High-dose methotrexate (HDMTX) is a cornerstone of contemporary treatment protocols for both pediatric and adult acute lymphoblastic leukemia (ALL); however, up to 4% of children and 15% of adults develop renal toxicity with severely delayed MTX elimination (DME). Evidence-based guidance on re-exposure after DME is lacking, and omission of further HDMTX may compromise anti-leukemic efficacy and potentially increase the risk of relapse. This study, conducted within the Ponte di Legno International Toxicity Working Group, aimed to evaluate the safety of HDMTX re-challenge in pediatric patients after DME. National investigators from 12 countries provided case-level data on initial DME events and subsequent HDMTX re-exposures via structured questionnaires. Data from 189 patients treated for ALL who experienced DME were analyzed, of whom 143 were subsequently re-exposed to HDMTX. Clinical toxicities after the initial DME included gastrointestinal complications (vomiting, diarrhea, mucositis), infections, and neurological events (encephalopathy, seizures, MTX stroke-like syndrome). Laboratory toxicities comprised cytopenias and hepatic abnormalities. Two patients transiently required dialysis. DME led to chemotherapy modifications in 73% of the patients. After re-exposure, toxicities were similar in spectrum, self-limited, and non-fatal. Twenty children (14%) developed recurrent DME, including three with two additional episodes. Recurrent DME could neither be predicted by clinical, pharmacokinetic, or demographic variables, nor by uniform MTX dose reduction during re-exposure. In conclusion, re-exposure to HDMTX following DME is feasible and generally well tolerated, although the risk of recurrence is increased. Re-challenge should be considered once renal function has normalized, with careful monitoring and individualized dose adjustment.

Abstract T-cell lymphoblastic lymphoma (T-LBL) and T-cell acute lymphoblastic leukemia (T-ALL) originate from thymic T-cell precursors, with ongoing debate on whether they are variants of the same disease or distinct entities. For 211 patients, including pediatric and adult T-ALL and T-LBL cases, targeted next-generation sequencing and SNP-arrays were performed, and single-nucleotide variants, indels and copy-number variants (CNVs) were analyzed. We aimed to assess genetic differences between T-ALL and T-LBL across age. Generally, mutational landscape analysis identified mutated PHF6 being associated with higher, NOTCH1 with lower age at diagnosis for both T-LBL and T-ALL. Association of CNVs with higher age was evident for T-ALL, but not T-LBL. Analysis of clonal evolution revealed that CNVs – especially deletions and LOH in chromosome 9 (LOH_in_9p) – were observed as first mutational event in both pediatric T-ALL and T-LBL. The sequence of genetic events, starting with LOH_in_9p followed by mutations in NOTCH1, was significantly more frequent in pediatric T-ALL and T-LBL. Detailed evaluation of the patients’ individual clonal evolution indicated that the proportion of malignant cells without NOTCH MT determines the risk of relapse (hazard ratio 1.032, p = 4.65*10−5). In T-ALL, aside from MRD, validated molecular markers for risk-group stratification remain limited. Our data suggest that molecular metrics analogous to those in T-LBL may help refining risk stratification in T-ALL as well.

BACKGROUND:Auer rods (AuRs) are prominent intracellular structures found almost exclusively in myeloid cell malignancies, such as acute myeloid leukemia (AML), chronic and juvenile myelomonocytic leukemia and myelodysplastic syndrome. Extremely rare AuRs have been reported in patients with acute lymphoblastic leukemia (ALL) or among ambiguous lineage leukemia patients with a dominantly lymphoblastic immunophenotype. PROCEDURE:We report diagnostic and follow-up data of an international cohort of 11 children suffering from leukemias with AuRs and with significant presence of T and myeloid markers, majority of whom categorized as early T-cell precursor (ETP, n = 7); or T-ALL (ETP status unknown, n = 2), ALAL (acute leukemia of ambiguous lineage, n = 1), and AML reclassified from ALAL (n = 1). We described other diagnostic details and treatment types and responses. Moreover, we summarize previously published data. RESULTS:Among the four patients who started and remained on ALL-type therapy, all were in the first complete remission, whereas both patients who started and remained on AML-type therapy relapsed and died. Of the patients who followed either a combined ALL/AML protocol (Interfant 06) or who switched from one of the two types of therapy to the other, one patient died, and the remaining four were in first complete remission at the most recent follow-up. We also searched for similar cases in the literature and found only three additional children with nonmyeloid leukemia and AuRs and 10 adults with this type of leukemia. CONCLUSIONS:Briefly, ALL- or combined ALL/AML-type therapy may be effective for treating AuR-positive leukemia patients with a lymphoid immunophenotype.

