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A significant proportion of events in paediatric acute myeloid leukaemia (AML) are caused by resistant disease (RD). We investigated clinical and biological characteristics in 66 patients with RD from 1013 children with AML registered and treated according to the NOPHO-AML 93, NOPHO-AML 2004, DB AML-01 and NOPHO-DBH AML 2012 protocols. Risk factors for RD were age10 years or older and a white-blood-cell count (WBC) of 100 × 10 9 /L or more at diagnosis. The five-year overall survival (OS) was 38% (95% confidence interval [CI]: 28%-52%). Of the 63 children that received salvage therapy with chemotherapy, 59% (N = 37) achieved complete remission (CR) with OS 57% (95% CI: 42%-75%) compared to 12% (95% CI: 4%-35%) for children that did not achieve CR. Giving more than two salvage chemotherapy courses did not increase CR rates. OS for all 43 patients receiving allogeneic haematopoietic stem cell transplantation (HSCT) was 49% (95% CI: 36%-66%). Those achieving CR and proceeding to HSCT had an OS of 56% (95% CI: 41%-77%, N = 30). This study showed that almost 40% of children with primary resistant AML can be cured with salvage therapy followed by HSCT. Children that did not achieve CR after two salvage courses with chemotherapy did not benefit from additional chemotherapy.

The clinical presentation of chronic myeloid leukemia (CML) at diagnosis differs in children compared to adults. At younger age, anemia appears to be frequent at diagnosis, but its prevalence and its impact on prognosis are not well known. In the International Registry of Childhood CML, we selected children and adolescents in chronic phase at diagnosis of CML and treated upfront with imatinib. We examined their hemoglobin level at diagnosis according to the WHO grades to assess the prevalence of anemia and its impact on response to tyrosine kinase inhibitors (TKIs). Data on 430 patients were included. Anemia at diagnosis was observed in 350 patients (81%), with a mean hemoglobin level of 96.4 g/l (SD 23.6). Among them, 182 patients (52%) presented with moderate anemia and 110 (31%) with severe anemia while 58 (17%) had mild anemia. Compared with mild and no anemia, moderate and severe forms were significantly associated with younger age at diagnosis, asthenia, splenomegaly, and increased leukocyte and basophil counts. Delays in achieving major and deep molecular responses were significantly increased for patients with moderate and severe anemia, and also failure of imatinib treatment was more frequent in these two sub-cohorts. However, hemoglobin level was not significantly associated with survival. Anemia at diagnosis of pediatric CML was frequent and may be considered as a prognostic factor.

Background: Analysis of measurable residual disease (MRD) is increasingly being implemented in the clinical care of children and adults with acute myeloid leukemia (AML). However, MRD methodologies differ and discordances in results lead to difficulties in interpretation and clinical decision-making. The aim of this study was to compare results from reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiparameter flow cytometry (MFC) in childhood AML and describe the kinetics of residual leukemic burden during induction treatment. Methods: In 15 children who were treated in the NOPHO-AML 2004 trial and had fusion transcripts quantified by RT-qPCR, we compared MFC with RT-qPCR for analysis of MRD during (day 15) and after induction therapy. Eight children had RUNX1::RUNX1T1, one CBFB::MYH11 and six KMT2A::LLT3. Results: When ≥0.1% was used as cut-off for positivity, 10 of 22 samples were discordant. The majority (9/10) were MRD positive with RT-qPCR but MRD negative with MFC, and several such cases showed presence of mature myeloid cells. Fusion transcript expression was verified in mature cells as well as in CD34 expressing cells sorted from diagnostic samples. Conclusions: Measurement with RT-qPCR suggests slower response kinetics than indicated from MFC, presumably due to the presence of mature cells expressing fusion transcript. The prognostic impact of early measurements with RT-qPCR remains to be determined.

Inborn hemolytic anemia requiring frequent blood transfusions can be a life-threatening disease. Treatment, besides blood transfusion, includes iron chelation for prevention of iron accumulation due to frequent blood transfusions. We present the results of a clinical investigation where the proband was diagnosed with severe hemolytic anemia of unknown origin soon after birth. Transfusion was required every 4–6 weeks. After whole exome sequencing of the proband and his parents as well as a healthy sibling, we established that the proband had a compound heterozygous state carrying two rare variants in the erythrocytic spectrin gene, SPTA1. The maternal allele was a stop mutation (rs755630903) and the paternal allele was a missense mutation (rs375506528). The healthy sibling had the paternal variant but not the maternal variant. These rare variants of SPTA1 most likely account for the hemolytic anemia. A severely reduced osmotic resistance in the erythrocytes from the proband was demonstrated. Splenectomy considerably improved the hemolytic anemia and obviated the need for blood transfusion despite the severe clinical presentation.

PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide). MATERIALS AND METHODS MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10−4), and high (≥ 5 × 10−4). RESULTS EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively ( P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively ( P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively ( P < .0001), while for myeloid-style–treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9). CONCLUSION This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.

Background: Chronic myeloid leukaemia (CML) is a rare disease in children. The frequency and outcome of children evolving to accelerated phase (AP) or blastic phase (BP) under treatment with imatinib is unknown. The aim of the current study is to assess the inci-dence of progression from CML in chronic phase with imatinib frontline in a paediatric setting and describe the management and outcome of these patients.Patients and methods: In the I-CML-Ped Study database (www.clinicaltrials.gov, #NCT01281735), 19 of 339 paediatric patients in chronic phase treated with imatinib in the frontline evolved to CML-AP or CML-BP.Results: With a median follow-up of 38 months (range: 2-190 months), the cumulative inci-dence of progression at 1 and 3 years was 3% (confidence interval [CI] 95%: 1-5%) and 7% (CI 95%: 4-11%), respectively. We observed a large predominance of lymphoid-BP (70%) over myeloid-BP (30%) with imatinib in frontline therapy. Sixteen patients underwent haemato-poietic stem cell transplantation, and eight were treated with a tyrosine kinase inhibitor after transplant. Only the transplanted patients are alive. The 5-year overall survival rate of children with CML-AP/BP is 44%, with no statistical difference between the lymphoid-BP and myeloid-BP outcome.Conclusion: Children evolving to AP or BP under treatment with imatinib have a very poor prognosis with an overall survival under 50%, much worse than children with advanced phase at diagnosis. (c) 2020 Elsevier Ltd. All rights reserved.

