of acute lymphoblastic leukemia treatment by protocol ALL IC-BFM 2002

Background. Аcute lymphoblastic leukemia (ALL) in children is not only the most common but also potentially curable disease in 8590% cases. The other side of high effectiveness of modern treatment protocols their toxicity. Inspite of skin and mucosal toxicity is not a life threatening condition, it requires a supportive care to prevent infectious complications, which prolong hospitalization, administration of antibacterial, antifungal and in some cases antiviral drugs.
 Aim. To study skin and mucosal toxicity in patients with ALL, treated by ALL IC-BFM 2002 protocol.
 Materials and methods. One hundred and nineteen pediatric patients with primary diagnosed ALL were enrolled the study. All the patients were treated by ALL IC-BFM 2002 protocol. Toxicity assessment was performed on each step by the scale of National Cancer Institute (NCI) USA, 2d version.
 Results. The most often variants of skin and mucosal toxicity during ALL IC-BFM 2002 protocol were found on protocol mM/M, based on high-dosed methotrexate. In 42.1% stomatitis 2st. was diagnosed on methotrexate dose 5000 mg/m2, 3st. 15.8%, 4st. 5,3%. Methotrexate dosed 2000 mg/m2 coused stomatitis 3 st. in 6.3%, in other patients stomatitis was 12st. Exfoliative dermatitis was in 1 (1%) case with prolonged methotrexate elimination. Block polychemotherapy used in high risk group of patients was complicated with stomatitis 34 st. in 90%. In 20% naso-gastral catheter was performed and in 25% used partial/hole parenteral nutrition support. Alopecia was reversible and observed in 100% patients. Protocols I, II and maintenance treatment were free of clinically significant skin and mucosal toxicity.
 Conclusion. The skin and mucosal toxicity profile of ALL IC-BFM 2002 protocol was tolerable. Hole volume of supportive care, preventing and treatment of dermatological toxicity of chemotherapy can prevent of severe skin and mucosal toxicity (soft tissue infections, sepsis, metabolic disorders).

Toxicity assessment of acute lymphoblastic leukemia treatment protocol ALL IC-BFM 2002

Background. Treatment results of acute lymphoblastic leukemia in children on protocol, developed by one of the leading research study group BFM (Berlin-Frankfurt-Munster) are impressive - longstanding overall survival rate comprise 93.4 %. The basis of success is a differential approach based on prognostic factors. In some local issues BFM protocols receive criticism because of high toxicity, but trying to find results of complex protocol toxicity assessment by modern scales in local literature, it was absent.Aim. To study a toxicity of acute lymphoblastic leukemia treatment by ALL IC-BFM 2002 protocol.Materials and methods. 119 patients with primary diagnosed acute lymphoblastic leukemia were enrolled the study. All the patients were treated by ALL IC-BFM 2002 protocol. Toxicity assessment was performed by the scale of National Cancer Institute (NCI) USA, 2nd version.Results. The most often variants of toxicity during treatment according to the ALL IC-BFM 2002 protocol were myelo-suppression, infections and hepatotoxicity of I-IV degrees of severity. Clinically significant toxicity (grade IV) was myelosuppression and necessity for transfusions in 76.8-100 % (depending on prognostic risk group and as such protocol arm). Nephro- and hepatotoxicity described on high-dosed methotrexate (2000 mg/m2 or 5000 mg/m2) were I-II grade in 89.5 % patients. Stomatitis grade I-II was in 93.7 % patients of standard and intermediate risk groups, but in the most (90 %) patients from high risk group it was higher - grade III-IV. Mortality on protocol ALL IC-BFM 2002 caused by infection complications was 1.6 %. It should be noted, that supportive care, prescribed in ALL IC-BFM 2002 protocol help to prevent and correct severe toxicity effectively.Conclusion. The toxicity profile of ALL IC-BFM 2002 protocol, analyzed by frequency of toxicity grade III-IV with whole supportive care approaches, is acceptable. The noted variants of toxicity were fully resolved without irreversible consequences for the patients.