In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-M & uuml;nster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371(pos)) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371(pos). This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371(pos) BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371(pos). In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371(pos) is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Steroids are a mainstay in the treatment of acute lymphoblastic leukaemia (ALL) in children and adolescents; however, their use can cause clinically significant steroid-related neuropsychiatric symptoms (SRNS). As current knowledge on SRNS during ALL treatment is limited, we mapped the phenotypes, occurrence and treatment strategies using a database created by the international Ponte di Legno Neurotoxicity Working Group including data on toxicity in the central nervous system (CNS) in patients treated with frontline ALL protocols between 2000 and 2017. Ninety-four of 1813 patients in the CNS toxicity database (5.2%) experienced clinically significant SRNS with two peaks: one during induction and one during intensification phase. Dexamethasone was implicated in 86% of SRNS episodes. The most common symptoms were psychosis (52%), agitation (44%) and aggression (31%). Pharmacological treatment, mainly antipsychotics and benzodiazepines, was given to 87% of patients while 38% were hospitalised due to their symptoms. Recurrence of symptoms was reported in 29% of patients and two previously healthy patients required ongoing pharmacological treatment at the last follow up. Awareness of SRNS during ALL treatment and recommendation on treatment strategies merit further studies and consensus.

Pneumocystis jirovecii can cause life-threatening pneumonia (PjP), and patients with haematological malignancies are at high risk of this infection. Prophylactic measures have significantly decreased morbidity and mortality, but there is a paucity of contemporary data on the incidence and clinical course of PjP in well-defined and homogenous patient populations, such as children suffering from acute lymphoblastic leukaemia (ALL). In the multi-international trial AIEOP-BFM ALL2009, PjP was diagnosed in six children (incidence 1/1000) and was associated with insufficient prophylaxis in five of them. Although none of the patients died of PjP, the long-term impact of the infection is unclear.

Background: T-cell lymphoblastic lymphoma (T-LBL) and acute lymphoblastic leukemia (T-ALL) originate from the malignant transformation of similar subsets of immature T-cell precursors in the thymus. Unique genetic and epigenetic aberrations lead to distinct clinical presentations. Both are distinguished by bone marrow infiltration. Patient's age at diagnosis is associated with the distribution of molecular subgroups in T-ALL (Neumann, et al. Leukemia, 2024). Despite sharing many clinical features and molecular alterations, there is ongoing debate about whether T-LBL and T-ALL represent a spectrum of a disease or are distinct entities. Recent papers identified TRB::NOTCH1 fusions exclusively in T-LBL and associated with high relapse risk (te Vrugt, et al. Blood, 2024). Both diseases are characterized by poor survival in resistant or relapsed cases, highlighting a clear unmet medical need. Understanding varying therapy resistance and molecular pathogenesis across ages is crucial. Clonal evolution analysis can help reconstruct tumor development, explore treatment responses, and identify reasons for therapy failure (Sandmann, et al. Int J Environ Res Public Health, 2023). This study compares DNA samples from pediatric and adult T-LBL and T-ALL patients to uncover genetic differences and tackle therapy resistance. Methods: DNA samples include material from initial diagnosis, corresponding germline, and, if applicable, relapse. Subcohorts include 87 pediatric T-LBL cases (27 relapsed, 60 non-relapsed), 36 pediatric T-ALL (12 relapsed, 24 non-relapsed), 47 adult T-LBL (of which 3 corresponding relapse samples were available), and 41 adult T-ALL, (of which 4 corresponding relapse samples were available). Besides targeted sequencing, CNV analysis is performed by Illumina Infinium Global Screening Array v3.0. Results and Discussion: For T-LBL and T-ALL, there were no significant differences in the average number of single nucleotide variants (SNV) per sample, either within or between the groups. For CNVs, a clear increase with age was observed for both T-LBL and T-ALL, except deletions and LOH affecting chromosome 9, which were more frequent in pediatric cases and decreased with age. Detailed analysis revealed subgroup-specific characteristics in initial samples. In adult T-LBL vs pediatric T-LBL samples, variants in PHF6 (34% vs 13%), JAK1 (9% vs 0%), TP53 (6% vs 0%), NOTCH3 (11% vs 5%), JAK3 (11% vs 5%), STAT5B (11% vs 5%), TET2 (6% vs 1%), and NRAS (9% vs 3%) were more prevalent. NOTCH1 (60% vs 49%), PIK3CA (10% vs 2%), PTEN (15% vs 9%), PIK3R1 (8% vs 3%), CCND3 (6% vs 0%), and KMT2C (6% vs 0%) were more frequent in pediatric T-LBL than adult T-LBL patients. In the T-ALL cohort, some variants, such as DNM2 (17%) and RUNX1 (10%) were almost exclusively found in adult cases. The increased prevalence of TP53 mutations in pediatric relapse samples underscored the critical role of TP53 in treatment resistance and disease progression (T-ALL: 8% primary vs 29% relapse; T-LBL: 0% primary vs 13% relapse). Clonal evolution analysis showed that CNVs appeared early, especially deletions or LOH affecting chromosome 9. Subsequently, additional subclones with point mutations evolved. Pediatric cases revealed differential timing and frequency of mutations in T-LBL vs in T-ALL: MYB (nested level 4.6 vs 1.0), SMARCA4 (4.7 vs 1.5), or TET2 (4.0 vs 1.0). In pediatric T-LBL, deletions and LOH on chromosome 9 occurred equally in primary samples of patients who suffered/did not suffer relapse. In relapse samples, the clonal population with CNV increased, indicating a growth advantage. Frequent CNVs in chromosome 9, encompassing genes like CDKN2A/B and MTAP, underscored the importance of this region in both T-LBL and T-ALL. These CNVs, involving tumor suppressor genes such as p16INK4a and p14ARF, were early events in clonal evolution. Conclusion: Our integrated genomic analysis highlights differences and similarities between pediatric and adult T-LBL and T-ALL cases. The analysis of CNVs revealed their role in clonal evolution, particularly with frequent deletions and LOH affecting chromosome 9 in pediatric cases. This emphasize the importance of genomic profiling for each subgroup. By understanding distinct genetic landscapes and mutation patterns, we aim to discover potential novel targets to improve risk group stratification of patients with T-LBL and T-ALL.

