Intermediate Risk Acute Lymphoblastic Leukemia Research Articles

Capizzi Vs HDMTX in the Intermediate Maintenance Phase in Patients with Intermediate-Risk ALL in a 3rd Level Hospital in Northeastern Mexico

INTRODUCTION: Methotrexate is a fundamental drug in the treatment of acute lymphoblastic leukemia (ALL), playing an important role in CNS prophylaxis. Two strategies of methotrexate intensification have been studied: high-dose regimens (with leucovorin salvage) and Capizzi regimen (escalating doses, without salvage, with addition of L-asparaginase). Fifty-five percent of pediatric patients with ALL in Mexico are treated in general hospitals, with limited hospital bed availability, which may represent a deferral of treatment. We wanted to explore a feasible, economical and accessible ambulatory scheme, which may represent an option for those centers that do not have the infrastructure for maintenance with HDMTX. OBJECTIVE: Evaluate the efficacy of methotrexate/L-asparaginase combination therapy based on the Capizzi scheme versus standard high-dose methotrexate (HDMTX) in the intermediate maintenance phase of a cohort of patients diagnosed with intermediate-risk B- ALL. METHODS: From January 2017 to June 2023, all patients diagnosed with ALL between 0 and 15 years of age, at a single center in northeastern Mexico, were collected in a prospective hospital registry. Based on risk stratification, patients received an adjusted treatment protocol with variations in intensity and duration for the different phases, based on the BFM. The type of intermediate maintenance received (Capizzi vs HDMTX scheme), duration time of the intensive phase, patients' overall survival and relapse events were evaluated. Demographic data were reported by distribution analysis and the Kaplan-Meier method was used to analyze and predict OS and EFS. RESULTS: Forty-four pediatric patients with a diagnosis of B-ALL and intermediate risk were included, 32 patients from the Capizzi group (G1) and 12 from the HDMTX group (G2). The median age at diagnosis was 7.5 years, 60% of patients were male. No statistically significant differences were found with respect to the duration of the intensive phase of treatment between the groups (p=0.58). Two deaths were recorded (Capizzi 1, HDMTX 1), with no differences in the type of treatment used (p=0.90). The risk of relapse according to the type of scheme used was not statistically significant (Capizzi 3 vs HDMTX 2, p=0.46, OR 0.51, 95% CI 0.07-3.55) (Table 1). Overall survival of patients during the study time (intensive treatment phase) was 90.9%, being 90.6% in the Capizzi group and 91.7% in the HDMTX group (p=0.851) (Figure 1). CONCLUSIONS: According to our results, no difference was found regarding the temporality of the 2 schemes (p=0.58), risk of relapse (p=0.46) or mortality (p=0.90). It is useful in our center because it provides us with an ambulatory, feasible and economical option that equals the results of the standard risk treatment. Due to the limited number of patients included in the study, it is recommended to increase the population in order to corroborate these results.

Impact of the homozygous mutation in Nudix hydrolase 15 on myelosuppression with 6-mercaptopurine in a European girl with acute lymphoblastic leukemia: A case report

A 6-year-old girl diagnosed with intermediate-risk acute lymphoblastic leukemia (ALL) presented with severe myelotoxicity and multiple infections during phase IB induction treatment with 6-mercaptopurine (6-MP). In the subsequent treatment phases, which included 6-MP, the patient continued to show bone marrow aplasia and neutropenia, necessitating numerous dose adjustments and interruptions. The recommended dose was eventually reduced to 5 %. A pharmacogenetic analysis, conducted in induction phase IB, detected three single-nucleotide polymorphisms (SNPs) of the thiopurine S-methyltransferase (TPMT) gene, and the phenotype of a normal metabolizer was observed. As a result of a second pharmacogenetic analysis, pathological polymorphisms were revealed in Nudix hydrolase 15 (NUDT15), which may explain the patient’s myelotoxicity. Hence, a pharmacogenetic analysis performed in advance would have been able to prevent her from suffering severe toxicity and/or treatment failure.

A pharmacoeconomic study of pegaspargase in the treatment of pediatric acute lymphoblastic leukemia in Russia