Evaluation of early-onset cardiotoxic effects of anthracyclines used during the treatment of childhood acute lymphoblastic leukemia by speckle-tracking echocardiography

Anthracyclines are widely used in the treatment of acute lymphoblastic leukemia (ALL). However, cardiotoxicity is the most critical side effect that requires dose limitation. It is thought to occur at first exposure, but the clinical presentation may occur years later. In this study, we aimed to determine the time of initial damage and cardiotoxicity that develops in children with ALL. In this prospective study, 13 patients with newly diagnosed intermediate-risk precursor B cell ALL treated with the ALL-IC BFM 2009 protocol were included. Conventional echocardiography, tissue Doppler imaging (TDI), and speckle-tracking echocardiography (STE) were performed in all the patients before chemotherapy, after completing the induction phase, and at the end of the reinduction phase. The mean age of the patients was 7.8±4.6 (3.1-16.3) years. Myocardial velocity during systole (Sm) determined by TDI at the interventricular septum significantly decreased during the induction phase. Despite a decrease in STE parameters, a statistically significant reduction was determined in the global longitudinal strain rate at both left and right ventricles at the end of the induction. Nevertheless, a statistically significant increase was observed among the conventional echocardiographic findings in the left ventricular end-diastolic diameter at the end of the reinduction. During the treatment of ALL, subclinical anthracycline-associated cardiotoxicity develops in the early stages of treatment. The findings detected by TDI and STE could be missed by conventional echocardiography. We recommend evaluating patients with these newly developed techniques to detect subclinical cardiotoxicity at an early stage and starting appropriate therapy on time.

Clinical impacts of copy number variations in B-cell differentiation and cell cycle control genes in pediatric B-cell acute lymphoblastic leukemia: a single centre experience

BackgroundIKZF1 gene deletions have been identified as a poor prognostic factor in pediatric B-cell acute lymphoblastic leukemia (B-ALL), especially in the presence of co-occurring deletions (IKZF1plus profile). This study aimed to determine the frequency of IKZF1 deletions and deletions in other B-cell differentiation and cell cycle control genes, and their prognostic impact in Slovenian pediatric B-ALL patients.Patients and methodsWe studied a cohort of 99 patients diagnosed with B-ALL from January 2012 to December 2020 and treated according to the ALL IC-BFM 2009 protocol. Eighty-eight bone marrow or peripheral blood samples were analysed for copy number variations (CNVs) using the SALSA MLPA P335 ALL-IKZF1 probemix.ResultsAt least one CNV was detected in more than 65% of analysed samples. The most frequently altered genes were PAX5 and CDKN2A/B (30.7%, 26.1%, and 25.0%, respectively). Deletions in IKZF1 were present in 18.2% of analysed samples and were associated with an inferior 5-year event-free survival (EFS; 54.8% vs. 85.9%, p = 0.016). The IKZF1plus profile was identified in 12.5% of the analysed samples, and these patients had an inferior 5-year EFS than those with deletions in IKZF1 only and those without deletions (50.8% vs. 75.0% vs. 85.9%, respectively, p = 0.049). Overall survival (OS) was also worse in patients with the IKZF1plus profile than those with deletions in IKZF1 only and those without deletions (5-year OS 76.2% vs. 100% vs. 93.0%, respectively). However, the difference between the groups was not statistically significant.ConclusionsOur results are in concordance with the results obtained in larger cooperative clinical trials. Copy number variations analysis using the SALSA MLPA kit is a reliable tool for initial diagnostic approach in children with B-ALL, even in smaller institutions in low- and middle-income countries.