Background: Anthracyclines have contributed to the development of effective antileukemic treatment of ALL. Modern ALL therapy, however, is shifting its attention towards reduction of acute and late toxicities while maintaining the same cure rate. Acute and late sequelae of anthracyclines are of major concern, thus, this trial aimed to clarify their role in low-risk patients (pts). Patients and Methods: The randomization R1 was included in trial AIEOP-BFM ALL 2009 for pediatric pts (1 to < 18 years of age) with newly diagnosed ALL with the aim to safely reduce the daunorubicin (DNR) dose in induction with non-inferior event-free survival (EFS) and reduced toxicity. Pts were eligible for R1 if they had non-high-risk B-ALL (i.e. good response to the prednisone pre-phase, absence of hypodiploidy or KMT2A::AFF1 rearrangement) with either positivity for ETV6::RUNX1 or rapid response to the first two weeks of induction treatment (flow cytometry d15 with < 0.1% marrow blasts). After two weeks of induction therapy including 2 DNR doses (30 mg/m2 on days 8 and 15), pts were randomly assigned to receive either a 3rd and 4th DNR dose (on days 22 and 29, control arm, CA) or no further DNR during induction (experimental arm, EA). Other treatment components have been previously described (Rizzari C et al, Hemasphere 2023, 7:e893). Depending on minimal residual disease (MRD, evaluated by real-time quantitative PCR) at the end of induction (EoI) and consolidation (EoC), pts were finally stratified into standard risk (SR), medium risk (MR), or high risk (HR) (Conter V. et al, J Clin Oncol 2023, 42:915-926). Pts who were treated as assigned by randomization R1 were included in the primary analyses on EFS, cumulative incidence of relapse (CIR), and overall survival (OS). Secondary outcome analyses were done by intention to treat (ITT). Incidences of protocol-defined adverse reactions (AR) of special interest, occurring during the randomized treatment phase (from day 22 of induction to start of consolidation) were analyzed in the as-treated population. Results: Out of 6136 pts enrolled in AIEOP-BFM ALL 2009, 2514 pts (41.0 %) were eligible for R1. Of those, 2079 pts (82.7 %) were randomized to receive either the EA (n=1040) or the CA (n=1039). Forty randomized pts were not included in the treated-as-assigned population because they were either retrospectively not eligible (n=5), died before day 22 (n=1) or did not receive the assigned arm (switch from CA to EA (n=13) or vice versa (n=19) or 3rd DNR dose given by mistake in EA (n=2)) resulting in 1016 pts in EA and 1023 pts in CA. Patient characteristics were equally distributed between the arms. With a median observation time of 7.0 years, the probability of 5-year EFS in the treated-as-assigned population was 92.7% (standard error, SE 0.8%) in CA and 92.2% (SE 0.9%) in EA. The lower limit of the one-sided 95% confidence interval for the difference was -2.3%, well above the pre-defined non-inferiority margin of -4%. CIR at 5 years was 5.6% (SE 0.7%) in CA compared to 5.9% (SE 0.8%) in EA. Five-year OS was 97.4% (SE 0.5%) in CA and 97.7% (SE 0.5%) in EA. Additional analyses in various subgroups (gender, age, ETV6::RUNX1 status, MRD status at EoI or at EoC, final risk groups SR, MR, or HR) did not reveal any differences in outcome between the randomization arms. Outcome results of ITT analyses were almost identical: 5y-EFS 92.7% (SE 0.8%) in CA, and 92.3% (SE 0.8%) in EA. AR of special interest were observed in 7.4% and 6.1% of pts in CA and EA, respectively (p=0.25). Life-threatening AR occurred in 1.4% in CA and 1.1% in EA (p=0.45), and were fatal in 0.7% and 0.5% of pts, respectively. Analyses of AR types revealed a trend towards a higher incidence of infection-related AR in CA than in EA (2.9% vs 1.7%, p=0.061) due to a four times higher incidence of invasive fungal infections (IFI) in CA than in EA (1.5% vs 0.4%, p=0.0076). Conclusion: The results of this unique prospective trial in a large patient cohort clearly show that a 50% reduction of the cumulative DNR dose during induction did not compromise the outcome. Although the two different schedules showed almost no difference in the overall incidence of serious toxicity, the risk of IFI could be significantly mitigated by reducing the DNR dose. Our study demonstrates, that one can safely recommend reduced anthracyclines in ALL induction therapy if patients are selected by favorable prognostic factors, such as presence of ETV6::RUNX1, or very fast early treatment response.