Pegaspargase is an L-asparaginase that is conjugated to monomethoxypolyethylene glycol. It is currently approved for use in children and adults for acute lymphoblastic leukemia (ALL) in the Russian Federation, is included in the recommendations of clinical practice guidelines and is recommended for inclusion in the Vital and Essential Drug List. The aim of this research is to perform a pharmacoeconomic analysis of pegaspargase in the treatment of pediatric patients with ALL in the Russian healthcare setting. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Ministry of Healthcare of the Russian Federation. The target population of the study were pediatric patients with intermediate-risk ALL (according to the Moscow–Berlin protocol) who had not previously received treatment. We compared two treatment scenarios: therapy according to the Moscow–Berlin protocol with and without pegaspargase. A decision tree model was used to analyze the costs of ALL treatment strategies. The costs of first-line therapy, relapse therapy (in accordance with the ALL-REZ-MB-2014 protocol), hematopoietic allogenic stem cell transplantation, and third-line therapy with blinatumomab (in patients with B-cell ALL) were considered. Costs were calculated separately for patients receiving different treatments and were weight-averaged according to the total number of intermediate-risk patients. The size of the target population was determined by the incidence of ALL in 2020 and risk group distribution data obtained from the Moscow-Berlin protocol study. The costs were calculated for a cohort of patients available for therapy in 2023. The use of pegaspargase leads to a decrease in the average discounted cost of therapy for children with intermediate risk ALL of 93,273 rubles due to a decrease in the need for costly relapse therapy. Estimates show that up to 418 new patients are diagnosed with intermediate-risk ALL annually and belong the target population of this study. If 100% of ALL patients receive pegaspargase on the third day of remission induction therapy, the total cost of therapy for the target patient population will decrease by 38.99 million rubles. The use of pegaspargase on the third day of remission induction therapy in children with intermediate-risk ALL leads to a reduction in the relapse rate (superior efficacy) and lower costs, compared with the treatment regimen without pegaspargase.

P328: DYNAMIC EVOLUTION OF TCF3-PBX1 LEUKEMIAS AT THE SINGLE-CELL LEVEL UNDER CHEMOTHERAPY PRESSURE

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While research has been focused on high-risk patients, the biology of low-to-intermediate-risk patients has so far been inadequately investigated and can lead to overtreatment with severe side effects and under treatment with risk of relapse. The translocation t(1;19) codes for chimeric fusion protein TCF3-PBX1, which is associated with intermediate risk ALL. Using our previous generated TCF3-PBX1 conditional knock-in mice, we established a model to study in vivo chemotherapy resistance. Aims: We hypothesize that chemotherapy and microenvironment play a crucial role in development of resistance in TCF3-PBX1 leukemias by influencing transcriptional regulation, activation of signaling pathways and the hierarchical structure of leukemic cells. In this project we aim to characterize dynamic changes of TCF3-PBX1 leukemia cells in different tissues under chemotherapy pressure. Methods: Recipient C57/BL6 healthy mice were sub-lethally irradiated, transplanted with mouse TCF3-PBX1 leukemia cells and treated with vehicle (n=15), prednisolone (n=13) and daunorubicin (n=15) for 20 days. Mice were monitored for signs of disease regularly and circulating GFP+ TCF3-PBX1 leukemia cells were assessed by flow cytometry. Sick mice were sacrificed, leukemia cells were isolated from five different organs (bone marrow, spleen, lymph nodes, spinal cord and brain) and characterized by immunophenotyping, sanger sequencing, bulk RNA sequencing (bulk RNAseq) of GFP+ sorted cells, single cell RNA sequencing and mass cytometry (CYTOF). Results: All mice transplanted with TCF3-PBX1 leukemia cells and treated with vehicle succumbed to disease with a median survival of about 70 days. We optimized chemotherapy drug concentration and transplanted cell dose, so 60% of mice treated with prednisolone or daunorubicin survived at least 150 days. Leukemic infiltration was showed by histological stainings in analyzed tissues including central nervous system (CNS) (spinal cord, brain) and GFP+ leukemia cells were quantified by flow cytometry. No major differences were observed in immunophenotype of TCF3-PBX1 leukemia and variant allele frequency (VAF) of the known PTPN11 mutation in analyzed tissues or depending on in vivo treatments. Bulk RNAseq of FACS-sorted GFP+ TCF3-PBX1 leukemia cells were clustered and CNS cells separated from other tissues. scRNAseq revealed additional heterogeneity within each tissue based on signaling pathway and cell cycle activity. Interleukin signaling, regulation of apoptosis pathways, and signaling by the B cell receptor (BCR) were regulated based on pathway analysis in CNS compared to other tissues. Hence, we elucidated the hierarchical structure of hematopoietic cells, interaction of leukemic cells with the microenvironment, and changes in signaling response to chemotherapy in vivo treatment by mass spectrometry (CyTOF) to validate the identified altered signaling pathway activities at protein level. Summary/Conclusion: We have developed a mouse model in order to characterize in vivo chemotherapy resistance depending on niche. Global transcriptomics and phospho-proteomics at the single-cell level might elucidate novel mechanisms of chemotherapy resistance suitable for pharmacological therapies.