Background The outcome of infants with KMT2A-germline ALL is much better than of infants with KMT2A-rearranged ALL, but still worse than of non-infant ALL patients. Here, we describe the outcome and prognostic factors for infants with KMT2A-germline ALL treated on Interfant-06 protocol. Methods 167 infants with KMT2A-germline ALL were enrolled in Interfant-06. Univariate analysis on prognostic factors (age, WBC at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI) (n=163). Bone marrow minimal residual disease (MRD) was measured in 73 patients, by real-time quantitative PCR of immunoglobulin genes and/or T-cell receptor genes, at various time points (EOI, n= 68, end of consolidation (EOC) n= 56, and before OCTADAD (n=57)). MRD results were classified as negative, intermediate (< 5*10 -4), and high (≥5*10 -4). Genetic data on NUTM1 rearrangements and MRD were available in 53 patients. Results The 6-year event free survival [SE] and overall survival [ SE] was 73.9% (3,6) and 87.2% (2.7). 28 of 31 (90%) relapses occurred early, within 2 years of diagnosis. Treatment related mortality was 3.6%. Age 6 months of age (p=0.04). Of the MRD timepoints, MRD at end of induction (EOI) was most prognostic for outcome. At EOI, 76.5% (n=52/68) of patients were either MRD negative (41.2%, n=28) or intermediate MRD (35.3%, n=24). 23.5% (n=16) had high EOI MRD, which was associated with significantly lower 6-year DFS (SE), compared to patients with intermediate or negative EOI MRD (61.4% (12.4), 76.4% (11.3) and 87.9% (6.6), respectively; p=0.02, Figure 1a). At EOC, 55.4% (n=31/56) of patients were MRD negative. Outcome by MRD levels at EOC was not significantly different (p=0.24); the 6-year DFS (SE) of negative and intermediate EOC MRD patients was 89.0% (6.0) and 72.7 % (10.6), respectively, while only one of the 5 patients with high EOC MRD relapsed in BM and CNS (Figure 1b). MRD data for both EOI and EOC were available for 55 patients. Of these patients, 18 were MRD negative at EOI and EOC, with a 6-year DFS 93.3% (SE, 6.4). Five patients had negative EOI MRD, but showed intermediate EOC MRD levels; none of these patients relapsed. There were 12 of 55 patients who were MRD positive at EOI and became MRD negative at EOC. These patients had a 6-year DFS of 82.5 % (SE, 11.3). Patients with detectable disease at both timepoints had a 6-year DFS of 68.3% (SE,10.8) (n=20, Figure 1c). At the end of MARMA (TP5), 77.2% (n=44/57) of patients were MRD negative. MRD at TP5 was significantly related to DFS (Figure 1d); the 6-year DFS was 89.6% (SE, 5.0) for MRD negative patients, compared to 65.6% (SE, 14.0) for patients with intermediate MRD levels (p= 0.039). In the current study, NUTM1 status was known in 53 patients with MRD data. Of them, 13 harbored a NUTM1-rearrangement. Seven out of 11 (63.6%) were aged< 6 months and 6 out of 42 (14.3%) were older. None of them relapsed, despite positive EOI MRD detected in 8 cases. Conclusion We conclude that young age at diagnosis and low EOI MRD are favorable prognostic factors in infants with KMT2A-germline ALL. However, the prognostic value of MRD is not as strong as in infants with KMT2A-rearranged ALL or older children with ALL. This can partly be explained by the differences in genetic makeup of infants with KMT2A-germline ALL, thus supporting the hypothesis that in the future a combined MRD- and genetic-based stratification of KMT2A-g infants might be considered Figure 1 Figure 1. Disclosures Biondi: Colmmune: Honoraria; Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Silverman: Takeda, Servier, Syndax, Jazz Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Schrappe: Novartis: Honoraria, Other: research support; SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Servier: Honoraria, Other: research support.

Infants with acute lymphoblastic leukaemia (ALL) have a high frequency of central nervous system (CNS) involvement. Flow cytometric analysis of cerebrospinal fluid (CSF) was recently demonstrated to be a sensitive method for detecting CNS involvement in childhood ALL. In the present study, CSF from 14 infants was collected at routine lumbar punctures and analysed by multicolour flow cytometry. At initial diagnosis, leukaemic blasts were detected in CSF by flow cytometry in 11 patients (78·6%) compared to seven patients (50%) by cytospin. Larger studies are needed to determine if CSF flow cytometry has prognostic value in infant ALL.

Purpose Despite improved treatment and care, children and adolescents diagnosed with cancer continue to die, while many of those cured are burdened by treatment-related sequelae. The best clinical management of children and adolescents with cancer depends on healthcare professionals with various skills and expertise. Complex treatment, care and rehabilitation require collaboration between healthcare professionals. The purpose of this scoping review is to identify and evaluate existing interprofessional education in paediatric cancer. Methods We utilised the scoping review methodology and searched PubMed, Scopus and Education Resources Information Center. Inclusion criteria were postgraduate studies targeting more than one profession and evaluation of the educational intervention. We applied Kirkpatrick’s modified interprofessional education outcomes model to systematise outcomes. Results Of 418 references, nine studies fulfilled the inclusion criteria. The design, strategy and content of all the studies were heterogeneous. None of the interprofessional educations systematically evaluated knowledge, skills, attitudes or the effects on patient outcomes or quality of care. Conclusion There is a lack of well-structured, interprofessional education in paediatric cancer that has undergone evaluation. Paediatric cancer may benefit from systematic education and evaluation frameworks since interprofessional education could potentially strengthen the treatment, care and rehabilitation for children and adolescents with cancer. Electronic supplementary material The online version of this article (10.1007/s00520-019-04856-4) contains supplementary material, which is available to authorized users.

Liposomal amphotericin-B (L-AmB) prophylaxis is used in children with leukemia when azoles are contraindicated, but its effect is debated. We reviewed cases of invasive aspergillosis despite L-AmB 2.5 mg/kg twice weekly in children with high-risk leukemia during 2012-2019. Ten (16%) of 62 children had proven or probable aspergillosis. Thus, L-AmB prophylaxis offered insufficient protection for Aspergillus, in particular for Aspergillus flavus.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease composed of clinically relevant subtypes defined by recurrent cytogenetic aberrations. The majority of the aberrations used in risk grouping for treatment decisions are extensively studied, but still a large proportion of pediatric AML patients remain cytogenetically undefined and would therefore benefit from additional molecular investigation. As aberrant epigenetic regulation has been widely observed during leukemogenesis, we hypothesized that DNA methylation signatures could be used to predict molecular subtypes and identify signatures with prognostic impact in AML. To study genome-wide DNA methylation, we analyzed 123 diagnostic and 19 relapse AML samples on Illumina 450k DNA methylation arrays. We designed and validated DNA methylation-based classifiers for AML cytogenetic subtype, resulting in an overall test accuracy of 91%. Furthermore, we identified methylation signatures associated with outcome in t(8, 21)/RUNX1-RUNX1T1, normal karyotype, and MLL/KMT2A-rearranged subgroups (p &lt, 0.01). Overall, these results further underscore the clinical value of DNA methylation analysis in AML.

Overexpressed genes may be useful for monitoring of measurable residual disease (MRD) in patients with childhood acute myeloid leukemia (AML) without a leukemia-specific target. The normal expression of five leukemia-associated genes (SPAG6, ST18, MSLN, PRAME, XAGE1A) was defined in children without hematologic disease (n = 53) and children with suspected infection (n = 90). Gene expression at AML diagnosis (n=50) and during follow-up (n = 21) was compared with child-specific reference values. At diagnosis, 34/50 children (68%) had high expression of at least one of the five genes, and so did 16/31 children (52%) without a leukemia-specific target. Gene expression was quantified in 110 peripheral blood (PB) samples (median, five samples/patient; range, 1 to 10) during follow-up in 21 patients with high expression at diagnosis. All nine patients with PB sampling performed within 100 days of disease recurrence displayed overexpression of SPAG6, ST18, PRAME, or XAGE1A at a median of 2 months (range, 0.6 to 9.6 months) before hematologic relapse, whereas MSLN did not reach expression above normal prior to hematologic relapse. Only 1 of 130 (0.8%) follow-up analyses performed in 10 patients in continuous complete remission had transient expression above normal. SPAG6, ST18, PRAME, and XAGE1A expression in PB may predict relapse in childhood AML patients and facilitate MRD monitoring in most patients without a leukemia-specific target.

Acute myeloid leukemia (AML) accounts for 15% to 20% of childhood leukemias. Because of high-intensive therapy, up to 5% of patients suffer from treatment-related mortality (TRM). Abdominal complications are frequent, however, literature on this subject is sparse. We aimed to characterize severe abdominal pain (AP) and hyperbilirubinemia experienced by pediatric AML patients treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO)-AML 2004 protocol (n=313). Patients were censored at hematopoietic stem cell transplantation and relapse. Toxicity information was collected prospectively. Additional information was requested retrospectively from the treating centers. Sixteen episodes of hyperbilirubinemia and 107 episodes of AP were reported. The treating centers deemed infection (30%) and typhlitis (18%) as the most frequent causes of AP. Six patients developed appendicitis (2%). Patients experiencing concurrent AP and sepsis had a high risk of TRM (36%, n=4). Eighty percent of episodes with hyperbilirubinemia fulfilled the European Society for Bone and Marrow Transplantation criteria for sinusoidal obstruction syndrome. In conclusion, abdominal complications were frequent with infection considered the predominate cause. Most patients with hyperbilirubinemia fulfilled the criteria for sinusoidal obstruction syndrome. AML treatment might be associated with appendicitis. Patients suffering from concurrent AP and sepsis had a high risk of TRM indicating that high awareness of abdominal complications is essential to reduce mortality, especially during sepsis.

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence ofMRD was measured in 249 infants by DNA-based polymerase chain reaction of rearrangedEOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively (This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9%

Acute lymphoblastic leukemia (ALL) is a rare disease in infants. Asparaginase is an essential part of the treatment, and there Acute is a need to evaluate the efficiency and safety of this drug in this age group. We evaluated the pharmacokinetics of intramuscularly administered native E. coli asparaginase (Asparaginase Medac®) and PEG-asparaginase (Oncaspar®) as well as hypersensitivity reactions during treatment in Interfant-06 ( www.clinicaltrials.gov : NCT01025804). All patients without hypersensitivity had sufficiently high enzyme activity levels during treatment with both preparations. Patients with hypersensitivity reactions during treatment, characterized by the presence of either or not of clinical symptoms and no measurable enzyme activity, received ineffective therapy. For optimization of the bad prognosis in infant ALL, therapeutic drug monitoring should be performed for identification of patients who should be switched to a different asparaginase preparation because of inactivation of the drug.

Data on occurrence, genetic characteristics and prognostic impact of complex and monosomal karyotype (CK/MK) in children with acute myeloid leukaemia (AML) are scarce. We studied CK and MK in a large unselected cohort of childhood AML patients diagnosed and treated according to Nordic Society for Paediatric Haematology and Oncology (NOPHO)-AML protocols 1993-2015. In total, 800 patients with de novo AML were included. CK was found in 122 (15%) and MK in 41 (5%) patients. CK and MK patients were young (median age 2·1 and 3·3 years, respectively) and frequently had FAB M7 morphology (24% and 22%, respectively). Refractory disease was more common in MK patients (15% vs. 4%) and stem cell transplantation in first complete remission was more frequent (32% vs. 19%) compared with non-CK/non-MK patients. CK showed no association with refractory disease but was an independent predictor of an inferior event-free survival (EFS; hazard ratio [HR] 1·43, P = 0·03) and overall survival (OS; HR 1·48, P = 0·01). MK was associated with a poor EFS (HR 1·57, P = 0·03) but did not show an inferior OS compared to non-MK patients (HR 1·14, P = 0·62). In a large paediatric cohort, we characterized AML with non-recurrent abnormal karyotype and unravelled the adverse impact of CK and MK on prognosis.

Genetic rearrangements involving the KMT2A gene (KMT2A-R) are seen in around 10% of acute leukemia overall. KMT2A-R occurs in all ages and usually correlates with high-risk clinical features, in particular in infants aged 0-12 months of age with acute lymphoblastic leukemia (ALL). To uncover age- and leukemia-subtype specific molecular patterns in KMT2A-R ALL and acute myeloid leukemia (AML), we performed whole genome (WGS), whole exome (WES), and RNA-sequencing on a well-annotated Nordic KMT2A-R cohort of 104 patients, including infant ALL (n=33), childhood ALL (n=18), adult ALL (n=15), and pediatric AML (n=38) patients. For 77 patients, we performed WGS (40x) at diagnosis and remission as well as WES (140x) on the diagnostic sample, and remaining patients underwent WES only (n=27). RNA-sequencing was performed on 58 cases with available RNA. Twenty-two genes were recurrently altered and remarkably, NRAS, KRAS, FLT3, PAX5, TP53, CDKN2A/B and IKZF1 accounted for 70% of mutations. The landscape of mutations suggested the presence of leukemia and age-specific associations with MYST4, PTPN11, and SETD2 uniquely altered in AML and PIK3CD, DNAH11, NOTCH1, CSMD3 and CDKN2A/B in ALL. Some genes were mutated in both KMT2A-R ALL and AML, but were more common in one disease, such as FLT3 and KRAS in AML and PAX5, TP53 and IKZF1 in ALL. Moreover, age-associated patterns were seen in ALL with NRAS more frequently mutated than KRAS in infant ALL (26% vs 15%), and KRAS more frequently mutated than NRAS in childhood ALL (24% vs 18%), with adult ALL having fewer such mutations (NRAS 13%; KRAS 7%). Alterations of CDKN2A/B and TP53 were absent in infant ALL, detected in childhood and adult ALL only. PAX5 alterations were primarily detected in childhood ALL (22%, 9% infant ALL, 7% adult ALL), with all three PAX5-altered infant cases having the KMT2A-MLLT3 fusion gene. Finally, KMT2A-R pediatric AML had the highest fraction of FLT3 mutations (24%, 9% infant ALL, 11% childhood ALL, 0% adult ALL) and all but one mutation occurred in KMT2A-MLLT3 rearranged cases and most were kinase domain point mutations. We next expanded our analysis to include non-recurrent alterations. PI3K/RAS pathway alterations were detected across ages and subtypes with the highest fraction in pediatric AML (63%) and the lowest in adult ALL (27%, 43% infant ALL, 41% childhood ALL). Further, cell cycle related genes were primarily mutated in childhood (39%) and adult ALL cases (33%) and rarer in infant ALL (12%) and pediatric AML (16%) and genes within the B-cell pathway were more commonly altered in childhood ALL (29%) than in infant ALL (9%). Finally, in line with our previous study (Andersson et al, Nat Genet 2015) epigenetic mutations were absent in infant ALL, but present in 20-35% of the other patients. RNA-sequencing identified the KMT2A-fusion in 56/58 cases, with low exonic coverage preventing detection of the fusion in two cases. The reciprocal KMT2A fusion was only expressed in 13/39 cases where it was predicted to be expressed based on karyotype or whole genome sequencing data with 11/13 cases having the KMT2A-AFF1 fusion gene. In addition, RNA-sequencing identified 6 in-frame and 12 out-of-frame fusion genes that had formed either as part of the KMT2A-R itself or that were independent genetic events. Further, a novel in-frame KMT2A-ACIN1 fusion was identified in a child aged 1 year with B-precursor ALL. ACIN1 encodes Apoptotic Chromatin Condensation Inducer 1 and the fusion was formed through an insertion of 14q11 into 11q23. ACIN1 is also rearranged as part of the ACIN1-NUTM1 that we identified in an infant with ins(15;14)(q22;q11.2q32.1) (Andersson et al Nat Genet 2015). To study the ability of KMT2A-ACIN1 to induce leukemia in mice, we injected retrovirally transduced mouse bone marrow cells containing the fusion into syngeneic mice and KMT2A-MLLT3 was used as control. All mice succumbed to disease at an average of 112 days for KMT2A-ACIN1 (n=12) and 63 days for KMT2A-MLLT3 (n=5) and mice displayed splenomegaly and leukocytosis with an immunophenotype indicative of AML. Primary leukemia cells isolated from moribund mice gave rise to leukemia in sublethally irradiated recipients with reduced disease latency. In conclusion, these results highlight the differential molecular patterns in KMT2A-R leukemia across infancy to adulthood thereby providing novel pathogenetic insight. Disclosures No relevant conflicts of interest to declare.

Close surveillance of measurable residual disease (MRD) following completion of therapy for acute myeloid leukemia (AML) enables early detection of relapse in time for pre-emptive treatment. Today this is available only for the fraction of patients with recurrent targets quantifiable with standardized RT-qPCR. Patient-tailored MRD monitoring based on leukemia-specific mutations may be an attractive option in patients lacking such aberrations. In this population-based study, we used whole exome sequencing (WES) to identify leukemia-specific mutations suitable for MRD monitoring, and targeted deep sequencing (deep seq) and droplet digital PCR (ddPCR) for quantitative monitoring in children with AML. We investigated the clinical applicability of patient-tailored deep seq and ddPCR for early detection of AML relapse in peripheral blood (PB). All children (0-18 years) with de novo AML diagnosed between March 2013 and December 2018 who achieved complete remission after treatment according to the NOPHO-DBH AML 2012 protocol (EudraCT number: 2012-002934-35) in Sweden, Denmark, the Netherlands, Norway and Finland were eligible for the study (n = 179). In the study, patients had PB samples drawn at monthly intervals and biobanked for retrospective analyses of leukemia-specific mutations. In patients with a marker for RT-qPCR, samples were also analyzed with RT-qPCR with results reported back to the clinic. Included patients (n = 134) had PB samples drawn starting one month after last consolidation course or date of allogeneic stem cell transplantation and until relapse or end of follow-up (12-18 months following therapy completion), (median 11 samples/patient, range 1-28). Out of the 34 patients who experienced relapse during the study period, sufficient biological material for WES at diagnosis was available from 26 patients (21 hematologic and 5 molecular relapses as determined by RT-qPCR in patients with a suitable marker). For these 26 patients, the median time from end of therapy to relapse (hematologic or molecular) was 6.7 months (range 3.1-23.7). In 24/26 patients, leukemia-specific mutations could be identified at diagnosis. For 22/24 patients, leukemia-specific mutations were verified to be present at relapse (hematologic or molecular) either by WES or deep seq. In the remaining two patients with molecular relapse, leukemia-specific mutations were not verified at molecular relapse. In the 22 patients where leukemia-specific mutations could be detected, 55 mutations (median 3/patient, range 1-4) were quantified with deep seq (limit of detection [LoD] 0.02% variant allele frequency [VAF]) in 111 PB samples (263 analyses) with a sampling interval of 0.9 months (range 0.2-2.6). Parallel ddPCR analyses of 43 mutations from 20 of these patients were performed in 100 PB samples (206 analyses) with a median LoD of 0.04% VAF (range 0.003-0.1%). Deep seq and ddPCR were concordant in 187/206 (91%) of analyses and 84/100 (84%) of analyzed samples. Median VAF in the 113 paired analyses with MRD level above LoD was 0.34% (range 0.02-47.23) with deep seq and 0.38% (range 0.02-45.97) with ddPCR (P = 0.7). Obtained VAFs correlated strongly between the two methods (Spearman's ρ 0.91, P < 0.0001). Nineteen of the analyzed patients experienced hematologic relapse and in 17 of these, at least one mutation was detected in blood before relapse. The median lead time from MRD positivity to hematologic relapse was 3.2 months (range 0.6-7.9) when analyzed with deep seq (n = 17) and 2.6 months (range 0.5-7.9) when analyzed with ddPCR (n = 15). Lead times in patients analyzed with both deep seq and ddPCR showed comparable Kaplan-Meier curves (Fig. 1A). Additional comparison of the lead times achieved with deep seq/ddPCR and RT-qPCR of WT1 expression (n = 6) and recurrent genetic aberrations (n = 5) showed similar Kaplan-Meier curves (Fig. 1B-C). As assessed by deep seq in patients who were not pre-emptively treated, relapse kinetics varied greatly between patients; median VAF doubling time was 12 days with a range of 4-43 (n = 17). In conclusion, highly sensitive quantification of leukemia-specific mutations in a patient-tailored manner using deep seq or ddPCR enables post-treatment monitoring for the majority of children with AML. Frequent assessment in blood may allow for timely identification of pending relapses to initiate pre-emptive treatment strategies. Figure 1 Figure 1. Disclosures Abrahamsson: wedish Children´s Cancer Foundation. Research grants and 50% senior research position for clinical research on pediatric leukemia: Research Funding.

Introduction: CML is a rare disease in children and adolescents, accounting for 2-3% of leukemia cases in this population. Pediatric CML is more frequent in the 2 nd decade of life but data on adolescents are limited. Only response to treatment has been analyzed in few studies and seems to be poorer in this age cohort. The International Registry of Childhood Chronic Myeloid Leukemia gave us the opportunity to analyze the characteristics and outcome in a large cohort of adolescents. Aims and objectives: To report on clinical features and response to treatment in adolescents (15-18 years of age at diagnosis) with CML in chronic phase (CML-CP). Material and methods: The International Registry of Childhood CML (I-CML-Ped Study, registered at www.clinicaltrials.gov NCT01281735) enrolled patients less than 18 years of age at diagnosis of CML in all phases according to the criteria of the European Leukemia Net (ELN). Data from this registry collected from Jan 2011 - Mar 2021 into the I-CML-Ped Study (Poitiers, France) were retrospectively analyzed. Results: Out of 614 patients (pts) registered in the I-CML-Ped Study, we identified 144 (23.4%) adolescents (15-18 years of age at diagnosis) with sufficient available data. Among them, according to the ELN criteria, 132 (92%), 7 (5%), and 5 (3%) pts presented with CML-CP, accelerated phase (AP), and blastic phase (BP), respectively. The median age of the cohort comprising 132 adolescents with CML-CP was 16.2 years [range, 15-18]. Ratio male/female was 1.75. Splenomegaly was reported in 66% of the pts with a median spleen size of 11.5 cm below the costal margin [range, 1-32]. Hepatomegaly was reported in 31% of the pts with a median liver size of 2.5 cm below the costal margin [range, 1-19]. Pain, asthenia and weight loss were the most frequent symptoms at diagnosis in 35%, 33%, and 20% of pts, respectively. The performance status according to the OMS and Karnofsky scores were 0 and 100% in 81% and 57% of the pts, respectively. At diagnosis, median leukocyte count was 181 G/L [range, 7-820]; median platelet count was 516 G/L [range, 102-2619], and median hemoglobin level was 10.45 g/dL [range, 4-17]. BCR-ABL1 transcript type b3a2 was present in 53% of the assessable pts. Additional chromosomal abnormalities and variants were found in 5% of the pts. 9% and 40% of pts were considered at high-risk according to the ELTS score and to the Sokal score (for pts less than 45 years), respectively. The majority of pts (97%) were treated with imatinib as first line treatment. After 12 months of treatment, in pts with data available, rate of complete cytogenetic response (CCyR) was 74/109 (68%) pts with imatinib first line. Overall, rate of CCyR was 101/109 (93%) pts with imatinib first line [88/109 (81%) pts receiving imatinib only, 13/109 (12%) pts after switching to another TKI, 8/109 (7%) pts did not achieve CCyR]. Median time to achieve CCyR was 8.2 months [range, 2.7-106.7]. The cumulative rate of MMR at month 12 was 40/111 (36%) pts. Overall rate of MMR was 91/111 (82%) pts [72/111 (65%) pts with first line imatinib only; 19/111 (17%) pts with another TKI]. Median time to achieve first MMR was 14.5 months [range, 0.9-63.1]. Out of all patients on first line imatinib, 11/123 (9%) pts progressed: 3/11 pts progressed to AP and 8/11 pts to BP. Further progress to BP was observed in 2 of the 3 pts with AP. BP phenotype was myeloid in 5/10 pts, lymphoid in 3/10 pts and bilineage in 1/10 pts (no data for 1/10 pts). With imatinib first line, deaths occurred in 5/123 (4%) pts, among them 4/5 due to complications associated with hematopoietic stem cell transplantation. With a median follow up of 37 months [range, 0.9-231], the overall survival rate was 89.2% [83.6%; 94.7%] for pts treated with imatinib first line (N = 123). Conclusion: Our results are in line with publications describing adolescents and young adults (AYAs) as a risk population for a poorer treatment outcome: when comparing AYAs (15-29 yrs) to adults (>30 yrs), CCyR and MMR were inferior in AYAs (cumulative CCyR 84% vs 93%, cumulative MMR 75% vs 86%, respectively) [Pemmaraju N et al., Haematologica 2012] and rates of progression to AP and BP were 8.7% in AYAs (16-29 yrs) but only 5.3% and 6.1% in adults (45-59 yrs and >60 yrs, respectively) [Kalmanti L et al. , Ann Hematol. 2013]. Additional research is required in adolescents with CML to shed light on the cause(s) for the observed differences and to further improve the outcome. Disclosures No relevant conflicts of interest to declare.

Background In the adult population with newly diagnosed chronic myeloid leukemia (CML), variant translocations are usually not considered to be impairing the prognosis, whereas some additional cytogenetic abnormalities (ACAs) are associated with a negative impact on survival. Because of the rarity of CML in the pediatric population, such abnormalities have not been investigated in a large group of children with CML. Methods The prognostic relevance of variant t(9;22) and ACAs at diagnosis was assessed in 301 children with CML in the chronic phase who were enrolled in the International Registry for Chronic Myeloid Leukemia in Children and Adolescents. Results Overall, 19 children (6.3%) presented with additional cytogenetic findings at diagnosis: 5 children (1.7%) had a variant t(9;22) translocation, 13 children (4.3%) had ACAs, and 1 had both. At 3 years, for children with a classic translocation, children with ACAs, and children with a variant t(9;22) translocation who were treated with imatinib as frontline therapy, the probability of progression-free survival (PFS) was 95% (95% confidence interval [CI], 91%-97%), 100%, and 75% (95% CI, 13%-96%), respectively, and the probability of overall survival (OS) was 98% (95% CI, 95%-100%), 100% (95% CI, 43%-98%), and 75% (95% CI, 13%-96%), respectively. No statistical difference was observed between the patients with classic cytogenetic findings and those with additional chromosomal abnormalities in terms of PFS and OS. Conclusions In contrast to adults with CML, additional chromosomal abnormalities observed at diagnosis do not seem to have a significant prognostic impact. Cancer 2017;123:3609-16. © 2017 American Cancer Society.

Given that 30-40% of children with acute myeloid leukaemia (AML) relapse after primary therapy it is important to define prognostic factors and identify optimal therapy. From 1993 to 2012, 543 children from the Nordic countries were treated according to two consecutive protocols: 208 children relapsed. The influence of disease characteristics, first line treatment, relapse therapy and duration of first remission on outcome was analysed. Second complete remission (CR2) was achieved in 146 (70%) patients. Estimated 5-year overall survival (OS5y ) was 39 ± 4% for the whole group and 43 ± 4% for the 190 patients given re-induction therapy, of whom 76% received regimens that included fludarabine, cytarabine (FLA) ± anthracyclines, 18% received Nordic Society for Paediatric Haematology and Oncology (NOPHO) upfront blocks and 5% received other regimens. Late relapse ≥1 year from diagnosis, no allogeneic stem cell transplantation (SCT) in first remission and core binding factor AML were independent favourable prognostic factors for survival. For the 128 children (124 in CR2) that received SCT as consolidation therapy after relapse, OS5y was 61 ± 5%. Four of 19 children (21%) survived without receiving SCT as part of relapse therapy. Our data show that intensive re-induction followed by SCT can give cure rates of 40% in children with relapsed AML.

Background: Imatinib, a tyrosine kinase inhibitor (TKI) is currently proposed as first line therapy in children with chronic myeloid leukemia (CML) in chronic phase (CP). Studies in adults with CML demonstrated that 40 to 50% of patients with prolonged deep molecular response under TKI could discontinue TKI permanently without molecular relapse. However, data regarding TKI discontinuation in children with CML are limited. Methods: Using the ELN criteria we identified in the International Registry of Childhood Chronic Myeloid Leukemia 18 patients less than 18 years of age at diagnosis with CML in CP exhibiting under imatinib treatment sustained deep molecular response >MR4.0 (DMR) for ≥ 2 years and then discontinued the TKI. We retrospectively analyzed outcome of these patients and treatment-free remission rate (TFR) at various time points. Treatment with imatinib was resumed in case of molecular relapse defined as loss of major molecular response (MMR). Results: There were 11 boys and 7 girls. From diagnosis in CP until TKI discontinuation the 18 children showed no progression, resistance, warning or suboptimal response or switch to another TKI before discontinuation. Median age at diagnosis of CML was 11.9 years (range, 2.3 to 15.8 years) and median age at discontinuation of TKI was 16 years (range, 9 to 24 years). Median overall follow-up from diagnosis of CML was 107 months (range, 67-209 months). DMR was achieved after a median time of 12 months (range, 3 - 50 months) on imatinib. Before discontinuation median treatment duration of imatinib was 73.25 months (range, 32 to 109 months) and median duration of MR4.0 was 46.2 months (range, 23.9 to 98.6 months). Seven patients experienced molecular relapse 4.1 months (range, 1.9-6.4 months) after stopping and restarted imatinib. Two patient resumed imatinib 3.6 and 3.4 months after discontinuation because of increased in transcript level (from 0.001% to 0.01 and 0.012, respectively) but without loss of MMR. The median molecular follow up after discontinuation was 116 months (range, 71 to 209 months) for the patients without molecular relapse. The proportion of patients maintaining molecular free remission was 61% (95% CI, 38%-83%), 56% (95% CI, 33%-79%) and 56% (95% CI, 33%-79%) at 6, 12, and 36 months, respectively (Figure 1). Six of the 7 children who experienced molecular relapse after discontinuation again achieved MR4.0 at median of 4.7 months (range, 2.5-18 months) after restart of imatinib; the remaining patient achieved MMR but not DMR and was switched to Dasatinib. No withdrawal syndrome was observed in this cohort of 18 patients. In univariate analysis, age, sex, Sokal and ELTS scores, imatinib treatment duration before discontinuation and duration of DMR until imatinib discontinuation had no influence on treatment free remission. Conclusion: These data indicate that imatinib could be safely discontinued in children younger than 18 years of age at diagnosis of CML with sustained MR4.0 for at least 2 years under imatinib. Larger studies of TKI discontinuation in children with CML are needed in order to identify factors predicting treatment free remission. Disclosures Dalle: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medac: Consultancy, Honoraria; Orchard: Consultancy, Honoraria; Bellicum: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION Chronic myeloid leukemia (CML) is rare in the first two decades of life comprising only 3% of newly diagnosed pediatric and adolescent leukemias. We studied the epidemiologic and clinical features of patients with CML diagnosed at younger than 3 years of age and evaluated treatment and long-term outcome. METHOD Data from the International Pediatric I-BFM/CML Registry were retrospectively analyzed using the European LeukemiaNet criteria of the year 2006. Characteristics and treatment outcome of patients

Background: Relapse remains a serious event and main obstacle to permanent cure in childhood acute myeloid leukemia (AML). Reduction of measurable/minimal residual disease (MRD) assessed by real-time quantitative PCR (qPCR) during therapy is predictive of outcome in adults but persistent MRD positivity may be observed despite long-term remission and hamper accurate risk assignment. Sequential MRD determinations, rather than analysis at single landmark time points, may better capture the dynamics of leukemia eradication and identify relapse at molecular levels when applied in the post-therapy setting. We investigated the post-induction qPCR MRD kinetics in peripheral blood (PB) and bone marrow (BM) in a large cohort of childhood AML patients and demonstrate the utility of serial assessments for disease surveillance after therapy completion. Methods: We collected the results from qPCR MRD analyses of 774 samples (BM 347, PB 427) from 75 children with AML harboring recurrent fusion transcripts (32 RUNX1-RUNX1T1, 24 CBFB-MYH11, 16 KMT2A-MLLT3 and 3 KMT2A-ELL). Patients were treated according to The Nordic Society for Paediatric Haematology and Oncology (NOPHO)-AML 2004 or NOPHO-DBH AML 2012 protocols (2004 - 2016), or AML-BFM 2012 protocol (2014 - 2016). Only patients who achieved complete remission (CR) during induction therapy and received standard-risk consolidation without allografting were eligible for the study. Risk of relapse as a function of time of MRD positivity in sequential samples from BM or PB during consolidation therapy was modeled using restricted cubic splines. Considering shifting from MRD negative to positive in PB and MRD increments in BM above 5x10-4 as time-dependent variables, the Mantel-Byar method was applied to evaluate the prognostic impact of MRD kinetics during follow-up. Results: Risk of relapse was independent of MRD persistence in BM during therapy, but showed a strong correlation with time of MRD positivity in PB where 8/8 patients with detectable MRD after first consolidation course relapsed (Figure 1A and 1B). At therapy completion, MRD level in BM did not correlate to outcome, HR=0.64/MRD log reduction, 95% confidence interval (CI):0.32 - 1.26 (P=0.19), and there was no difference in 3-year cumulative incidence of relapse (CIR) according to MRD status (P=0.51, Figure 2A). Four patients remained MRD positive throughout consolidation therapy and all subsequently relapsed, whereas 7/28 patients who were MRD negative in PB at the end of therapy suffered relapse, 3-year CIR=27%, CI:14% - 49% (P Shifting from negative to positive in PB during follow-up predicted subsequent relapse in 10/10 patients. All 253 PB samples collected during follow-up from 36 patients in continuous CR were MRD negative. In core binding factor (CBF) AML, persistent low-level MRD positivity in BM was frequent (detected in 38% of patients tested within six months of therapy completion) but an increment to above 5x10-4 heralded subsequent relapse in 12/12 patients. Pre-relapse MRD kinetics were delineated in 16 patients and revealed a median log increment/30 days of 0.8 (range:0.4 - 1.5) in CBF patients versus 2.2 (range:1.1 - 3.7) in patients with KMT2A-MLLT3 (P=0.008). Perspectives: This study demonstrates that qPCR MRD monitoring in PB, rather than BM, represents an accurate discriminator of prognosis and is a highly informative tool for disease surveillance in childhood AML. Early relapse detection during follow-up may facilitate preemptive therapy strategies of molecular relapse, possibly improving relapse treatment outcomes. Disclosures No relevant conflicts of interest to declare.

This international study aimed to assess the effect of imatinib discontinuation in paediatric patients with chronic myeloid leukaemia (CML) after deep molecular remission (DMR) had been achieved and maintained for at least 2 years. The primary endpoint of this analysis was the molecular relapse-free survival, estimated by the non-parametric Kaplan-Meier method. Major endpoint was the estimated rate of patients without molecular relapse at 6 months. Fourteen patients were enrolled; 4 patients maintained DMR with a follow-up of 24 (two patients), 34 and 66 months, respectively, whereas 10 patients relapsed. All molecular relapses occurred within 6 months (median 3 months, range 1-6) after imatinib discontinuation. The overall probability of maintaining DMR at 6 months was 28 center dot 6%. No parameters associated with molecular relapse could be identified. Keeping in mind the rarity of paediatric CML, which contributed to the small size of the cohort, our findings illustrate that imatinib cessation after sustained DMR is successful in only limited numbers of patients, whereas much higher rates are reported in adult patients. Further research is needed to extend the cohort of paediatric CML patients who might achieve treatment-free remission with an ideal prerequisite of predicting the occurrence of molecular relapse l after imatinib cessation.

Within the International Registry of Childhood Chronic Myeloid Leukemia (CML), we identified 18 patients less than 18 years old at diagnosis of CML who were in the chronic phase and exhibiting a sustained deep molecular response (DMR) to imatinib defined as BCR-ABL1/ABL1 &lt, 0.01% (MR4) for at least two years followed by discontinuation of imatinib. Before discontinuation, the median duration of imatinib was 73.2 months (range, 32–109) and the median duration of MR4 was 46.2 months (range, 23.9–98.6). Seven patients experienced loss of major molecular response (MMR) 4.1 months (range, 1.9–6.4) after stopping and so restarted imatinib. The median molecular follow-up after discontinuation was 51 months (range, 6–100) for the nine patients without molecular relapse. The molecular free remission rate was 61% (95% CI, 38–83%), 56% (95% CI, 33–79%) and 56% (95% CI, 33–79%) at 6, 12 and 36 months, respectively. Six of the seven children who experienced molecular relapse after discontinuation regained DMR (median, 4.7 months, range, 2.5–18) after restarting imatinib. No withdrawal syndrome was observed. In univariate analysis, age, sex, Sokal and ELTS scores, imatinib treatment and DMR durations before discontinuation had no influence on treatment free remission. These data suggest that imatinib can be safely discontinued in children with sustained MR4 for at least two years.

Trisomy 8 (+8) is a common cytogenetic aberration in acute myeloid leukemia (AML); however, the impact of +8 in pediatric AML is largely unknown. We retrospectively investigated 609 patients from t ...

PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by KMT2A ( MLL) gene rearrangements and coexpression of myeloid markers. The Interfant-06 study, comprising 18 national and international study groups, tested whether myeloid-style consolidation chemotherapy is superior to lymphoid style, the role of stem-cell transplantation (SCT), and which factors had independent prognostic value. MATERIALS AND METHODS Three risk groups were defined: low risk (LR): KMT2A germline; high risk (HR): KMT2A-rearranged and older than 6 months with WBC count 300 × 109/L or more or a poor prednisone response; and medium risk (MR): all other KMT2A-rearranged cases. Patients in the MR and HR groups were randomly assigned to receive the lymphoid course low-dose cytosine arabinoside [araC], 6-mercaptopurine, cyclophosphamide (IB) or experimental myeloid courses, namely araC, daunorubicin, etoposide (ADE) and mitoxantrone, araC, etoposide (MAE). RESULTS A total of 651 infants were included, with 6-year event-free survival (EFS) and overall survival of 46.1% (SE, 2.1) and 58.2% (SE, 2.0). In West European/North American groups, 6-year EFS and overall survival were 49.4% (SE, 2.5) and 62.1% (SE, 2.4), which were 10% to 12% higher than in other countries. The 6-year probability of disease-free survival was comparable for the randomized arms (ADE+MAE 39.3% [SE 4.0; n = 169] v IB 36.8% [SE, 3.9; n = 161]; log-rank P = .47). The 6-year EFS rate of patients in the HR group was 20.9% (SE, 3.4) with the intention to undergo SCT; only 46% of them received SCT, because many had early events. KMT2A rearrangement was the strongest prognostic factor for EFS, followed by age, WBC count, and prednisone response. CONCLUSION Early intensification with postinduction myeloid-type chemotherapy courses did not significantly improve outcome for infant ALL compared with the lymphoid-type course IB. Outcome for infant ALL in Interfant-06 did not improve compared with that in Interfant-99.

The prognosis of children with acute myeloid leukemia (AML) is improving but still unsatisfactory. We and others have shown that measurable residual disease (MRD) as measured with multiparameter flow cytometry (MFC) is a very strong prognostic factor, however this technique cannot reliably identify low-risk cases. An alternative approach for MRD analysis is quantification of leukemia-specific transcripts using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). This approach has shown strong prognostic value in adult AML but its significance in childhood AML is less studied. In this retrospective study, we evaluated early treatment response with RT-qPCR and MFC in parallel to determine treatment kinetics and the relationship between results of the two methods. The study included 15 children (7 females, 8 males, median age 6 years (range 1-16)), diagnosed 2004-2011 with de novo AML with a quantifiable fusion transcript (8 with RUNX1-RUNX1T1, one with CBFB-MYH11 and 6 with KMT2A-MLLT3), all treated in the NOPHO AML-2004 trial. MRD analyses were performed on samples from day 15 from start of the first induction course and just before the first consolidation course. MRD analysis with MFC was performed in bone marrow samples by identifying leukemia-associated immunophenotypes (LAIP), and reported as percent of viable cells. Fusion transcripts were analyzed with RT-qPCR in bone marrow and blood according to the Europe against cancer program, and reported as percentage of diagnostic level in bone marrow. Results of all MRD analyses were reported to clinicians but not used for treatment decisions. All patients achieved complete remission. 9 patients relapsed (median time from diagnosis 12 months, range 8-16); 5 with RUNX1-RUNX1T1 and 4 with KMT2A-MLLT3. When we compared results of RT-qPCR in blood and bone marrow day 15 and before consolidation, there was a high correlation; 0.93, p0.1%. These analyses confirmed the absence of immature cells with LAIP and detected either more mature myeloid cells or immature cells without LAIP. We hypothesize that fusion transcripts can be present in mature cells not detected as MRD using MFC. This was supported by analyzing sorted immature and mature cells from bone marrow samples at diagnosis of AML (n=4, adults). In CD34-CD117+ cells, leukemic transcripts (RUNX1-RUNX1T1/CBFB-MYH11/mutated NPM1) were 67±10% (mean±SEM) of the level in the most immature cells, CD34+CD117+, from the same patients and in mature granulocytes (CD34-CD117-, high SSC) 88±9%. In conclusion, RT-qPCR and MFC provide different information during induction treatment. RT-qPCR has a stronger ability to detect remaining leukemic burden, but whether this burden is clinically relevant remains to be shown. The impact of fusion transcript levels on relapse risk is currently investigated in the NOPHO-DBH AML-2012 trial in which MFC is used for risk stratification. Disclosures No relevant conflicts of interest to declare.

Background: Even though children with acute myeloid leukemia (AML) receive a very intensive chemotherapy and most achieve a complete remission (CR) ~30% of patients suffer from relapse. Post-treatment monitoring of measurable residual disease (MRD) can allow detection of a re-emerging leukemic clone several months before clinical relapse, and studies are in progress that aim at treating children with a molecular relapse. Specific genetic aberrations can be used for disease monitoring after therapy completion, but even though oncogenic fusion transcripts are more common in childhood than adult AML, NPM1 mutation is much rarer and consequently MRD measurements based on these aberrations are clinically applicable in only ~40% of childhood AML patients. Thus, a considerable fraction of patients do not have a suitable leukemia-specific molecular MRD target, but genes with an abnormally high expression in the leukemic cells might be candidate MRD targets in those patients. WT1 overexpression in childhood AML is well described, and if distinctly overexpressed at diagnosis, serves as a suitable MRD target in a large proportion of patients. Gene expression profiling has identified several other genes with an abnormally high expression in the leukemic blasts compared to normal hematopoietic cells. We investigated the applicability of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) as targets for early detection of relapse in peripheral blood (PB) in a Danish cohort of childhood AML patients, defined child-specific reference values of gene expression based on a large material of PB and BM samples from hematologically healthy children, and investigated gene expression levels under the presence of infection. Methods: We investigated the expression of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) in hematologically healthy children (n=53) and during suspected infection in febrile but otherwise healthy children (n=90). Gene expression in de novo AML at diagnosis (n=50) and during follow-up (n=20) was compared with child-specific reference values. We defined the 95th percentile of expression levels in hematologically healthy children as the upper limit of normal expression. RT-qPCR analyses were performed in compliance with EAC protocols and due to concordant qPCR efficiencies the ΔΔCq method for relative quantification could be applied. Results: At AML diagnosis, 64% had high expression of at least 1 of the 4 genes defined as >20-fold overexpression compared to hematologically healthy children. Nine out of 10 patients (90%) without established molecular MRD targets or high WT1 expression had high expression of at least 1 of the 4 genes. All 7 children with t(9;11) had GAGED2>1000-fold overexpressed. Gene expression was quantified in 99 PB samples (163 RT-qPCR analyses) during follow-up in 20 patients with distinct overexpression at diagnosis. All 10 patients with PB sampling performed within 100 days of disease recurrence displayed expression above normal by a median of 1.6 months (range 0.5-6 months) before hematological relapse. Patients with CBF-AML had a significantly longer interval between molecular relapse and hematological relapse than patients with non-CBF-AML (2.5 months (range 0.8-6 months) vs. 0.9 months (range 0.5-2 months), p=0.047). One patient with PB sampling performed only once at 119 days prior to hematological relapse did not show any molecular evidence of disease recurrence before hematological relapse. Only 1 of 96 (1%) post-therapy follow-up analyses performed in 9 patients in continuous CR for >5 years after diagnosis had expression above normal. In this case, a 9-year-old girl in continuous CR had an increase in ST18 expression, however the increase was transient and returned to normal level in the following samples. We found no clinically relevant influence of fever on gene expression levels, except for GAGED2, where 21% of febrile children had expression above normal. Conclusions: Sequential post-therapy monitoring of overexpressed genes in PB can predict relapse in childhood AML patients and facilitates molecular MRD monitoring in 90% of patients without a leukemia-specific target or WT1 overexpression. Frequent PB sampling (every 4-6 weeks) is necessary to detect an upcoming relapse, however in the post-treatment follow-up setting PB serves as an attractive and easily accessible source of preference compared to BM aspiration. Disclosures No relevant conflicts of interest to declare.

Dear EditorWe read Hearn et al.’s article (Hearn et al. 2019) with great interest and appreciate the invitation for a wider debate on what patient-centred means in medical education.We are developi...

Background: Studies on adolescents and young adults with acute lymphoblastic leukemia suggest better results when using pediatric protocols for adult patients, while corresponding data for acute my ...

The World Health Organization (WHO) classification of myeloid leukaemia was revised in 2008. It incorporates newly recognized entities and emphasizes the pivotal role of cytogenetic abnormalities. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population-based paediatric acute myeloid leukaemia (AML) cohort. We included children diagnosed with de novo AML, 0-18 years of age from the Nordic countries and Hong Kong from 1993 to 2012. Data were retrieved from the Nordic Society for Paediatric Haematology and Oncology AML database and patients classified according to the WHO 2008 classification. A successful karyotype was available in 97% of the cases. AML with recurrent genetic abnormalities were present in 262 (41%) and 94 (15%) were classified as AML with myelodysplasia-related changes (AML-MDS). WHO classifies patients with monosomy 7 and del(7q) into one group. We found that -7 (n = 14) had significantly poorer outcome than del(7q) (n = 11); 5-year event-free survival 26% vs. 67%, (P = 0·02), and 5-year overall survival 51% vs. 90%, (P = 0·04). The largest group was the highly heterogeneous AML not otherwise specified (NOS) (n = 280) (44%). In conclusion, the WHO classification allocated 15% to AML-MDS, 44% to NOS and grouped together entities with clearly different outcome, therefore limiting the applicability of the current WHO classification in children with AML.

A total of 156 patients (age range 1.3-18.0 years, median 13.2 years; 91 (58.3%) male) with newly diagnosed CML (N = 146 chronic phase (CML-CP), N = 3 accelerated phase (CML-AP), N = 7 blastic phase (CML-BP)) received imatinib up-front (300, 400, 500 mg/m 2 , respectively) within a prospective phase III trial. Therapy response, progression-free survival, causes of treatment failure, and side effects were analyzed in 148 children and adolescents with complete data. Event-free survival rate by 18 months for patients in CML-CP (median follow-up time 25 months, range: 1-120) was 97% (95% CI, 94.2-99.9%). According to the 2006 ELN-criteria complete hematologic response by month 3, complete cytogenetic response (CCyR) by month 12, and major molecular response (MMR) by month 18 were achieved in 98, 63, and 59% of the patients, respectively. By month 36, 86% of the patients achieved CCyR and 74% achieved MMR. Thirty-eight patients (27%) experienced imatinib failure because of unsatisfactory response or intolerance (N = 9). In all, 28/148 patients (19%) underwent stem cell transplantation (SCT). In the SCT sub-cohort 2/23 patients diagnosed in CML-CP, 0/1 in CML-AP, and 2/4 in CML-BP, respectively, died of relapse (N = 3) or SCT-related complications (N = 2). This large pediatric trial extends and confirms data from smaller series that first-line imatinib in children is highly effective.

The membrane transporter P-glycoprotein, encoded by the ABCB1 gene, influences the pharmacokinetics of anti-cancer drugs. We hypothesized that variants of ABCB1 affect outcome and toxicity in childhood acute lymphoblastic leukemia (ALL). We studied 522 Danish children with ALL, 93% of all those eligible. Risk of relapse was increased 2.9-fold for patients with the 1199GA variant versus 1199GG (P = 0.001), and reduced 61% and 40%, respectively, for patients with the 3435CT or 3435TT variants versus 3435CC (overall P = 0.02). The degree of bone marrow toxicity during doxorubicin, vincristine and prednisolone induction therapy was more prominent in patients with 3435TT variant versus 3435CT/3435CC (P = 0.01/P less than 0.0001). We observed more liver toxicity after high-dose methotrexate in patients with 3435CC variant versus 3435CT/TT ( P = 0.03). In conclusion, there is a statistically significant association between ABCB1 polymorphisms, efficacy and toxicity in the treatment of ALL, and ABCB1 1199G greater than A may be a new possible predictive marker for outcome in childhood ALL. Funding Agencies|Swedish Cancer Society; Swedish Research Council; County Concil in Ostergotland; Danish Childhood Cancer Foundation; University Hospital Rigshospitalet, Denmark

BACKGROUND: Children with acute myeloid leukemia (AML) treated similarly show different toxicity and leukemic responses. We investigated associations between neutrophil recovery time after the first induction course, infection and relapse in children treated according to NOPHO-AML 2004 and DB AML-01.PROCEDURE: Newly diagnosed patients with AML with bone marrow blast RESULTS: Neutrophil recovery time after the first induction course was positively associated with neutrophil recovery time after the remaining courses, and longer neutrophil recovery time (≥25 days) was associated with increased risk of grade 3-4 infections (hazard ratio 1.4, 95% confidence interval [CI], 1.1-1.8). Longer neutrophil recovery time after the first induction (>30 days) was associated with the increased risk of relapse (5-year cumulative incidence: 48% vs. 42%, hazard ratio 1.7, 95% CI, 1.1-2.6) for cases not treated with hematopoietic stem cell transplantation in first complete remission.CONCLUSION: Longer neutrophil recovery time after the first induction course was associated with grade 3-4 infections and relapse. If confirmed, this knowledge could be incorporated into risk stratification strategies in pediatric AML.

The t(7;12)(q36;p13) (MNX1/ETV6) is not included in the WHO classification but has been described in up to 30% of acute myeloid leukemia (AML) in children2 years and associated with a poor prognosis. We present the clinical and cytogenetics characteristics of AML cases with t(7;12)(p36;p13). A literature review identified 35 patients with this translocation, published between 2000 and 2015. Outcome data were available in 22 cases. The NOPHO-AML (Nordic Society for Pediatric Hematology and Oncology) database contained 651 patients with AML from 1993 to 2014 and seven (1.1%) had the translocation. The t(7;12) was only present in patients2 years of age (median age 6 months) but none was diagnosed as newborn. These patients constituted 4.3% of the patients2 years of age. There was a strong association with trisomy 19 (literature: 86%, NOPHO: 100%) and +8 (literature: 19%, NOPHO: 14%). Seventeen of 22 patients from the literature with t(7;12) and four of seven patients from the NOPHO database suffered from relapse. The patients with t(7;12) had a 3-year event free survival of 24% (literature) vs. 43% (NOPHO) and a 3-year overall survival of 42% (literature) vs. 100% (NOPHO). None of the NOPHO patients was treated with hematopoietic stem cell transplantation (HSCT) in first complete remission. Relapse was frequent but the salvage rate using HSCT was high. We conclude that t(7;12)(q36;13) is a unique subgroup of childhood AML with presentation before 2 years of age with most cases being associated with +19.

Hyperleucocytosis in paediatric acute myeloid leukaemia (AML) is associated with increased morbidity and mortality. We studied hyperleucocytosis in 890 patients with AML aged 0-18 years registered in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) registry, with special focus on very high white blood cell counts (WBC >200 × 10/l). Eighty-six patients (10%) had WBC 100-199 × 10(9) /l and 57 (6%) had WBC ≥200 × 10(9) /l. Patients with WBC ≥200 × 10(9) /l had a high frequency of t(9;11) and a paucity of trisomy 8. Due to the high frequency of deaths within the first 2 weeks (30% vs. 1% for all others), overall survival in this group was inferior to patients with WBC

The EUTOS Long-Term Survival score was tested in 350 children with chronic myeloid leukemia in first chronic phase treated with imatinib and registered in the International Registry for Childhood Chronic Myeloid Leukemia. With a median follow up of 3 years (range, 1 month to 6 years) progression and/or death (whichever came first) occurred in 23 patients. For the entire cohort of patients the 5-year progression-free survival rate was 92% (95% CI: 87%-94%) and the 5-year survival accounting for chronic myeloid leukemia deaths was 97% (95% CI: 94%-99%). Of the 309 patients allocated to low (n=199), intermediate (n=68) and high (n=42) risk groups by the EUTOS Long-Term Survival score, events (progression and/or death) occurred in 6.0%, 8.8% and 26.2%, respectively. Estimates of the 5-year progression-free survival rates according to these three risk groups were 96% (95% CI: 92%-98%), 88% (95% CI: 76%-95%) and 67% (95% CI: 48%-81%), respectively. Differences in progression-free survival according to these risk groups were highly significant (P