Background Characterization of clinical phenotypes in context with tumor and host genomic information can aid in the development of more effective and less toxic risk-adapted and targeted treatment strategies. To analyze the impact of therapy-related hyperbilirubinemia on treatment outcome and to identify contributing genetic risk factors of this well-recognized adverse effect we evaluated serum bilirubin levels in 1547 pediatric patients with acute lymphoblastic leukemia (ALL) and conducted a genome-wide association study (GWAS). Patients and methods Patients were treated in multicenter trial AIEOP-BFM ALL 2000 for pediatric ALL. Bilirubin toxicity was graded 0 to 4 according to the Common Toxicity Criteria (CTC) of the National Cancer Institute. In the GWAS discovery cohort, including 650 of the 1547 individuals, genotype frequencies of 745,895 single nucleotide variants were compared between 435 patients with hyperbilirubinemia (CTC grades 1-4) during induction/consolidation treatment and 215 patients without it (grade 0). Replication analyses included 224 patients from the same trial. Results Compared to patients with no (grade 0) or moderate hyperbilirubinemia (grades 1-2) during induction/consolidation, patients with grades 3-4 had a poorer 5-year event free survival (76.6 ± 3% versus 87.7 ± 1% for grades 1-2, P = 0.003; 85.2 ± 2% for grade 0, P < 0.001) and a higher cumulative incidence of relapse (15.6 ± 3% versus 9.0 ± 1% for grades 1-2, P = 0.08; 11.1 ± 1% for grade 0, P = 0.007). GWAS identified a strong association of the rs6744284 variant T allele in the UGT1A gene cluster with risk of hyperbilirubinemia (allelic odds ratio (OR) = 2.1, P = 7 × 10 − 8 ). TT-homozygotes had a 6.5-fold increased risk of hyperbilirubinemia (grades 1-4; 95% confidence interval (CI) = 2.9-14.6, P = 7 × 10 − 6 ) and a 16.4-fold higher risk of grade 3-4 hyperbilirubinemia (95% CI 6.1-43.8, P = 2 × 10 − 8 ). Replication analyses confirmed these associations with joint analysis yielding genome-wide significance (allelic OR = 2.1, P = 6 × 10 − 11 ; 95% CI 1.7-2.7). Moreover, rs6744284 genotypes were strongly linked to the Gilbert’s syndrome-associated UGT1A1 *28/*37 allele (r 2 = 0.70), providing functional support for study findings. Of clinical importance, the rs6744284 TT genotype counterbalanced the adverse prognostic impact of high hyperbilirubinemia on therapy outcome. Conclusions Chemotherapy-related hyperbilirubinemia is a prognostic factor for treatment outcome in pediatric ALL and genetic variation in UGT1A aids in predicting the clinical impact of hyperbilirubinemia. Trial registration http://www.clinicaltrials.gov ; #NCT00430118.

The international multicenter frontline therapy trial AIEOP-BFM ALL 2017 prospectively randomizes CD19-targeting T-cell engager blinatumomab (BLN) against standard of care (SOC) to improve outcomes in pediatric patients (pts) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) stratified for high (HR) and medium (MR) risks of relapse. In HR arm BLN is given earlier, directly after intensified HR therapy block, while in the MR arm, it is given towards the end of chemotherapy, before maintenance. The immune composition prior to BLN might thus differ between both treatment groups (HR vs MR) which may impact BLN-mediated anti-leukemic activity. Within the Germany-wide immune monitoring program accompanying the AIEOP-BFM ALL 2017 trial we addressed this question and studied a total of 1056 longitudinal peripheral blood samples collected between 11/2018 and 05/2022 from 381 BCP-ALL pts. Samples were prospectively analyzed with multicolor flow cytometry to study immune cell composition (Panel 1) at the time of BLN application (d0) and under ongoing immunotherapy within first 24h (d1), at day 14 (d14), at the end of the cycle I (d28) and end of cycle 2 (day28_II), if applicable (Figure 1A). In-depth T-cell profiling was performed in the BLN arm, utilizing 4 additional 12-color panels concentrated on markers associated with T-cell functionality (measured with standardized cytometer settings). Data were analyzed using classical 2D-manual gating strategies (> 2000 variables) and completed with R-based machine learning algorithms; namely partial Least-Squares Discriminant Analysis (PLS-DA), a multivariate dimensionality-reduction tool for identifying HR- and MR-defining immune signatures and PhenoGraph algorithm for unsupervised T-cell cluster detection. T-cell receptor excision circles (TRECs) and TR-repertoire analysis were also performed among paired samples of 117 BLN-treated pts. Baseline immune composition differed notably between HR and MR pts directly prior to BLN therapy, as shown by PLS-DA analysis on manually gated immune subsets. HR pts showed higher neutrophil counts and lower lymphocyte and monocyte counts compared to MR pts. Interestingly, MR pts had higher levels of B- and T-cells, which are crucial for BLN-mediated T-cell activation. HR pts displayed an elevated CD4+/CD8+ ratio, shifted naïve/memory T-cell phenotypes and increased proportions of immunosuppressive T-regulatory cells. These results suggest that more intensive therapy protocols directly prior to BLN influence the immune composition towards compromised states in the HR arm, potentially affecting BLN-mediated T-cell leukemia control. BLN effectively depleted CD19+ cells in 221 out of 224 pts. We observed significantly stronger T-cell activation/expansion in MR pts compared to HR pts under BLN therapy. This expansion that was primarily attributed to naïve CD8+ T-cell populations, resulting from clonal T-cell proliferation independent of thymic contribution, as supported by TREC and TR-repertoire analysis. The PhenoGraph clustering also identified this expanding naïve CD8+ T-cell populations, surprisingly marked by loss of adhesion molecule DNAM1 (panel 1), normally involved in NK and T-cell mediated cytotoxicity and by gaining the expression of TIM-3 (panel 3), an inhibitory molecule usually co-expressed with PD1 on exhausted T-cells with memory phenotypes. In this setting however, TIM3 was not co-expressed with PD1 (Figure 1B) and its expression was correlated with the B-cell levels at baseline and was strongest in MR pts under BLN therapy. Our data suggest an until now undescribed association between DNAM1 and TIM3 with BLN-mediated T-cell activation, hinting that they may serve as a measure of T-cell activation strength or an early T-cell exhaustion marker. In conclusion our study provides clinically relevant insights in how chemotherapy prior BLN might affect the patients´ immune system, possibly influencing the BLN anti-leukemic activity, and helps to determine the optimal place of immunotherapy with BLN in multimodal treatment schedules. Here, we report until know undescribed effect of BLN on T-cell compartment. Upcoming clinical data, which will be correlated with obtained immune profiles will answer the open question regarding the clinical relevance of these findings, and shed light into the mechanisms determining response to BLN, for its maximum benefit in childhood ALL.