Роль молекулярно-генетичних транслокацій у первинній відповіді на лікування дітей, хворих на гостру лімфобластну лейкемію, за програмою ALLIC BFM 2009

The correlation analysis between the number of blast cells in bone marrow and peripheral blood was performed, and the dependence of blast percentage on the presence of molecular genetic translocations (AF4/MLL, BCR/ABL1, TEL/AML, E2A/PBX1) in patients with acute lymphoblastic leukemia (ALL) under the conditions of ALLIC-BFM 2009 cytostatic therapy was researched. The purpose of the study was to establish a relationship between the number of blast cells in bone marrow and peripheral blood depending on the presence of molecular genetic translocations for early detection of induction treatment by ALLIC BFM 2009. Materials and methods. The survey group consisted of 105 children aged 12 months to 16 years (median age was 6 years). Among those surveyed were 62 boys (59.0%) and 43 girls (41.0%). All patients were diagnosed with acute lymphoblastic leukemia. Results and discussion. Correlation analysis revealed a high degree of correlation between the number of blast cells in the bone marrow and peripheral blood, as the correlation coefficient (r) is 0.87. It is shown that the increase in the number of blast cells depends on the presence of chromosomal translocations. The highest number of blasts was observed in patients with BCR/ABL1 and E2A/PBX1 translocations, in whom the content of blasts in bone marrow was 97 and 96%, respectively, and in peripheral blood - 67 and 73%, respectively. It was found that treatment under the ALLIC BFM 2009 program leads to a decrease in the number of blast cells in the bone marrow and blood with minimal values on the 33rd day of treatment. It has been shown that the highest levels of blast cells during chemotherapy are observed in patients with chromosomal translocations BCR/ABL1 and E2A/PBX1. In patients with AF4/MLL translocation, the efficacy of therapy was the highest because no blast cells in the bone marrow were visualized on day 33 of treatment. The study of the primary response of patients with acute lymphoblastic leukemia to induction treatment according to the ALLIC BFM 2009 program revealed the dependence of the level of blast cells of bone marrow and blood on the type of chromosomal aberration. Patients with BCR/ABL1 and E2A/PBX1 have the highest resistance to chemotherapy with molecular genetic translocations, and patients with AF4/MLL and TEL/AML have the lowest resistance, as evidenced by the presence and absence of blast cells in the peripheral blood, respectively. Conclusion. Establishing the relationship between cytogenetic and molecular genetic features of the tumour clone will help determine the degree of malignancy of the process, as well as the risk group for the course of the disease. Determining the dependence of acute leukemia on molecular genetic translocations will make it possible to further modify the treatment program

PTEN Alterations Are Related to Unfavorable Prognosis in Children with T-Cell Acute Lymphoblastic Leukemia Treated According to ALL IC-BFM Protocols