Administration and Toxicity Profile of the Capizzi Interim Maintenance—Retrospective Study from a Tertiary Care Cancer Centre

Abstract Introduction The Capizzi-style methotrexate (MTX) is an integral part of acute lymphoblastic leukemia (ALL) treatment. The escalating dose of MTX originally used in the United Kingdom and Children’s Oncology Group protocols along with L-asparaginase has been modified in the Indian Childhood Collaborative Leukemia (ICiCLe) group protocol where L-asparaginase has been omitted. The data regarding the incidence of toxicities and ease of administration on the Capizzi-style interim maintenance is not robust. Objectives We have compiled our experience with administration and toxicity profile in children with intermediate-risk ALL. Materials and Methods A retrospective data collection of all children diagnosed with intermediate-risk ALL as per the ICiCLe risk stratification in the year 2019 was included in the analysis. Each cycle of MTX was started after ensuring an absolute neutrophil count of >750/mm3 and transaminases <2 upper limit of normal. As a unit protocol, pre- and post-MTX hydration was administered in all our children. No urine pH or midcycle biochemical parameter monitoring was done. Statistical analysis was done using Microsoft Excel and SPSS version 24 IBM Corp. in Armonk, New York, United States. Results Forty-six children were included in the study. The median age of children in our study was 6 years (range: 1 year 2 months–12 years). Undernutrition was associated with a significant increase in toxicity (p = 0.02). Fifty-two percent of children had evidence of toxicity, elevated transaminases being the most common. There were recurring symptoms resulting in 53 episodes of toxicities overall. Incidence of toxicity was more in the early cycles (<3). Conclusion The pre- and post-MTX hydration is an effective way to reduce toxicities with the Capizzi-style MTX and this course can be administered with ease on outpatient basis with minimal need for monitoring or admission.

Peg-Asparaginase Administration on Day 3 of Remission Induction Accelerates MRD Response in Children with B-Cell Precursor Acute Lymphoblastic Leukemia

To improve the outcome of children with standard and intermediate risk acute lymphoblastic leukemia (ALL) by the addition of PEG-asparaginase (PEG) on day 3 of remission induction was one of the key aims of trial ALL MB-2008 (J. Roumjantseva et al, Pediatriya, 2009). Therapy was designed based on previous MB-group studies (A. Karachunsky et al, Leukemia, 2008). Aim of the present study was to evaluate the influence of day 3 PEG on the end-induction MRD-status which is one of the significant predictors of outcome in children with B-cell precursor ALL (BCP-ALL).Patients and methods. In total, 474 patients (242 boys and 232 girls aged 1 to 17 years (median 5 years 8 months) were studied. Inclusion criteria were: BCP-ALL, treatment according to protocol ALL MB-2008, compliance with standard risk group (SRG, n=266) and intermediate risk group (ImRG, n=208) criteria according to the protocol stratification system, and availability of end-induction (day 36) bone marrow (BM) samples. Remission induction of ALL MB-2008 consisted of oral dexamethasone (6mg/m2 daily), 5 injections of vincristine (1,5 mg/m2), 0-2 injections of daunorubicin (DNR, 45 mg/m2) and 6 triple intrathecal injections. Patients were randomized in respect to PEG administration on day 3. SRG patients were divided into three groups (PEG+DNR+, PEG+DNR- and PEG-DNR+); ImRG patients were split into two (PEG+DNR+ and PEG-DNR+). MRD detection was performed by multicolor flow cytometry (MFC) in three reference laboratories. Samples with a tumor cell level above 0.01% were considered MRD-positive. Outcome parameters were event-free survival (EFS) and cumulative incidence of relapse (CIR). Median of follow up was 5 years.Results. Outcome in 474 study group patients was not significantly different from outcome of 2310 children with BCP-ALL enrolled in SRG and ImRG of ALL MB-2008 trial in whom MRD was not studied: EFS was 0.86±0.02 and 0.84±0.01, respectively, p=0.14, and CIR was 0.12±0.02 and 0.09±0.01, p=0.38.MFC MRD data obtained at day 36 had a significant impact on prognosis in SRG patients. EFS as well as CIR were worse for patients with MRD >0.1% (n=26; EFS 0.47±0.15, CIR 0.49±0.17) compared with 248 patients with low/negative MRD (EFS 0.94±0.2, CIR 0.05±0.02, p<0.001). Nevertheless, the distribution of MRD levels was completely different dependent on randomization arms: in arm PEG-DNR+ (n=92) 18.5% of patients belonged to the group with unfavorable MRD compared with only 6.2% in arm PEG+DNR- (n=80, p=0.017) and 4.3% in arm PEG+DNR+ (n=94, p=0.002). Interestingly, in two PEG+ arms the number of patients with high MRD was not significantly different (p=0.558). Despite the lower proportion of high MRD levels in PEG+ arms MRD maintained its prognostic significance also in these patients. EFS was 0.65±0.16 and CIR 0.35±0.18 in 9 children with MRD ≥0.1 compared with EFS 0.94±0.02 and CIR 0.05±0.02 in 165 patients of the low MRD group (p<0.001 for both EFS and CIR).In ImRG, MFC MRD data disclosed a prognostic value, as well. EFS was 0.92±0.03 and CIR 0.06±0.02 for 143 patients with MRD-negativity on day 36; in contrast, outcome of 78 MRD-positive patients was significantly inferior (EFS 0.73±0.06, CIR 0.25±0.06, p<0.001 for both comparisons). Like in SRG, in ImRG the distribution of MRD levels was different between two randomization arms. In PEG-DNR+ group (n=93) only 54.8% of patients were MRD-negative at the end of remission induction. In contrast, in PEG+DNR+ group (n=115) the proportion of patients with favorable MRD results was significantly higher (76.5%, p=0.001). Again even with low number of MRD-positive cases in PEG+ arm, day 36 MRD disclosed a significant influence on outcome: In 88 MRD-negative patients EFS was 0.93±0.03 and CIR 0.05+0.03, significantly better than in 27 MRD-positive patients (EFS 0.80+0.08 and CIR 0.17+0.08, p=0.025 and p=0.019, respectively).Conclusions. Administration of PEG at day 3 of remission induction leads to a more rapid MRD response in children both with standard-risk and intermediate-risk BCP-ALL. In SRG, rapidity of MRD clearance in PEG+ arms was independent of the additional administration of daunorubicin. We conclude that end-induction MRD data are independent predictors of outcome despite the small proportion of MRD-positive patients after the implementation PEG during induction. DisclosuresNo relevant conflicts of interest to declare.