Background: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Even though novel therapeutic strategies and stratification led to a ~90% long-term cure, there is still a proportion of patients experiencing relapse or therapy-related toxicities. Recently, the IKZF1plus profile, defined as the presence of a deletion in IKZF1 together with one or more additional deletions in PAX5 and/or CDKN2A/B and/or PAR1 and the absence of a deletion in ERG, was characterized as a very poor prognostic marker specifically in MRD-positive patients based on retrospective analyses of the AIEOP-BFM ALL 2000/2009 trials (PMID 29498923). Dependent on MRD at day 33, IKZF1plus is used for high-risk stratification in the ongoing AIEOP-BFM ALL 2017 trial. Aims: This retrospective study aimed to decipher the underlying molecular complexity of the IKZF1plus profile by reanalyzing patients from the AIEOP-BFM ALL 2000 and 2009 trials with optical genome mapping (OGM), a molecular cytogenetic approach which allows the parallel detection of all kinds of structural variants including copy number variations, translocation as well as aneuploidies. Methods: In total, 142 patients with IKZF1 deletion (73 with IKZF1del, 69 with IKZF1plus profile) as determined by MLPA in previous analyses were re-analyzed by means of OGM. Based on our findings seven patients were not eligible and eight patients were reclassified from IKZF1del to IKZF1plus or vice versa (94.1% concordance with MLPA). Results were validated by comparison to previously collected data and in case of new fusions by RT-PCR/RNA-Seq. Next, genetic markers were correlated with patient outcome (5-year event-free survival (EFS), overall survival and cumulative incidence of relapse). Results: In 45.9% of the analyzed patients either known prognostic markers (18/135; 13.3% with ETV6::RUNX1, high hyperdiploidy or iAMP21) or other gene fusions (44/135; 32.6%) were identified. These fusions were evenly distributed among the subgroups (21 IKZF1del and 23 IKZF1del) and categorized into: ABL-class fusions (12; 8.9%), PAX5 fusions (9; 6.7%), JAK2 fusions (10; 7.4%), ZNF384 fusions (4; 3.0%) and other fusions (9; 6.7%). Investigating the patient outcome, we reproduced the initial results (PMID 29498923) and show that IKZF1del and especially IKZF1plus positives did much worse compared to a cohort 845 other patients from the AIEOP-BFM ALL 2000 and 2009 trials without an IKZF1 deletion (EFS 73.1±5.3 vs 60.6±6.3 vs 87.1±1.2). The dismal outcome was even more pronounced when we excluded patients with an established good prognostic marker from both groups. (EFS IKZF1del vs IKZF1plus; 68.3±6.2 vs 62.0±6.4). IKZF1-deleted patients with a favorable prognostic marker (high hyperdiploidy and ETV6::RUNX1) had an EFS of 100% and were exclusively found in the IKZF1del but not the IKZF1plus subgroup. Furthermore, IKZF1del/plus patients had an inferior 5-year EFS in the presence of a gene fusion (55.9±7.6), especially with ABL-class (41.7±14.2), JAK2 (60.0±15.5) and PAX5 (50.0±17.7) fusions when compared to the absence of fusions (70.5±5.4). Validation of our results in an independent cohort is ongoing. Summary/Conclusion: By using OGM, we show that the genomic landscape in ALL is complex and that ~46% of the IKZF1del/plus patients carry an established marker or other gene fusions which may be the underlying leukemogenic driver and contribute to an (un-) favorable outcome. Thus, a comprehensive genetic characterization could be valuable to further improve risk-adapted treatment stratification. Keywords: Acute lymphoblastic leukemia, Gene fusion, Ikaros, Pediatric