▪Background: Resistant or relapsed T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis and is usually fatal. Thus, patients with a very high risk of treatment failure need to be considered for alternative treatments. To identify these patients, clinical parameters and response to therapy are currently in use as prognostic markers. These markers, however, do not allow for reliable identification of all high-risk patients early during induction treatment. In search for genetic markers that inform treatment strategies early after leukemia diagnosis, we evaluated the prognostic significance of a panel of 25 established T-ALL oncogenes or tumor suppressor genes, in children treated according to ALL IC-BFM protocols.Patients and Methods: The study group included 162 children with de novo T-ALL, treated according to I-BFM protocols ALL-IC BFM 2002 (n=91; 56%) and ALL-IC BFM 2009 (n=71; 44%). Mutations in NOTCH1 (exons 26, 27, 34) , FBXW7 (exons 9 and 10) , PTEN (exon 5, 6, 7) , WT1 (exon 7 and 9) , IL7R (exon 6) , STAT5B (exon 16) , FLT3 (exons 11-12) , RUNX1 (exons 1, 2, 3, 6) and DNMT3A (exons 8-23) were detected by either high resolution melt analysis (HRM), Sanger sequencing, or both. Copy number alterations (CNAs) in SIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, CDKN2A/B, MLLT3, NUP214-ABL1, LMO1, LMO2, NF1, SUZ12, PTPN2, PTEN, PHF6 were detected by multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA P383 T-ALL probe mix (MRC Holland) and a custom made probes for exons 2, 3, 4, 5 of PTEN . MRD was assessed by multicolor flow cytometry (FC-MRD at d15, 33, 78) and ‘FC-MRD positive’ result was defined as >=0.01%. Statistical analyses: Fisher exact test, Cochran-Mantel-Haenszel test (CMH), Kaplan-Meier estimators and log-rank test. Probability of relapse-free survival (pRFS) was calculated using the time from diagnosis to the date of relapse or the last follow-up in complete remission.Results: Of all 25 genes analyzed, PTEN alterations significantly predict for the risk of relapse in 162 children treated according to ALL IC-BFM protocols. The strength of the study is combined analysis of CNAs and point mutations that enabled us to identify PTEN alterations (PTEN.ALT), resulting from both loss-of function mutations (PTEN.MUT) and/or genomic deletions (PTEN.DEL). Patients with PTEN.ALT were at higher risk of relapse with pRFS of 60% vs. 83% in non-mutated patients, p=0.0053. Moreover,in the separate analysis of each of PTEN alterations (PTEN.DEL or PTEN.MUT), both were associated with worse pRFS (58% in patients with PTEN.DEL vs. 82% in non-mutated, p=0.01; and pRFS of 60% in patients with PTEN.MUT vs. 82% in non-mutated, p=0.06). However, in the analysis of PTEN status (PTEN.ALT, PTEN.MUT or PTEN.DEL) in each of the treatment protocols separately, the significant impact on outcome was retained only in the ALL IC-BFM 2009 protocol. Remarkably, long-term negative impact of PTEN alterations was reflected in the poorer early response to treatment. The odds ratios for the occurrence of unfavorable prednisone response (≥ 1000 blasts/mL) were: 3.06 (95%CI: 1.22-7.67, p=0.02) among patients with PTEN.ALT; 3.49 (95%CI: 1,21-10,03, p=0.02) among patients with PTEN.DEL and 3.99 (95%CI: 1,13-13,36, p=0.03) among patients with PTEN.MUT. In addition, in a stratified analysis for both ALL-IC protocols, we found patients with PTEN.MUT to be associated with positive FC-MRD at day 78 (OR: 11,13, 95%CI: 2.38-51.92, p=0.0033). With respect to characteristics at diagnosis,patients with PTEN.DEL or PTEN.ALT status more often presented with higher WBC counts (>=20 000) than patients with wild-type PTEN (p=0.01 and p=0.02, respectively) and those with PTEN.MUT status were predominantly males (P=0.03).Conclusion: Out of the 25 established T-ALL driver genes analyzed in 162 children treated according to ALL IC-BFM schemes, PTEN abnormalities predict worse relapse-free survival and poorer early response to treatment and are candidates for genetic risk markers in pediatric T-ALL.Research funded by National Science Centre, Poland grant 2013/11/N/NZ5/03730 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017. DisclosuresNo relevant conflicts of interest to declare.

Relationship of genetic deviations with laboratory indicators of peripheral blood and bone marrow in children with acute lymphoblastic leukemia