Adverse effects with intravenous methotrexate in children with acute lymphoblastic leukemia/lymphoma: a retrospective study.

Methotrexate (MTX) forms the backbone of maintenance cycles in childhood acute lymphoblastic leukemia (ALL) chemotherapy, including interim maintenance. There is sufficient published data describing toxicities of high dose MTX(HD-MTX), but toxicities with escalating doses of MTX (Capizzi regimen) is not well documented. Capizzi regimen is thought to be relatively safe; we contend that even low escalating doses of MTX have significant toxicities. Our study intends to characterise such events with Capizzi MTX in comparison tothat seenwith HD-MTX. The retrospective study was conducted at a tertiary care centre of North India. We looked for the presence of six main toxicities: febrile neutropenia, thrombocytopenia, mucositis, hepatic toxicity, renal toxicity and skin toxicityfrom the clinical records of children with newly diagnosed acute lymphoblastic leukemia and lymphoma (intermediate and high risk disease), treated at our centre from November 2013 to July 2018. Intermediate risk ALL(IR-ALL) received Capizzi MTX, whereas high risk ALL (HR-ALL/T-NHL), received HD-MTX. Both these regimens do not use L-asparaginase. A total of 237 cycles of Capizzi escalating MTX and 151 cycles of HD-MTX (B cell: 3gm/m2 and T cell ALL/T-NHL: 5gm/m2) during interim maintenance were studied in 93 children. Fifty-four (54) childrenwereof IR (all B cell ALL) and 39 ofHR-ALL (21 B-ALL, 18 T-ALL/T-NHL). The combined incidence of toxicities, were similar between the two groups: 68/237 cycles (28.7%) of Capizzi MTX and 45/151 cycles (29.8%) of HD-MTX (P = 0.815). However, mucositis was more commonly witnessed in the later group at 22/151 cycle (14.6%) versus 13/237 cycles (5.5%) in Capizzi MTX (P = 0.002). Nephrotoxicity and skin toxicity was seen only in the HD-MTX group. There was no difference in the severity of toxicity, graded using NCI CTCAE v 5.0,between the two groups. There was no mortality directly attributable to methotrexate toxicity (Grade V toxicity). Serum MTX levels were available in 69/151 (45.7%) cycles of HD-MTX and showed no association with toxicity in this group. Also, there was no difference in the incidence of combined toxicities between groups with (19/69 cycles) or without (26/82 cycles) available serum MTX levels in the HR group (P = 0.577). Male gender, lower baseline ANC and lower BMI had significant association with toxicity. Methotrexate related toxicity is common with both Capizzi and HD-MTX schedule in childhood ALL with a correlation of lower BMI, baseline ANC and male gender. However, it is possible to administer Capizzi as well as HD-MTX in lower middle income countries, with manageable toxicity. Further studies will be required to substantiate our findings and determine the predictors of such events.

Body Mass Index (BMI) and Infectious/Febrile Episodes in Children with Intermediate Risk Acute Lymphoblastic Leukemia (IR ALL)

The incidence of treatment related mortality in children with acute lymphoblastic leukemia (ALL) is reported to be between 2% and 4% with infections being the leading cause.Aim: To establish a relationship between body mass index at diagnosis (BMI 0), after protocol I therapy completion (BMI I) and the incidence rate ratio (IRR) of infectious/febrile episodes in children with ALL intermediate risk.Methods: Thirty one consecutive patients (2–18 years old, with a male to female ratio of 19/12) with newly diagnosed ALL that were treated uniformly according to ALL IC 2009 protocol were included in this analysis.Results: A BMI decrease of at least 5% during protocol I therapy and BMI 1 under 15th percentile score corresponds significantly with higher IRR (with P-values 0.04 and 0.006 respectively) during the whole intensive therapy.Conclusion: Some relationships between BMI reduction and higher IRR in ALL patients were found, but their significance is limited by the size of the group analyzed.

Clinical Analysis of Deep Venous Thromboembolism in Children with Acute Lymphocytic Leukemia

To explore clinical characteristics and outcome of deep vein thrombosis(DVT) in children with acute lymphoblastic leukemia (ALL). A tatol of 266 patients were diagnosed as ALL from January 1, 2010 to May 31, 2016. The clinical data of 12 cases of patients with DVT were retrospectively analyzed, 183 cases diagnosed before January 1, 2015 were received chemotherapy with the scheme of SCMC-05. The other cases were treated by the scheme of CCCG. All the patients received central venous catheter. The DVT happened in 12 cases including 10 cases of limb DVT and 2 cases of intacranial venous sinus thrombosis. The DVT mostly occured in intermediate risk ALL patients, the infection and coagulopathy existed in most patients. They were treated with low molecular heparin(LWHP), among them 5 cases were given extubation; the thrombus disappeared in 6 cases after 1 week; the intracranial venous sinus thrombosis in 1 case did not obviously improved after 6 months of treatment. The ALL children with DVT were treated with LWHP when using L-ASP, as a result no thrombuses happened. Centralvenous catheter and chemotherapeutic drugs were the major cause of DVT. Abnormal coagulation, infection, and risk stratification are another risk factors for thrombosis. ALL children thrombosis are benefited from LWHP prevention when using L-ASP again.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia.

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

The treatment of childhood acute lymphoblastic leukemia in Guatemala: Biologic features, treatment hurdles, and results.

The National Pediatric Oncology Unit (UNOP) is the only pediatric hemato-oncology center in Guatemala. Patients ages 1 to 17 years with acute lymphoblastic leukemia (ALL) were treated according to modified ALL Intercontinental Berlin-Frankfurt-Münster (IC-BFM) 2002 protocol. Risk classification was based on age, white blood cell count, immunophenotype, genetics (when available), and early response to therapy. From July 2007 to June 2014, 787 patients were treated, including 160 who had standard-risk ALL, 450 who had intermediate-risk ALL, and 177 who had high-risk ALL. The induction death rate was 6.6%, and the remission rate was 92.9%. The rates of death and treatment abandonment during first complete remission were 4.8% and 2.5%, respectively. At a median observation time of 3.6 years, and with abandonment considered an event, the 5-year event-free survival and overall survival estimates ( ± standard error) were 56.2% ± 2.1% and 64.1% ± 2.1%, respectively, with a 5-year cumulative incidence of relapse of 28.9% ± 2.0%. Twenty-one of 281 patients (7.5%) investigated were positive for the ets variant 6/runt-related transcription factor 1 (ETV6/RUNX1) fusion. A well organized center in a low-middle-income country can overcome the disadvantages of malnutrition and reduce abandonment. Outcomes remain suboptimal because of late diagnosis, early death, and a high relapse rate, which may have a partly genetic basis. Earlier diagnosis, better management of complications, and better knowledge of ALL will improve outcomes. Cancer 2017;123:436-448. © 2016 American Cancer Society.

The influence of detoxification agents on the intensity of side effects caused by medium-high doses of methotrexate in children with acute lymphoblastic leukemia: case series

SUMMARY Objective The treatment of childhood acute lymphoblastic leukemia (ALL) in Serbia is conducted according to protocol ALL IC BMF-2009. The therapy includes the application of cytostatic drugs methotrexate and 6-mercaptopurine, and drug detoxifying Calcium Folinate. At the moment, 80% of affected children could be cured with current treatment, but resistance to the therapy and its toxic effects remain serious clinical problems. The aim of the study was to investigate the influence of detoxification agents (Calcium Folinate, silymarin and ursodeoxycholic acid) on the side effects of methotrexate, applied in this protocol. Methods A modified acute toxicity form (GPOH) was used for side effects monitoring. The research included children with either standard or intermediate risk ALL in the consolidation therapy phase, who were hospitalised at the Institute for Child and Youth Health Care of Vojvodina in Novi Sad during the period from July 2010 to February 2011. Results The most frequent side effect after 40 applications of methotrexate in ten children was bone marrow depression. Methotrexate caused: leukopenia in 10 patients, thrombocytopenia in 5 patients; after the use of folic acid, platelet count grew in 8 patients, leukocyte in 2 patients. Less frequent side effects: an increase serum transaminase activity, the state of fever, bronchopneumonia, diarrhoea with mild cramps and hypercalcaemia. Conclusion The application of Calcium Folinate, silymarin and ursodeoxycholic acid prevented the occurrence of severe adverse effects caused by medium-high doses of methotrexate. Observed adverse effects were of mild to moderate intensity, reversible and did not significantly disturb the quality of life in treated patients.

FOXM1 Mediates Drug-Resistance and Represents a Therapeutic Target in Pre-B Acute Lymphoblastic Leukemia

Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL.Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice.Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Augmented post-remission therapy for a minimal residual disease-defined high-risk subgroup of children and young people with clinical standard-risk and intermediate-risk acute lymphoblastic leukaemia (UKALL 2003): a randomised controlled trial

No randomised study has shown whether stratification of treatment by minimal residual disease (MRD) response improves outcome in children and young people with acute lymphoblastic leukaemia (ALL). We assessed whether children and young people with clinical standard and intermediate-risk ALL who have persistent MRD at the end of induction therapy benefit from augmented post-remission therapy. Between Oct 1, 2003, and June 30, 2011, we enrolled eligible patients aged 1-24 years and initially categorised them into clinical standard-risk, intermediate-risk, and high-risk groups on the basis of a combination of National Cancer Institute criteria, cytogenetics, and early morphological response to induction therapy. Clinical standard-risk and intermediate-risk patients with MRD of 0·01% or higher at day 29 of induction (MRD high risk) were randomly assigned (1:1) to standard therapy (treatment regimens A and B) or augmented post-remission therapy (regimen C). Compared with standard therapy, the augmented treatment regimen (regimen C) included an additional eight doses of pegylated asparaginase, 18 doses of vincristine, and escalated-dose intravenous methotrexate without folinic acid rescue during interim maintenance courses. Computer randomisation was used for treatment allocation and was balanced for sex, age (<10 years vs ≥10 years), and white blood cell count at diagnosis (<50 × 10(9)/L vs ≥50 × 10(9)/L) by minimisation. Patients, clinicians, and data analysts were not masked to treatment allocation. The primary outcomes were event-free survival and overall survival. Analyses were by intention to treat. This trial is registered with Current Controlled Trials, number ISRCTN07355119. 533 MRD high-risk patients were randomly assigned to receive standard (n=266) or augmented (n=267) post-remission therapy. After a median follow-up of 70 months (IQR 52-91), 5-year event-free survival was better in the augmented treatment group (89·6% [95% CI 85·9-93·3]) than in the standard group (82·8% [78·1-87·5]; odds ratio [OR] 0·61 [95% CI 0·39-0·98], p=0·04). Overall survival at 5 years was numerically, but not significantly, higher in the augmented treatment group (92·9% [95% CI 89·8-96·0]) than in the standard therapy group (88·9% [85·0-92·8]; OR 0·67 [95% CI 0·38-1·17], p=0·16). More adverse events occurred in the augmented treatment group than in the standard group (asparaginase-related hypersensitivity in 18 [6·7%] in the augmented group vs two [0·8%] in the standard group and asparaginase-related pancreatitis in eight [3·0%] vs one [0·4%]; intravenous methotrexate-related mucositis in 11 [4·1%] vs three [1·1%] and methotrexate-related stomatitis in 48 [18·0%] vs 12 [4·5%]). Our findings suggest that children and young people with acute lymphoblastic leukaemia and 0·01% or more MRD at the end of remission induction therapy could benefit from augmented post-remission therapy. However, the asparaginase and intravenous methotrexate used in the augmented treatment regimen is associated with more adverse events than is the standard post-remission treatment regimen. Medical Research Council and Leukaemia and Lymphoma Research.

Neuropsychological functioning of children treated for acute lymphoblastic leukemia: impact of whole brain radiation therapy.

To provide one of the first prospective reports examining neuropsychological outcomes for children treated with 1800 cGy whole brain radiotherapy (WBRT) and prophylactic chemotherapy versus prophylactic chemotherapy alone for acute lymphoblastic leukemia (ALL). Acute and long-term neuropsychological toxicities associated with WBRT are compared. This multisite study included 188 children, ages 4-21 years at enrollment, who were assessed with standardized neuropsychological tests at 9, 21, and 48 months after diagnosis with intermediate risk ALL. All participating children were receiving treatment on a parent study CCG105. Verbal intelligence (VIQ) scores for children receiving WBRT was significantly lower than VIQ for prophylactic chemotherapy at the 48-month time point (p < 0.05). A significant cross-level interaction between time since diagnosis and treatment condition was observed (p < 0.05). WBRT did not result in differences in PIQ; both groups of children demonstrated comparable increases in PIQ. Neuropsychological findings at 48 months after diagnosis indicated diminished performance in neuromotor, visual-motor coordination, and executive functioning for children receiving WBRT. Academic achievement was unaffected by WBRT at 4 years after diagnosis. The measurement of verbal and performance IQ as a primary endpoint in ALL clinical trials is critical to characterizing neuropsychological late effects. A trajectory of decline in neuropsychological functioning, specifically verbal IQ, was observed. Missing data within the trial occurred at random and did not impact results observed. The impact of WBRT becomes evident at 48 months after diagnosis, suggesting the need for long-term follow-up beyond the time frame typically used in Phase III trials.

The influence of detoxification agents on the intensity of side effects caused by medium-high doses of methotrexate in children with acute lymphoblastic leukemia: Case series

Objective The treatment of childhood acute lymphoblastic leukemia (ALL) in Serbia is conducted according to protocol ALL IC BMF-2009. The therapy includes the application of cytostatic drugs methotrexate and 6-mercaptopurine, and drug detoxifying Calcium Folinate. At the moment, 80% of affected children could be cured with current treatment, but resistance to the therapy and its toxic effects remain serious clinical problems. The aim of the study was to investigate the influence of detoxification agents (Calcium Folinate, silymarin and ursodeoxycholic acid) on the side effects of methotrexate, applied in this protocol. Methods A modified acute toxicity form (GPOH) was used for side effects monitoring. The research included children with either standard or intermediate risk ALL in the consolidation therapy phase, who were hospitalised at the Institute for Child and Youth Health Care of Vojvodina in Novi Sad during the period from July 2010 to February 2011. Results The most frequent side effect after 40 applications of methotrexate in ten children was bone marrow depression. Methotrexate caused: leukopenia in 10 patients, thrombocytopenia in 5 patients; after the use of folic acid, platelet count grew in 8 patients, leukocyte in 2 patients. Less frequent side effects: an increase serum transaminase activity, the state of fever, bronchopneumonia, diarrhoea with mild cramps and hypercalcaemia. Conclusion The application of Calcium Folinate, silymarin and ursodeoxycholic acid prevented the occurrence of severe adverse effects caused by medium-high doses of methotrexate. Observed adverse effects were of mild to moderate intensity, reversible and did not significantly disturb the quality of life in treated patients.

Antithrombin deficiency after prolonged asparaginase treatment in children with acute lymphoblastic leukemia

Children with acute lymphoblastic leukemia (ALL) have several risk factors for deep venous thromboses (DVTs) such as central venous catheters and asparaginase (ASP), related antithrombin (AT) deficiency. After introduction of a new standard and intermediate-risk ALL treatment protocol with prolonged continuous ASP treatment, two symptomatic DVTs in 10 patients were observed at the Children's Hospital, Helsinki, Finland. To prevent further thrombotic complications yet ensuring continuous exposure to ASP, an AT substitution strategy was adopted in Helsinki. The same ALL treatment protocol is used without AT substitution in the other Nordic countries. In this retrospective study, we describe the effect of prolonged ASP treatment on AT and fibrinogen levels in children without AT substitution in Stockholm, Sweden (n = 39) and the AT substitution in children with AT activity below 0.55 kIU/l in Helsinki (n = 36, intervention group). The intervention group is compared with children treated similarly earlier in Helsinki without AT substitution (n = 10). The median lowest AT activity during the ASP treatment without AT substitution was 0.55 kIU/l. Fibrinogen level of 1.0 g/l or less was found in 14% of all routine samples during the ASP treatment. In the intervention group, 23 (64%) received AT concentrate. Two (20%) children had symptomatic DVT before initiation of the AT substitution and two (6%) thereafter. We conclude that most children are exposed to low AT activity during ASP treatment predisposing to thrombosis. The effect of prophylactic AT substitution remains unclear.

Minimal Residual Disease–Guided Treatment Deintensification for Children With Acute Lymphoblastic Leukemia: Results From the Malaysia-Singapore Acute Lymphoblastic Leukemia 2003 Study

To improve treatment outcome for childhood acute lymphoblastic leukemia (ALL), we designed the Malaysia-Singapore ALL 2003 study with treatment stratification based on presenting clinical and genetic features and minimal residual disease (MRD) levels measured by polymerase chain reaction targeting a single antigen-receptor gene rearrangement. Five hundred fifty-six patients received risk-adapted therapy with a modified Berlin-Frankfurt-Münster-ALL treatment. High-risk ALL was defined by MRD ≥ 1 × 10(-3) at week 12 and/or poor prednisolone response, BCR-ABL1, MLL gene rearrangements, hypodiploid less than 45 chromosomes, or induction failure; standard-risk ALL was defined by MRD ≤ 1 × 10(-4) at weeks 5 and 12 and no extramedullary involvement or high-risk features. Intermediate-risk ALL included all remaining patients. Patients who lacked high-risk presenting features (85.7%) received remission induction therapy with dexamethasone, vincristine, and asparaginase, without anthracyclines. Six-year event-free survival (EFS) was 80.6% ± 3.5%; overall survival was 88.4% ± 3.1%. Standard-risk patients (n = 172; 31%) received significantly deintensified subsequent therapy without compromising EFS (93.2% ± 4.1%). High-risk patients (n = 101; 18%) had the worst EFS (51.8% ± 10%); EFS was 83.6% ± 4.9% in intermediate-risk patients (n = 283; 51%). Our results demonstrate significant progress over previous trials in the region. Three-drug remission-induction therapy combined with MRD-based risk stratification to identify poor responders is an effective strategy for childhood ALL.

Intermediate-Risk Acute Lymphoblastic Leukemia (ALL) Patients with and without Relapse Differentially Depend on Survival Signals From Microenvironment

Abstract 752Risk stratification in the childhood ALL-BFM clinical trial has been based on molecular/cytogenetic markers and the in vivo response to treatment (blast reduction in peripheral blood after prednisone prephase, bone marrow clearance after induction treatment and minimal residual disease (MRD) after induction and induction consolidation). These criteria classify about 50% of ALL cases into standard-risk (SR) and high-risk (HR) groups. The intermediate-risk (IR) ALL group, showing moderate levels of MRD, provides the majority of relapses, but still lacks specific prognostic markers which could distinguish between those patients subsequently relapse and those remaining relapse-free (IR+r and IR-r). We established a bank of NOD/SCID ALL xenografts representing IR+r and IR-r subgroups (12 vs 13 patients) and characterized the samples in a broad series of functional assays. There was no difference in the sensitivity to drugs (dexamethasone, daunorubicin, L-asparaginase and vincristine) between the IR+r and IR-r groups, however, the rate of spontaneous apoptosis in vitro was significantly higher in the IR+r group than in the IR-r group (69 +/− 7% vs 39 +/− 6%, p=0.02). Given that in the presence of stromal cells the cell death level has been similar in both IR groups (9 +/− 5% vs 7 +/− 7%, p=0.8), these observations suggest that the IR+r ALL is more dependent on survival signals from microenvironment than IR-r ALL. In the NOD/SCID engraftment model, leukemic cells from IR+r patients engrafted more rapidly than cells from IR-r patients (93 +/− 12 days vs 170 +/− 24 days, p=0.006), pointing to a better in vivo cooperation between leukemia cells and microenvironment in the IR+r cases. In order to identify the underlying molecular pathways, gene expression changes induced in the presence and absence of stroma have been investigated at genome-wide level (Human Gene 1.0 ST Affymetrix array). Of the apoptosis-regulating genes, a caspase inhibitior from the IAP family, BIRC3, was found to be up-regulated significantly higher in the IR-r than in the IR+r group (3.7 +/− 1.1 vs 1.7 +/− 0.1-fold up-regulation, p=0.016). The adhesion molecule VCAM1 has been generally up-regulated by the co-incubation with stroma, with a tendency to a higher up-regulation in the IR-r (4.4 +/− 0.7-fold) than in the IR+r (2.7 +/− 1.0-fold) group. We further speculated that interaction with microenvironment is a mutual process which also implicates gene expression changes of the stroma. To this end, mouse stromal cells (MS5 cell line) were incubated with a series of ALL samples (n=3), purified by MACS sorting and investigated using Mouse Gene 1.0 ST Affymetrix arrays. The list of genes whose expression increased by at least 2-fold in all 3 cases (n=10), included the macrophage inflammatory peptide-1alpha (MIP1A/CCL3), a chemokine known to regulate chemotaxis of lymphocytes. Our data suggest that the cooperation of ALL with microenvironment may provide a promising approach to molecularly discriminate ALL cases with relapse and to identify potential relapse-associated targets within the IR-ALL group. Disclosures:No relevant conflicts of interest to declare.

The degree of myelosuppression during maintenance therapy of adolescents with B-lineage intermediate risk acute lymphoblastic leukemia predicts risk of relapse

Drug doses, blood levels of drug metabolites and myelotoxicity during 6-mercaptopurine/methotrexate (MTX) maintenance therapy were registered for 59 adolescents (>or=10 years) and 176 non-adolescents (<10 years) with B-cell precursor acute lymphoblastic leukemia (ALL) and a white blood cell count (WBC) <50 x 10(9)/l at diagnosis. Event-free survival was lower for adolescents than non-adolescents (pEFS(12y):0.71 vs 0.83, P=0.04). For adolescents staying in remission, the mean WBC during maintenance therapy (mWBC) was related to age (r(S)=0.36, P=0.02), which became nonsignificant for those who relapsed (r(S)=0.05, P=0.9). The best-fit multivariate Cox regression model to predict risk of relapse included mWBC and thiopurine methyltransferase activity, which methylates mercaptopurine and reduces the intracellular availability of cytotoxic 6-thioguanine nucleotides (coefficient: 0.11, P=0.02). The correlation of mWBC to the risk of relapse was more pronounced for adolescents (coefficient=0.65, P=0.003) than for non-adolescents (coefficient=0.42, P=0.04). Adolescents had higher mean neutrophil counts (P=0.002) than non-adolescents, but received nonsignificantly lower mercaptopurine and MTX doses during maintenance therapy. Red blood cell MTX levels were significantly related to the dose of MTX among adolescents who stayed in remission (r(S)=0.38, P=0.02), which was not the case for those who developed a relapse (r(S)=0.15, P=0.60). Thus, compliance to maintenance therapy may influence the risk of relapse for adolescents with ALL.