PURPOSE The AIEOP-BFM ALL 2009 protocol included, at the end of the induction phase, a randomized study of patients with high-risk (HR) ALL to investigate if an intensive exposure to pegylated L-asparaginase (PEG-ASNASE, 2,500 IU/sqm once a week × 4) on top of BFM consolidation phase IB allowed us to decrease minimal residual disease (MRD) and improve outcome. PATIENTS AND METHODS A total of 1,097 patients presented, from June 2010 to February 2017, with one or more of the following HR criteria: KMT2A::AFF1 rearrangement, hypodiploidy, prednisone poor response, poor bone marrow response at day 15 (Flow MRD ≥10%), or no complete remission (CR) at the end of induction. Of them, 809 (85.1%) were randomly assigned to receive (404) or not receive (405) four weekly doses of PEG-ASNASE. RESULTS By intention to treat (ITT) analysis, there was no significant difference in the proportion of patients with polimerase chain reaction MRD ≥5 × 10 −4 at the end of phase IB in the experimental versus control arm (13.9% v 17.0%, P = .25). The 5-year event-free survival (median follow-up 6.3 years) by ITT in the experimental and control arms was 70.4% (2.3) versus 75.0% (2.2; P = .18), and the 5-year overall survival was 81.5% (2.0) versus 84.0% (1.9; P = .25), respectively. The corresponding 5-year cumulative incidence of death in CR was 9.5% (1.5) versus 5.7% (1.2; P = .08), and that of relapse was 17.7% (1.9) versus 17.2% (1.9), respectively ( P = .94). Adverse reactions in phase IB occurred in 22.2% and 8.9% of patients in the experimental and control arm, respectively ( P < .001). CONCLUSION Additional PEG-ASNASE in phase IB did not translate into a benefit for decreasing relapse incidence but was associated with higher toxicity. Further improvements with conventional chemotherapy might be difficult in the context of intensive treatment protocols.

Background: One of the key questions in trial AIEOP-BFM ALL 2017 focused on the reduction of treatment-related complications by replacing parts of the highly intensive consolidation phase by two courses of Blinatumomab (Blina) in a prospective randomized trial. Herein, we report the toxicity profile of B-cell acute lymphoblastic leukemia (B-ALL) patients (pts) with high-risk (HR) characteristics pending final outcome data. Patients and Methods: 728 pts with HR B-ALL were enrolled from July 15, 2018 to October 31, 2022 in this multicenter trial run in 8 different countries. Patients were included in the HR group in view of the presence of at least one of the following criteria: induction failure; high levels of minimal residual disease (MRD) at day 15 of induction, or at the end of induction (EOI), or at the end of consolidation (EOC); presence of KMT2A::AFF1, of IKZF1+ and positive MRD at EOI (Stanulla et al, JCO 2018), TCF3::HLF, or hypodiploidy. B-ALL pts with one or several of these characteristics represented 20.8% of all pts aged 0-18 years of age with B-ALL. After a 4-drug induction phase, and two weeks of consolidation treatment, patients were randomized to receive Bortezomib in addition to standard consolidation (not part of this report). Before treatment continued with intensive consolidation block HR-1', MRD was analyzed in order to identify patients eligible for subsequent hematopoietic stem cell transplantation (HSCT) based on MRD results. After block HR-1', all B-ALL HR pts were randomized to receive either two courses of consolidation (blocks HR-2', and HR-3'), or two 28-day courses of Blina at 15µg/m 2/d administered by continuous IV infusion (with a 2-week treatment-free interval). Two intrathecal injections of methotrexate (MTX) for CNS prophylaxis were given on days 1 and 29 of each Blina course, respectively. Out of 728 HR B-ALL pts, 619 pts were eligible for randomization [reasons for non-eligibility were: Event (death or relapse) before randomization was due (26), Down syndrome (25; scheduled for a non-randomized intervention with Blina), presence of TCF3::HLF (3; could receive any alternative therapy including Blina), discontinuation/substantial change of preceding therapy (23) or other protocol exclusion criteria (32)]. 572 pts were randomized (92.4% of those eligible). One pt assigned to the experimental arm (EA) and 4 pts assigned to the control arm (CA) received the other arms, respectively. Results: Toxicity was evaluated in randomized pts according to treatment administered during the randomized phase, respectively, until next element started: 268 pts were treated in the control and 281 in the EA (see Table). 16 of them switched in/after the first Blina cycle to the HR blocks due to toxicity or poor response to Blina (due to increasing MRD, >=1x10 -4). Medically relevant adverse reactions of special interest (ARSI) according to MedDRA (specified in the protocol) were evaluated in the randomized treatment phases (CA: HR-2‘/HR-3‘ block, EA: 2 Blina cycles). ARSI were recorded in 61 of 268 pts in the CA (22.8%, 71 ARSI) and in 29 of 281 pts in the EA (10.3% [p<0.001], 33 ARSI); 3 of the 33 ARSI in the EA were related to HR blocks and were observed in 3 of the 16 pts that switched to HR blocks. Life-threatening SAR were seen in 14 pts of the control group (5.2%) and in no patient in the EA (p<0.001). Conclusions: Taken together, these results show for the first time in newly diagnosed pts with HR B ALL, the favorable toxicity profile previously reported with Blinatumomab in pediatric relapsed ALL (Locatelli F et al, JAMA 2021; Brown P et al, JAMA 2021). We have demonstrated that the toxicity profile of Blinatumomab is much more favorable as compared to the intensive chemotherapy approach using HR-blocks. If upcoming analyses of outcome data will show no inferiority of the EA, blinatumomab replacement of some of the intensive chemotherapy blocks will become the new standard of care for treatment if newly diagnosed patients with HR B-ALL.