The aim of this study was to evaluate the prognostic value of the relationship between genetic abnormalities and clinical and laboratory parameters of peripheral blood and bone marrow in children with acute lymphoblastic leukemia (ALL). Material and methods. 105 children diagnosed with ALL were examined (average age 6 years). To detect chromosomal translocations AF4/MLL t(4; 11) (q23; p23), BCR/ABL t (9; 22) (q34; q11), E2A/PBX1 t (1; 19) (q23; p13) and TEL/AML t(12; 21) (q13; q22) the method of polymerase chain reaction with reverse transcription (RT-PCR) was applicated. PCR was performed with specific primers for the appropriate chromosomal aberrations. Detection of PCR products was performed by electrophoresis in 2% agarose gel. Determination of minimal residual disease (MRD) was performed by multiparameter flow cytofluorimetry using monoclonal antibodies. Results. Among patients, the incidence of ALL is most pronounced in children aged 3 to 6 years - 37 people (35.2%) and aged 6 to 9 years - 26 people (24.8%). The highest accidence was found among patients with chromosomal translocation TEL/AML - 22 (21%) of patients with a median age of 5 years. In the second place, the frequency of mutations is the translocation of E2A / PBX1. BCR / ABL translocation was less common - 1.9% of patients, but the expression of this gene indicates a bad course of the disease, as patients after cytostatic therapy under the ALLIC BFM 2009 program had a recurrence. Recurrence has also been observed in patients with TEL/AML chromosomal translocation. Determination of MRD showed its increased level in patients with chromosomal aberrations BCR / ABL and TEL/AML throughout the treatment phase. In addition, patients in these groups were diagnosed with initial leukocytosis followed by leukopenia after a course of chemotherapy. Patients of all groups showed a decrease in hemoglobin. Conclusion. The most significant changes in clinical and laboratory parameters were found among patients with chromosomal translocations BCR/ABL and TEL/AML, as evidenced by the development of relapses in patients of these groups. The low level of association between karyotype disorders, with the formation of AF4/MLL and E2A/PBX1, and clinical and laboratory parameters in patients with GLL may indicate that the isolated clonal disorders are independent prognostic factors for the course of the disease.

Bone pain at leukemia diagnosis and other risk factors for symptomatic osteonecrosis in children with acute lymphoblastic leukemia.

Osteonecrosis is a major cause of acute and long-lasting complications of acute lymphoblastic leukemia (ALL) therapy in children. Our study aimed to evaluate the prevalence, characteristics, risk factors, and outcome of osteonecrosis in children with ALL. The cohort included 559 children aged 1-20years diagnosed with ALL between 2003 and 2018 at two tertiary medical centers in Israel and enrolled in two consecutive protocols: ALL-IC BFM 2002 and AIEOP-BFM ALL 2009. Symptomatic osteonecrosis was prospectively captured as an adverse event. Osteonecrosis occurred in 51 patients (9.1%). Ninety-four percent of the events were graded as moderate or severe (grades 3-4, Ponte di Legno Toxicity Working Group classification) and multiple bone involvement was common. Full resolution of osteonecrosis was documented in only 16% of the children (median follow-up 4.2years). Stepwise logistic regression identified five risk factors for osteonecrosis, with a high predictive value (AUC=0.88): older ageat ALL diagnosis, high-risk ALL group, T-cell immunophenotype, female gender, and a novel risk factor: bone pain at the time of leukemia diagnosis. In addition, osteonecrosis was less common among children of Arab ethnicity. Thrombophilia and an elevated age-adjusted body mass index were not confirmed as risk factors for osteonecrosis. Due to the low rates of osteonecrosis resolution and its debilitating long-term impact, the identification of patients at high risk for osteonecrosis is important for their inclusion in further studies evaluating potential therapeutic adjustments.

MOLECULAR AND GENETIC MECHANISMS OF DEVELOPMENT OF CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA AND THEIR CORRECTION ALLIC BFM 2009 BY CYTOSTATIC THERAPY

The aim of this study was to evaluate the prognostic value of the relationship between genetic abnormalities and clinical and laboratory parameters of peripheral blood and bone marrow in children with acute lymphoblastic leukemia (ALL).Material and methods. 105 children diagnosed with ALL were examined (average age 6 years). To detect chromosomal translocations AF4/MLL t(4; 11) (q23; p23), BCR/ABL t (9; 22) (q34; q11), E2A/PBX1 t (1; 19) (q23; p13) and TEL/AML t(12; 21) (q13; q22) the method of polymerase chain reaction with reverse transcription (RT-PCR) was applicated. PCR was performed with specific primers for the appropriate chromosomal aberrations. Detection of PCR products was performed by electrophoresis in 2% agarose gel. Determination of minimal residual disease (MRD) was performed by multiparameter flow cytofluorimetry using monoclonal antibodies.Results. Among patients, the incidence of ALL is most pronounced in children aged 3 to 6 years - 37 people (35.2%) and aged 6 to 9 years - 26 people (24.8%). The highest accidence was found among patients with chromosomal translocation TEL / AML - 22 (21%) of patients with a median age 5 years. In second place, the frequency of mutations is the translocation of E2A / PBX1. BCR / ABL translocation was less common - 1.9% of patients, but the expression of this gene indicates a bad course of the disease, as patients after cytostatic therapy under the ALLIC BFM 2009 program had a recurrence. Recurrence has also been observed in patients with TEL/AML chromosomal translocation.Determination of MRD showed its increased level in patients with chromosomal aberrations BCR / ABL and TEL/AML throughout the treatment phase. In addition, patients in these groups were diagnosed with initial leukocytosis followed by leukopenia after a course of chemotherapy. Patients of all groups showed a decrease in hemoglobin.Conclusion. The biggest changes in clinical and laboratory parameters were found between patients with chromosomal translocations BCR/ABL and TEL/AML, as evidenced by the development of relapses in patients of these groups. The low level of association between karyotype disorders, with the formation of AF4/MLL and E2A/PBX1, and clinical and laboratory parameters in patients with GLL may indicate that the isolated clonal disorders are independent prognostic factors for the course of the disease.

Імунофенотиповий профіль бластних клітин як маркер діагностики рецидивів гострої лімфобластної лейкемії у дітей

Immunophenotyping of leukemia cells was studied in this work; minimal residual disease was monitored among children with acute lymphoblastic leukemia under conditions of relapse and complete remission after the application of ALLIC-BFM 2009 cytostatic therapy. The study showed that after application of ALLIC-BFM 2009 therapy, 88% of children had complete remission, and 12% had relapses. Among patients with relapses, the number of blast cells in the bone marrow was at a high level (more than 6%). Monitoring of patients during therapy established an increase in the minimal residual disease level of more than 1% after treatment in patients with recurrent disease. Immunophenotyping of blast cells among patients with relapse showed the expression of linear independent antigens HLA (93%), Auti-TdT (91%), CD10 (78%), CD38 (91%) and CD34 (57%) and B-linear antigens: CD19, CD22, CD58, CD79a, the highest expression was found for the CD19 antigen. A low level of expression of CD45 (28%) was recorded with relapses of acute lymphoblastic leukemia and high (89%) level was with complete remission of the disease. We did not detect expression of antigens characteristic of T-cell acute lymphoblastic leukemia in bone marrow of patients with acute lymphoblastic leukemia, both with relapses and with remission. At the same time, the expression of myeloid antigens (CD33 and CD13) was noted among acute lymphoblastic leukemia patients. Among patients, the incidence of acute lymphoblastic leukemia was the most pronounced in children aged from 3 to 6 years – 37 patients (35.2%) and aged from 6 to 9 years – 26 (24.8%) patients. The highest accidence was found among patients with chromosomal translocation TEL / AML – 22 (21%) patients with a median age 5 years. In second place, the frequency of mutations is the translocation of E2A / PBX1. BCR / ABL translocation was less common. It was noted in 1.9% of patients, but the expression of this gene indicated a bad course of the disease, as patients after cytostatic therapy under the ALLIC BFM 2009 program had a recurrence. Recurrence was also observed in patients with TEL/AML chromosomal translocation. Determination of minimal residual disease showed its increased level in patients with chromosomal aberrations BCR / ABL and TEL/AML throughout the treatment phase. In addition, patients in these groups were diagnosed with initial leukocytosis followed by leukopenia after a course of chemotherapy. Patients of all groups showed a decrease in hemoglobin. The biggest changes in clinical and laboratory parameters were found between patients with chromosomal translocations BCR/ABL and TEL/AML, as evidenced by the development of relapses in patients of these groups. The low level of association between karyotype disorders, with the formation of AF4/MLL and E2A/PBX1, and clinical and laboratory parameters in patients with acute lymphoblastic leukemia may indicate that the isolated clonal disorders are independent prognostic factors for the course of the disease

Results of two consecutive treatment protocols in Polish children with acute lymphoblastic leukemia

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 < 0.001). The corresponding event-free survival rates were 82% and 84% (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.

Venous Thromboembolism and Its Risk Factors in Children with Acute Lymphoblastic Leukemia in Israel: A Population-Based Study.

Simple SummaryCurrent cure rates of childhood acute lymphoblastic leukemia (ALL) surpass 90% and thus the mitigation of chemotherapy-associated toxicity has become a major goal. Venous thromboembolism (VTE) is a major toxicity of ALL therapy, but its precise prevalence, risk factors, and optimal prophylaxis are yet to be determined. This study evaluated VTE in a cohort of 1191 children, including its rate, risk factors, and long-term sequelae. VTE was found in 7.5% of the cohort, 27% severe events. Most events resolved without late sequelae. We identified four risk factors associated with VTEs: Age >10 years, high-risk ALL group, severe hypertriglyceridemia during therapy, and inherited thrombophilia. Since children with these risk factors had significantly higher rates of severe VTE, a routine evaluation may identify potential candidates for prophylactic interventions.Venous thromboembolism (VTE) is a serious complication of acute lymphoblastic leukemia (ALL) therapy. The aim of this population-based study was to evaluate the rate, risk factors, and long-term sequelae of VTE in children treated for ALL. The cohort included 1191 children aged 1–19 years diagnosed with ALL between 2003–2018, prospectively enrolled in two consecutive protocols: ALL-IC BFM 2002 and AIEOP-BFM ALL 2009. VTEs occurred in 89 patients (7.5%). Long-term sequelae were uncommon. By univariate analysis, we identified four significant risk factors for VTEs: Severe hypertriglyceridemia (p = 0.005), inherited thrombophilia (p < 0.001), age >10 years (p = 0.015), and high-risk ALL group (p = 0.039). In addition, the incidence of VTE was significantly higher in patients enrolled in AIEOP-BFM ALL 2009 than in those enrolled in ALL-IC BFM 2002 (p = 0.001). Severe VTE occurred in 24 children (2%), all of whom had at least one risk factor. Elevated triglyceride levels at diagnosis did not predict hypertriglyceridemia during therapy. In a multivariate analysis of 388 children, severe hypertriglyceridemia and inherited thrombophilia were independent risk factors for VTE. Routine evaluation for these risk factors in children treated for ALL may help identify candidates for intervention.

GATA3 germline variant is associated with CRLF2 expression and predicts outcome in pediatric B-cell precursor acute lymphoblastic leukemia.

The germline variant at rs3824662 in GATA3 is a risk locus for Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), the biological subtype of B-cell precursor (BCP)-ALL defined by a distinct gene expression profile and the presence of specific somatic aberrations including rearrangements of CRLF2. In this study, we investigated whether rs3824662 in GATA3 associates with CRLF2 expression in leukemic cells and predicts prognosis in pediatric BCP-ALL patients treated according to the ALL Intercontinental Berlin-Frankfurt-Münster (IC BFM) 2009 (n = 645) and the ALL IC BFM 2002 (n = 216) protocols. High expression of CRLF2 was observed at both protein and mRNA levels (fourfold higher in AA than in CA + CC) among GATA3 AA variant carriers, independent of the presence of P2RY8-CRLF2 fusion. Additionally, the AA variant at rs3824662 was a significant factor affecting minimal residual disease level at the end of induction phase and overall survival regardless of the risk group and the protocol. The germline variant at rs3824662 in GATA3 is a prognostic factor which associates with CRLF2 expression in leukemic cells supporting the hypothesis that GATA3 may have a regulatory effect on the CRLF2 pathway in pediatric BCP-ALL.