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Introduction: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. Methods: In this study, we performed a genomic analysis of diagnosis–relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. Results: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. Conclusion: Comprehensive genomic characterization of diagnosis–relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Introduction: Since the beginning of COVID-19 pandemic, it is known that the severe course of the disease occurs mostly among the elderly, whereas it is rare among children and young adults. Comorbidities, in particular, diabetes and hypertension, clearly associated with age, besides obesity and smoke, are strongly associated with the need for intensive treatment and a dismal outcome. A weaker immunity of the elderly has been proposed as a possible explanation of this uneven age distribution. Thus, there is concern that children treated for cancer may allso be at risk for an unfavourable course of infection. Along the same line, anecdotal information from Wuhan, China, mentioned a severe course of COVID-19 in a child treated for leukaemia. Aim and methods: We made a flash survey on COVID-19 incidence and severity among children on anticancer treatment. Respondents were asked by email to fill in a short Web-based survey. Results: We received reports from 25 countries, where approximately 10,000 patients at risk are followed up. At the time of the survey, more than 200 of these children were tested, nine of whom were positive for COVID-19. Eight of the nine cases had asymptomatic to mild disease, and one was just diagnosed with COVID-19. We also discuss preventive measures that are in place or should be taken and treatment options in immunocompromised children with COVID-19. Conclusion: Thus, even children receiving anticancer chemotherapy may have a mild or asymptomatic course of COVID-19. While we should not underestimate the risk of developing a more severe course of COVID-19 than that observed here, the intensity of preventive measures should not cause delays or obstructions in oncological treatment.

The safety and efficacy of blinatumomab, a CD3/CD19-directed bispecific molecule, were examined in an open-label, single-arm, expanded access study (RIALTO). Children (.28 days and, 18 years) with CD191 relapsed/refractory B-cell precursor acute lymphoblastic leukemia (R/R B-ALL) received up to 5 cycles of blinatumomab by continuous infusion (cycle: 4 weeks on/2 weeks off). The primary end point was incidence of adverse events. Secondary end points included complete response (CR) and measurable residual disease (MRD) response within the first 2 cycles and relapse-free survival (RFS), overall survival (OS), and allogeneic hematopoietic stem cell transplant (alloHSCT) after treatment. At final data cutoff (10 January 2020), 110 patients were enrolled (median age, 8.5 years; 88% had ≥5% baseline blasts). A low incidence of grade 3 or 4 cytokine release syndrome (n = 2; 1.8%) and neurologic events (n = 4; 3.6%) was reported; no blinatumomab-related fatal adverse events were recorded. The probability of response was not affected by the presence of cytogenetic/molecular abnormalities. Median OS was 14.6 months (95% confidence interval [CI]: 11.0-not estimable) and was significantly better for MRD responders vs MRD nonresponders (not estimable vs 9.3; hazard ratio, 0.18; 95% CI: 0.08-0.39). Of patients achieving CR after 2 cycles, 73.5% (95% CI: 61.4%- 83.5%) proceeded to alloHSCT. One-year OS probability was higher for patients who received alloHSCT vs without alloHSCT after blinatumomab (87% vs 29%). These findings support the use of blinatumomab as a safe and efficacious treatment of pediatric R/R B-ALL. This trial was registered at www.clinicaltrials.gov as #NCT02187354.

Relapse of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may occur in the central nervous system (CNS). Most clinical trials of CAR T-cell therapy excluded patients with active CNS leukemia, partially for concerns of neurotoxicity. Here, we report an international study of fifty-five children and adolescents who received CAR T-cell therapy for relapsed BCP-ALL with CNS involvement at the time of referral. All patients received bridging therapy, 16 still having active CNS disease at the time of lymphodepletion. Twelve patients received CD28-based CAR T-cells, 9 being subsequently treated with allogeneic hematopoietic stem-cell transplantation (allo-HSCT). Forty-three patients received 4-1BB-based CAR T-cells. Cytokine-release syndrome (CRS) and neurotoxicity occurred in 65% and 38% of patients, respectively, more frequently following treatment with CD28-based CARs. Fifty-one of 54 evaluable patients (94%) achieved complete response following this therapy. Relapse occurred in 22 patients: 19/43 following 4-1BB-based CARs (12 CNS relapses), and 3/12 after CD28-based CARs with subsequent HSCT (no CNS relapse). Patients treated with tisagenlecleucel for an isolated CNS relapse had a high incidence of a subsequent CNS relapse (6 of 8). CAR T-cells were found to be effective in this cohort, though the risk of CNS relapse was not completely mitigated by this approach.

Despite improved outcomes achieved in the last decades for children with newly diagnosed leukaemia and lymphoma, treatment of patients with refractory/relapsed disease remains a challenge. The cure rate is still unsatisfactory and often achieved at the cost of significant morbidity. Exploring treatment with novel agents should offer less toxic therapeutic options, without compromising efficacy. Bispecific and antibody–drug conjugates targeting CD19 and CD22 (blinatumomab and inotuzumab ozogamicin) play an important role in the treatment of relapsed and refractory B-cell precursor acute lymphoblastic leukaemia (BCP-ALL); antibodies targeting CD123 and CD38 are also under investigation for acute myeloid leukaemia (AML) and T-ALL, respectively. Targeted therapy with small molecules is of primary importance for specific genetic subtypes, such as BCR-ABL-positive ALL, FLT3-ITD AML and anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma. KMT2A-directed targeted therapy with menin inhibitors holds promise to be of relevance in KMT2A-rearranged leukaemias, known to have dismal prognosis. Target inhibition in cellular pathways such as BCL-2, RAS, MEK, Bruton's tyrosine kinase, JAK-STAT or CDK4/CDK6 inhibition may be suitable for different diseases with common mutated pathways. Nevertheless, development and approval of new agents for paediatric cancers lags behind adult therapeutic options. New regulations were implemented to accelerate drug development for children. Considering the number of oncology medicinal products available for adults and the rarity of paediatric cancers, prioritisation based on scientific evidence and medical need, as well as international collaboration, is critical. Herein, we review the current status of drug development for children with leukaemia and lymphoma, excluding cellular therapy despite its well-known significance.

Background: Children aged younger than 3 years were excluded from the ELIANA phase 2 trial of tisagenlecleucel in children with acute lymphoblastic leukaemia. The feasibility, safety, and activity of tisagenlecleucel have not been defined in this group, the majority of whom have high-risk (KMT2A-rearranged) infant acute lymphoblastic leukaemia and historically poor outcomes despite intensification of chemotherapy, and for whom novel therapies are urgently needed. We aimed to provide real-world outcome analysis of the feasibility, activity, and safety of tisagenlecleucel in younger children and infants with acute lymphoblastic leukaemia. Methods: We did an international, multicentre, retrospective cohort study at 15 hospitals across ten countries in Europe. Eligible patients were children aged younger than 3 years at screening between Sept 1, 2018, and Sept 1, 2021, who were screened for tisagenlecleucel therapy for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia according to licensed indications. Patients received a single intravenous infusion of tisagenlecleucel. We tracked chimeric antigen receptor T-cell therapy outcomes using a standardised data reporting form. Overall survival, event-free survival, stringent event-free survival, B-cell aplasia, and toxicity were assessed in all patients who received a tisagenlecleucel infusion. Findings: 38 eligible patients were screened, of whom 35 (92%) received a tisagenlecleucel infusion. 29 (76%) of 38 patients had KMT2A-rearranged acute lymphoblastic leukaemia, and 25 (66%) had relapsed after previous allogeneic haematopoietic stem-cell transplantation (HSCT). Patients had previously received a median of 2 lines (IQR 2–3) of (non-HSCT) therapy. Seven (18%) of 38 patients had received inotuzumab and 14 (37%) had received blinatumomab. After a median of 14 months (IQR 9–21) of follow-up, overall survival at 12 months after tisagenlecleucel infusion was 84% (64–93; five patients had died), event-free s

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Outcome of 333 children with acute myeloid leukaemia relapsing after a first allogeneic haematopoietic stem cell transplantation was analyzed. Four-year probability of overall survival (4y-pOS) was 14%. 4y-pOS for 122 children receiving a second haematopoietic stem cell transplantation was 31% and 3% for those that did not (P = <0·0001). Achievement of a subsequent remission impacted survival (P = <0·0001). For patients receiving a second transplant survival with or without achieving a subsequent remission was comparable. Graft source (bone marrow vs. peripheral blood stem cells, P = 0·046) and donor choice (matched family vs. matched unrelated donor, P = 0·029) positively impacted survival after relapse. Disease recurrence and non-relapse mortality at four years reached 45% and 22%.

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

Outcome of 333 children with acute myeloid leukaemia relapsing after a first allogeneic haematopoietic stem cell transplantation was analyzed. Four-year probability of overall survival (4y-pOS) was 14%. 4y-pOS for 122 children receiving a second haematopoietic stem cell transplantation was 31% and 3% for those that did not (P = <0·0001). Achievement of a subsequent remission impacted survival (P = <0·0001). For patients receiving a second transplant survival with or without achieving a subsequent remission was comparable. Graft source (bone marrow vs. peripheral blood stem cells, P = 0·046) and donor choice (matched family vs. matched unrelated donor, P = 0·029) positively impacted survival after relapse. Disease recurrence and non-relapse mortality at four years reached 45% and 22%.

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Wound healing assays are extensively used to study tissue repair mechanisms; they are typically performed by means of physical (i.e., mechanical, electrical, or optical) detachment of the cells in order to create an open space in which live cells can lodge. Herein, an advanced system based on extensive photobleaching-induced apoptosis; providing a powerful tool to understand the repair response of lung epithelial tissue, consisting of a small injury area where apoptotic cells are still intact, is developed. Notably, the importance of epithelial mechanics and the presence of macrophages during the repair can be understood. The findings reveal that individual epithelial cells are able to clear the apoptotic cells by applying a pushing force, whilst macrophages actively phagocytose the dead cells to create an empty space. It is further shown that this repair mechanism is hampered when carbon nanotubes (CNTs) are introduced: formation of aberrant (i.e., thickening) F-actins, maturation of focal adhesion, and increase in traction force leading to retardation in cell migration are observed. The results provide a mechanistic view of how CNTs can interfere with lung repair.

Purpose Somatic deletions that affect the lymphoid transcription factor-coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 6 6% compared with 7965% in patients with IKZF1 deletion who did not fulfill the IKZF1plus definition and 87 ± 1% in patients who lacked an IKZF1 deletion (P ≤.001). Respective 5-year cumulative relapse incidence rates were 44±6%, 11±4%, and 10±1%(P ≤.001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1plus. The IKZF1plus prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1plus patients was 94 6 5% versus 40 6 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients (P ≤.001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% (P ≤.001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BF