The incidence of hypersensitivity reactions (HSRs) to PEG-asparaginase (PEG-ASNase) was evaluated in 6136 children with ALL enrolled in the AIEOP-BFM ALL 2009 study. Patients with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) were stratified as standard-risk/medium-risk (MR)/high-risk (HR) and those with T-ALL as non-High/HR. PEG-ASNase was administered intravenously at 2500 IU/sqm/dose. All patients received 2 PEG-ASNase doses in induction; thereafter non-HR versus HR patients received 1 versus 6 PEG-ASNase doses, respectively. After the single regular dose of PEG-ASNase at the beginning of delayed intensification, BCP-ALL-MR patients were randomized to receive 9 additional PEG-ASNase doses every 2 weeks (experimental arm [EA]) versus none (standard arm [SA]); HR patients were randomized to receive, in consolidation, 4 weekly PEG-ASNase doses (EA) versus none (SA). The HSR cumulative incidence (CI) was estimated adjusting for competing risks. An HSR occurred in 472 of 6136 (7.7%) patients. T-non- HR/BCP-Standard-Risk, BCP-MR-SA, BCP-MR-EA, HR-SA and HR-EA patients had 1-year-CI-HSR (±SE) rates of 5.2% (0.5), 5.2% (0.5), 4.0% (0.8), 20.2% (1.2), and 6.4% (1.3), respectively. The randomized intensification of PEG-ASNase did not significantly impact on HSR incidence in BCP-MR patients (1-y-CI-HSR 3.8% [0.8] versus 3.2% [0.6] in MR-EA versus MR-SA; P = 0.55), while impacted significantly in HR patients (1-y-CI-HSR 6.4% [1.3] versus 17.9% [1.8] in HR-EA and HR-SA, respectively; P < 0.001). The CI-HSR was comparable among non-HR groups and was not increased by a substantial intensification of PEG-ASNase in the BCP-MR-EA group whilst it was markedly higher in HR-SA than in HR-EA patients, suggesting that, in such a chemotherapy context, a continuous exposure to PEG-ASNase reduces the risk of developing an HSR.

Although initial central nervous system (CNS) involvement is rarely detected in childhood acute lymphoblastic leukemia (ALL), risk-adapted CNS-directed therapy is essential for all patients. Treatment intensity depends on the initial CNS status. In the AIEOP-BFM ALL 2009 trial, patients with cytomorphologic detection of leukemic blasts in initial cerebrospinal fluid were classified as CNS2 or CNS3 and received five intrathecal doses of methotrexate (MTX) in induction therapy compared to patients with CNS1 status (no blasts detected) who received three doses. The impact of additional intrathecal (IT) MTX on systemic toxicity in induction therapy is unknown. Between June 1st 2010 and February 28th 2017, a total of 6,136 ALL patients aged 1-17 years were enrolled onto the AIEOP-BFM ALL 2009 trial. The effect of three versus five doses of IT MTX during induction therapy on the incidence of severe infectious complications was analyzed. Among 4,706 patients treated with three IT MTX doses, 77 (1.6%) had a life-threatening infection during induction as compared to 59 of 1,350 (4.4%) patients treated with five doses (P<0.001; Odds Ratio 2.86 [95% Confidence Interval 1.99-4.13]). In a multivariate regression model, treatment with additional IT MTX proved to be the strongest risk factor for life-threatening infections (Odds Ratio 2.85 [1.96-4.14]). Fatal infections occurred in 16 (0.3%) and 38 (1.6%) patients treated with three or five IT MTX doses, respectively (P<0.001). As the relevance of additional intrathecal MTX in induction for relapse prevention in CNS2 patients is unclear, doses of intrathecal therapy have been reduced for these patients. (Clinicaltrials.gov identifiers: NCT01117441 and NCT00613457).

