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Background Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. Objectives In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. Methods Nine patients were enrolled and received at least two courses of ara-C (1 g/m(2)/2 h b.i.d. d1-5, i.e., a total of 10 g/m(2) per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m(2) d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. Results The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level 1.0 x 10(9)/L and to platelet recovery >50 x 10(9)/L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. Conclusion Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

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.

Ribonuclease inhibitor 1, also known as angiogenin inhibitor 1, encoded by RNH1, is a ubiquitously expressed leucine-rich repeat protein, which is highly conserved in mammalian species. Inactivation of rnh1 in mice causes an embryonically lethal anemia, but the exact biological function of RNH1 in humans remains unknown and no human genetic disease has so far been associated with RNH1. Here, we describe a family with two out of seven siblings affected by a disease characterized by congenital cataract, global developmental delay, myopathy and psychomotor deterioration, seizures and periodic anemia associated with upper respiratory tract infections. A homozygous splice-site variant (c.615-2A > C) in RNH1 segregated with the disease. Sequencing of RNA derived from patient fibroblasts and cDNA analysis of skeletal muscle mRNA showed aberrant splicing with skipping of exon 7. Western blot analysis revealed a total lack of the RNH1 protein. Functional analysis revealed that patient fibroblasts were more sensitive to RNase A exposure, and this phenotype was reversed by transduction with a lentivirus expressing RNH1 to complement patient cells. Our results demonstrate that loss-of-function of RNH1 in humans is associated with a multiorgan developmental disease with recessive inheritance. It may be speculated that the infection-induced deterioration resulted from an increased susceptibility toward extracellular RNases and/or other inflammatory responses normally kept in place by the RNase inhibitor RNH1.

Cytogenetic aberrations are recognized as important prognostic factors in adult acute lymphoblastic leukemia (ALL), but studies seldom include elderly patients. From the population-based Swedish ALL Registry, we identified 728 patients aged 18-95 years, who were diagnosed with ALL 1997-2015 and had cytogenetic information. Registry data were complemented with original cytogenetic reports.

Certain subtypes of acute myeloid leukemia (AML) in children have still inferior outcome. One of these AML subtypes has a translocation t(7;12)(q36;p13), always leading to high expression of MNX1 and often to MNX1::ETV6 fusion expression. Here we identified the transforming event in this AML and possible ways to target them. Only MNX1 was able to induce AML in mice, and this was observed using hematopoietic stem and progenitor cells derived from fetal origin but not from adult bone marrow. The restriction in the transforming capacity to cells from fetal liver origin is in concordance with the fact that t(7;12)(q36;p13) AML is mostly restricted to infants. Ectopic expression of MNX1 led to increase of H3K4me1, H3K4me2 and H3K4me3, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and different methyltransferases. MNX1 expression resulted in increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in leukemia development in AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.

Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome. One of these has a translocation t(7;12)(q36;p13) leading to aMNX1::ETV6fusion along with high expression of MNX1. Here we identified the transforming event in this AML and possible ways of treatment. Only MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by pretreatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) can be classified into subtypes according to the genetic aberrations they display. For instance, the translocation t(12;21)(p13;q22), representing the ETV6‐RUNX1 fusion gene (ER), is present in a quarter of BCP‐ALL cases. However, around 10% of the cases lack classifying chromosomal abnormalities (B‐other). In pediatric ER BCP‐ALL, rearrangement mediated by RAG (recombination‐activating genes) has been proposed as the predominant driver of oncogenic rearrangement. Herein we analyzed almost 1600 pediatric BCP‐ALL samples to determine which subtypes express RAG. We demonstrate that RAG1 mRNA levels are especially high in the ETV6‐RUNX1 (ER) subtype and in a subset of B‐other samples. We also define 31 genes that are co‐expressed with RAG1 (RAG1‐signature) in the ER subtype, a signature that also identifies this subset of B‐other samples. Moreover, this subset also shares leukemia and pro‐B gene expression signatures as well as high levels of the ETV6 target genes (BIRC7, WBP1L, CLIC5, ANGPTL2) with the ER subtype, indicating that these B‐other cases are the recently identified ER‐like subtype. We validated our results in a cohort where ER‐like has been defined, which confirmed expression of the RAG1‐signature in this recently described subtype. Taken together, our results demonstrate that the RAG1‐signature identifies the ER‐like subtype. As there are no definitive genetic markers to identify this novel subtype, the RAG1‐signature represents a means to screen for this leukemia in children., The recombination‐activating genes (RAG) may play a role in leukemogenesis. We determined, therefore, in around 1600 BCP‐ALL samples which subtypes express RAG and defined a set of co‐expressed genes, the RAG1‐signature, which was thereafter used to identify potential new subtypes. We demonstrate that the RAG1‐signature identifies the recently described ETV6‐RUNX1‐like BCP‐ALL.

The upcoming 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours is part of an effort to hierarchically catalogue human cancers arising in various organ systems within a single relational database. This paper summarizes the new WHO classification scheme for myeloid and histiocytic/dendritic neoplasms and provides an overview of the principles and rationale underpinning changes from the prior edition. The definition and diagnosis of disease types continues to be based on multiple clinicopathologic parameters, but with refinement of diagnostic criteria and emphasis on therapeutically and/or prognostically actionable biomarkers. While a genetic basis for defining diseases is sought where possible, the classification strives to keep practical worldwide applicability in perspective. The result is an enhanced, contemporary, evidence-based classification of myeloid and histiocytic/dendritic neoplasms, rooted in molecular biology and an organizational structure that permits future scalability as new discoveries continue to inexorably inform future editions.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Purpose: The current standard-of-care for front-line therapy for acute myeloid leukaemia (AML) results in short-term and long-term toxicity, but still approximately 40% of children relapse. Therefore, there is a major need to accelerate the evaluation of innovative medicines, yet drug development continues to be adult-focused. Furthermore, the large number of competing agents in rare patient populations requires coordinated prioritisation, within the global regulatory framework and cooperative group initiatives. Methods: The fourth multi-stakeholder Paediatric Strategy Forum focused on AML in children and adolescents. Results: CD123 is a high priority target and the paediatric development should be accelerated as a proof-of-concept. Efforts must be coordinated, however, as there are a limited number of studies that can be delivered. Studies of FLT3 inhibitors in agreed paediatric investigation plans present challenges to be completed because they require enrolment of a larger number of patients than actually exist. A consensus was developed by industry and academia of optimised clinical trials. For AML with rare mutations that are more frequent in adolescents than in children, adult trials should enrol adolescents and when scientifically justified, efficacy data could be extrapolated. Methodologies and definitions of minimal residual disease need to be standardised internationally and validated as a new response criterion. Industry supported, academic sponsored platform trials could identify products to be further developed. The Leukaemia and Lymphoma Society PedAL/EUpAL initiative has the potential to be a major advance in the field. Conclusion: These initiatives continue to accelerate drug development for children with AML and ultimately improve clinical outcomes.

Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed.The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact.Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care.[https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0.02% was achieved. The assay was linear in MRD range (0.03% to 1%) with good precision [CV, 4.1% (2.2% to 5.7%) at VAF 1% and 13.3% (8.8% to 19.4%) at VAF 0.1%], and low relative bias [7.9% (2.5% to 15.3%) at VAF 1%]. When applied to six childhood AML cases and compared with multiparameter flow cytometry for MRD analysis, deep sequencing showed concordance and superior sensitivity. Further high concordance was found with expression of fusion transcripts RUNX1-RUNX1T1 and KMT2A-MLLT10. The deep sequencing assay also detected mutations in blood when VAF in bone marrow exceeded 0.1% (n = 19). In conclusion, deep sequencing enables reliable detection of low levels of residual leukemic cells. Introduction of this method in patient care will allow for highly sensitive MRD surveillance in virtually every patient with AML.

Endothelin receptor type A (EDNRA) is known as a mediator of cell proliferation and survival. Aberrant regulation of EDNRA has been shown to play a role in tumor growth and metastasis. Using a global gene expression screen, we found that expression of Ednra was upregulated in murine leukemia inducing cells co-expressing Hoxa9 and Meis1 compared to cells only expressing Hoxa9. The aim of this study was to explore the role of Ednra in leukemogenesis further. In a murine bone marrow transplantation model, mice transplanted with cells overexpressing Ednra and Hoxa9 succumbed to leukemia significantly earlier than mice transplanted with cells overexpressing Hoxa9 only. Furthermore, overexpression of Ednra led to increased proliferation and resistance to apoptosis of bone marrow cells in vitro. We could also show that Meis1 binds to the Ednra promoter region, suggesting a regulatory role for Meis1 in Ednra expression. Taken together, our results suggest a role for Ednra in Hoxa9/Meis1-driven leukemogenesis.

Introduction: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. Methods: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. Results: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. Conclusion: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA. (Less)

The mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients. Funding Agencies|CancerfondenSwedish Cancer Society [CAN2018/623] Funding Source: Medline; Barncancerfonden [PR2017-0023, PR2019-0046] Funding Source: Medline

The Swedish national guidelines for treatment of acute myeloid leukemia (AML) recommend analysis of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in bone marrow in the routine clinical setting. The Swedish AML registry contains such MRD data in AML patients diagnosed 2011-2019. Of 327 patients with AML (non-APL) with MRD-results reported in complete remission after two courses of intensive chemotherapy 229 were MRD-negative (70%), as defined by Funding Agencies|Swedish Association of Local Authorities and Regions; Region Skane; Regional Cancer Center South

Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo. However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.

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.

Acute myeloid leukemia (AML) is a heterogeneous disease where selected subgroups of patients, linked by the presence of biological and clinical high-risk features, are candidates to receive allogenic hematopoietic stem cell transplantation HSCT) as post-remission consolidation treatment. The achievement of morphological complete remission (CR) before HSCT is an important pre-requisite to optimize the chance of successful post-transplant outcome. Minimal residual disease (MRD) assessment by quantitative polymerase chain reaction (q-PCR) has been shown to increase the ability to monitor therapy response in AML, improving prognostic accuracy and allowing to refine transplant strategies. Although MRD assessment was shown to have potential benefit when measured after induction and consolidation therapy courses, its role before HSCT remains to be fully elucidated. In order to contribute to better clarify this issue, we conducted a q-PCR I-BFM-AML collaborative study to measure MRD in bone marrow samples collected within 5 weeks prior to HSCT of 108 pediatric AML patients harboring one of the main recurrent AML aberrancies t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1q22)/t(16;16)(p13.1;q22); CBFB-MYH11, t(9;11)(p22;q23); KMT2A-MLLT3 or FLT3-ITD. Sixty patients underwent HSCT in first complete remission (CR1) with an overall survival (OS) of 84% versus 54% for the 48 transplanted in CR2 achieved after an initial relapse. Sixty patients showed q-MRD negativity (defined as a value lower than 2.1x10-4 calculated by ROC curve analysis with respect to diagnosis or relapse), whereas in 48 patients we detected q-MRD levels >2.1x10-4. Five-year OS after HSCT was 83% for patients with q-MRD negativity, while that of patients with q-MRD above the cutoff was 57% (p=0.012). As regards, cumulative incidence of relapse (CIR), q-MRD above the cutoff was associated with a high risk of recurrence (26% versus 10% for patients with q-MRD 2-log versus 73% for q-MRD2.1x10-4 and 2-log, HR). This combined stratification by q-MRD resulted into a better subdivision of the OS probability, which was 83%, 69% and 39% for LR, IR and HR respectively (p=0.004). Finally, a multivariate Cox regression model revealed that, together with CR status at time of the allograft (CR2, hazard ratio 4.4, p=0.001), q-MRD was an independent factor (hazard ratio 0.5, p=0.001) predicting HSCT outcome. In conclusion, this study supports the role of q-MRD pre-HSCT as a useful prognostic tool in childhood AML, able to provide information to tailor transplant strategies involving conditioning regimen intensity and graft-versus-host disease prophylaxis. Disclosures Reinhardt: AbbVie: Consultancy; Novartis: Consultancy, Other: Institutional Research Funding; Jazz: Consultancy, Other: Institutional Research Funding; Celgene: Consultancy, Other: Institutional Research Funding; bluebird bio: Consultancy; Roche: Consultancy, Other: Institutional Research Funding; Biotest: Other: Institutional Research Funding; Novo Nordisk: Other: Institutional Research Funding; Behring: Other: Institutional Research Funding. Merli:Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria; Sanofi-Genzyme: Honoraria; Atara Therapeutics: Honoraria.

Pediatric acute myeloid leukemia (AML) with the translocation t(7;12)(q36;p13) is a form of childhood leukemia associated with inferior outcome. In the current investigation we have studied the gene alterations that are induced by this chromosomal translocation, a gene fusion MNX1-ETV6 but also ectopic expression of the homeobox gene MNX1. In a murine bone marrow transplantation model, our results showed that ectopic expression of MNX1 was able to induce AML, but not the MNX1-ETV6 gene fusion. However, this transformation to AML was only seen using hematopoietic progenitor cells from fetal liver and not adult bone marrow cells. This was accompanied with an alteration of cell differentiation towards myeloid lineage, increased proliferation and colony formation capacity induced by MNX1. Our data also showed that ectopic expression of MNX1, and to a lower extend MNX1-ETV6 fusion, induced DNA damage and cell cycle arrest, where we saw an increased expression and binding of the MNX1 and the MNX1-ETV6 fusion to the gene CEP164 that has a critical role in cell cycle regulation and DNA damage response, and the transcriptional pioneer factor gene PBX1 that is important in development and differentiation. Furthermore, global histone modifications, specifically H3K4me3 and H3K27me, were induced by MNX1 but also the MNX1-ETV6 fusion. We could also see an enrichment of methylation/methionine proteins from mass spectrometry analysis of proteins associated with MNX1. In conclusion, these results suggest a possible mechanism by which MNX1 mediates its transforming effect through abnormal methylation of proteins that result in changed histone modifications and subsequent dysregulation of gene expression. Our finding that only fetal liver cells were able to transform into AML is also in concordance with the clinical finding that this AML subtype is only found in very young children.

Introduction The translocation t(12;21)(p13;q22) resulting in the fusion gene ETV6-RUNX1, is the most frequent gene fusion in childhood B lymphoblastic leukemia. In the Nordic Society of Paediatric Haematology and Oncology ALL-2008 treatment protocol, treatment stratification in B-lineage ALL is based on results of minimal residual disease (MRD) analysis with fluorescence-activated cell sorting (FACS). In this study, we determined whether RT-qPCR of the ETV6-RUNX1 fusion transcript can be a reliable alternative for MRD analysis. Methods Seventy-eight bone marrow samples from 29 children at diagnosis and day 15, 29, and 78 during treatment were analyzed for MRD with FACS and with quantitative reverse transcription polymerase chain reaction (RT-qPCR). Fusion transcript MRD was defined as the ETV6-RUNX1/GUSB ratio at the follow-up time point (day 15/29/78) divided with the ETV6-RUNX1/GUSB ratio at diagnosis (%). Results MRD analysis with FACS and with RT-qPCR of ETV6-RUNX1 fusion transcript showed strong correlation. All cases showed concordant results at the treatment stratifying time points day 29 and day 78, when comparing the two methods with a cutoff set to 0.1%. Conclusion RT-qPCR is a valuable addition and could also be an alternative to FACS in cases where FACS is not achievable for MRD analysis.

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.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD

HOXA9, MEIS1 and FLT3 are genes frequently upregulated in human acute myeloid leukemia. Hoxa9 and Meis1 also cooperate to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1-induced leukemogenesis. To define the role of Flt3 in AML with high Hoxa9/Meis1, we treated mice with Hoxa9/Meis1-induced AML with the Flt3 inhibitor AC220, used an Flt3-ligand (FL-/-) knockout model, and investigated whether overexpression of Flt3 could induce leukemia together with overexpression of Hoxa9. Flt3 inhibition by AC220 did not delay AML development in mice transplanted with bone marrow cells overexpressing Hoxa9 and Meis1. In addition, Hoxa9/Meis1 cells induced AML in FL-/- mice as rapid as in wild-type mice. However, FL-/- mice had reduced organ infiltration compared with wild-type mice, suggesting some Flt3-dependent effect on leukemic invasiveness. Interestingly, leukemic Hoxa9/Meis1 cells from sick mice expressed high levels of Flt3 regardless of presence of its ligand, showing that Flt3 is a passive marker on these cells. In line with this, combined engineered overexpression of Flt3 and Hoxa9 did not accelerate the progression to AML. We conclude that the Hoxa9- and Meis1-associated upregulation of Flt3 is not a requirement for leukemic progression induced by Hoxa9 and Meis1.

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

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.