Children with Down syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with lower survival than in non-DS-ALL. It is known that cytogenetic abnormalities common in childhood ALL are less frequent in DS-ALL, while other genetic aberrancies (ie, CRLF2 overexpression and IKZF1 deletions) are increased. A possible cause for the lower survival of DS-ALL that we herewith evaluated for the first time was the incidence and prognostic value of the Philadelphia-like (Ph-like) profile and the IKZF1plus pattern. These features have been associated with poor outcome in non-DS ALL and therefore introduced in current therapeutic protocols. Forty-six out of 70 DS-ALL patients treated in Italy from 2000 to 2014 displayed Ph-like signature, mostly characterized by CRLF2 (n = 33) and IKZF1 (n = 16) alterations; only 2 cases were positive for ABL-class or PAX5-fusion genes. Moreover, in an Italian and German joint cohort of 134 DS-ALL patients, we observed 18% patients positive for IKZF1plus feature. Ph-like signature and IKZF1 deletion were associated with poor outcome (cumulative incidence of relapse: 27.7 ± 6.8% versus 13 ± 7%; P = 0.04 and 35.2 ± 8.6% versus 17 ± 3.9%; P = 0.007, respectively), which further worsens when IKZF1 deletion was co-occurring with P2RY8::CRLF2, qualifying for the IKZF1plus definition (13/15 patients had an event of relapse or treatment-related death). Notably, ex vivo drug screening revealed sensitivity of IKZF1plus blasts for drugs active against Ph-like ALL such as Birinapant and histone deacetylase inhibitors. We provided data in a large setting of a rare condition (DS-ALL) supporting that these patients, not associated with other high-risk features, need tailored therapeutic strategies.

Background: Asparaginase (ASNase) has become a key component of treatment for acute lymphoblastic leukemia (ALL). The ability of this agent to eliminate leukemic blasts by utilizing the special feature that such cells are largely unable to synthesize the otherwise non-essential amino acid asparagine means that blasts cannot survive in an environment in which this amino acid has been depleted. Currently, there are three ASNase products commercially available sharing the same mechanism of action. The oldest one is the purified native enzyme extracted from E. coli. This product was further developed and refined by conjugation with polyethylene glycol (PEG-L-Asparaginase, PEG-ASNase) to prolong half-life and to reduce immunogenicity. A third ASNase product derived from the bacterium Erwinia chrysanthemi (Erwinase) is structurally different from the E. coli ASNase products. The main reason for limitation in the use of ASNase is the rather frequent occurrence of hypersensitivity reactions (HSR). Such reactions require the replacement of the ASNase product used with alternative preparations. The incidence of clinical HSR to ASNase varies with the type of ASNase product, the treatment schedule (number of ASNase doses, extended ASNase-free intervals) and the administration route (intravenously [IV] or intramuscularly [IM]). This retrospective study was primarily performed to demonstrate the dynamics and the interdependence when using ASNase in a complex multiagent treatment protocol. It aimed to evaluate the incidence of clinically apparent HSR in the ALL-BFM 2000 trial which was part of the cooperative trial AIEOP-BFM ALL 2000. Methods: Clinical hypersensitivity reactions (HSR) to native E. coli ASNase were retrospectively analyzed in a cohort of children with acute lymphoblastic leukemia (ALL) focusing on the impact of concurrent chemotherapy schedules in the risk-adapted and randomized treatment protocol and evaluating the prognostic relevance of asparaginase discontinuation. Patients: Patients with ALL aged 1-18 years were treated risk-stratified with standard-risk (SR), medium-risk (MR) and high-risk (HR) regimens in trial ALL-BFM 2000. Schedule of intravenously administered native E. coli ASNase varied by risk group and randomization arm. Randomizations compared the glucocorticoids prednisolone and dexamethasone in induction and different reintensification regimens (Möricke A et al, BLOOD 127(17): 2101-2112 (2016)). In this trial, native E. coli ASNase was administered IV as first-line product during the induction and reinduction phases. In the high-risk arm, the phase of intensified consolidation included additional E. coli ASNase doses. PEG-ASNase (IV) or Erwinase (IM) were used as second- or third-line products after HSR occurring with native E. coli ASNase or the respective second-line product. Results: HSR occurred in 40% (492/1207) of all evaluable patients. One-year cumulative incidence (CI-HSR±standard error) was 42.3±1.5% (40.6±2.5% in SR, 37.0±1.9% in MR, 65.2±3.8% in HR). Dexamethasone-treated SR and MR patients had significantly higher CI-HSR (48.5±4.3% and 45.9±3.2%) than prednisolone-treated patients (32.3±3.7% [p=0.007] and 31.2±2.9% [p<0.001]); no such difference was seen among HR patients who underwent multiple exposures to the drug (Fig. 1). Post-induction randomizations showed that one week of dexamethasone before asparaginase re-exposition in reinduction did not protect from clinical HSR in the SR/MR arm. Randomization in HR revealed an effect of the type of concurrent chemotherapeutic drugs on the risk of HSR. Discontinuation of ASNase occurred in 6.1% of all patients. No disadvantage in leukemia outcome could be detected for these patients. Conclusion: The results in our study demonstrate the complex interdependencies contributing to the risk of HSR to E. coli asparaginase in a multiagent chemotherapy protocol. The differential effects of dexamethasone and prednisolone in induction generated new hypotheses about the role of glucocorticoids in sensitization or achievement of immune tolerance to asparaginase. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal