The overall survival of children with newly diagnosed acute myeloid leukemia (AML) in high-income countries has increased to 80

BACKGROUND:Survival after relapse in pediatric acute myeloid leukemia (AML) remains poor, highlighting the critical importance of identifying prognostic factors to guide optimal relapse management. METHODS:We investigated the prognostic impact of multiparameter flow cytometry (MFC) measurable residual disease (MRD) in 188 patients with first relapse after initial treatment according to the NOPHO-DBH AML 2012 protocol. RESULTS:The 4-year overall survival (OS4y) was 44% (95% confidence interval [CI]: 36%-51%). OS4y was 61% (CI: 51%-69%) in 133/188 patients who received stem cell transplantation (SCT) after reinduction therapy. Nineteen patients treated at the time of molecular relapse showed an excellent OS4y of 84% (CI: 58%-95%). Patients with hematological relapse and MRD <0.1% after first reinduction course had a superior OS4y of 69% (CI: 51%-81%) compared to patients with MRD between 0.1% and 4.9% (OS4y 46%, CI:28%-63%) and MRD ≥5% (OS4y 16%, CI: 6%-30%), adjusted hazard ratio (HR) 2.2 for MRD 0.1%-4.9%; p = 0.04 and HR 6.0 for MRD ≥5%; p < 0.001. Patients in second complete remission after first reinduction course and MRD <0.1% after second reinduction course had an OS4y of 70% (CI: 52%-82%) compared to 46% (CI: 19%-70%) in patients with MRD ≥0.1%, HR 2.7; p = 0.048. OS4y was 71% (CI: 54%-82%) and 31% (CI: 13%-51%) for patients with MRD <0.1% or ≥0.1% prior to SCT, respectively, HR 3.1; p = 0.004. CONCLUSIONS:This study identifies MFC MRD during reinduction therapy and before SCT as novel and independent predictors of outcome in relapsed pediatric AML.

Background: Bone marrow (BM) aspirates during and after induction chemotherapy are established but cumbersome prognostic tools for assessing treatment response in pediatric acute myeloid leukemia (AML) (Creutzig 2014, Abrahamsson 2011, Kern 2003). Peripheral blood blast clearance (PBBC) assessment by morphology has shown prognostic value in adult AML and might offer a cost-effective, easily accessible alternative, especially in low-resource settings (Manabe 2008, Arellano 2012). Its utility in pediatric AML is not well-established. This study investigates early PBBC as a predictor of treatment response and outcomes in pediatric AML. Methods: Data on pediatric patients with de novo AML treated according to the NOPHO-DBH AML-2012 protocol were prospectively obtained. Participating countries included Belgium, Denmark, Israel, Latvia, The Netherlands, Norway and Sweden. The first 5 days of induction therapy consisted of etoposide, followed by 7 days of low-dose cytarabine and mitoxantrone or liposomal daunorubicin. Peripheral blood (PB) was assessed by morphology at the time of diagnosis, daily in the first week, and thrice weekly until D22 or until complete clearance of blasts occurred. BM at day 22 (D22) was assessed by morphology and flow cytometry. A receiver operating characteristic (ROC) analysis was performed to evaluate the predictive power of the number of days from starting chemotherapy to complete PBBC. The time to PBBC and rate of PBBC, defined as the daily percentage of peripheral blood blast count cleared from diagnosis to the last assessment, were correlated with BM response on D22, complete remission status (CR), refractory disease (RD), event-free survival (EFS), and overall survival (OS). Flow cytometry based measurable residual disease (flow-MRD) negativity was defined as the absence of detectable leukemic cells in the BM (<0.01%). Results: Between March 2013 and February 2023, a total of 319 patients were included, 241 of whom were eligible for analysis, due to missing or incomplete data (n=78). Day 9 after initiation of chemotherapy was identified as the most discriminating cut-off point for predicting BM D22 response (predicted probability of 89.3%; P<0.001). Patients who achieved PBBC ≤9 days were classified as early PBBC (n=159; 66%), while those with blast clearance >9 days were classified as delayed PBBC (n=82; 34%). For 217 patients (due to missing data in 24 cases), the rate of PBBC could be calculated, and a discriminative cut-off value of 11% was identified (predicted probability of 84%, P<0.001). Patients were categorized into a high rate of PBBC (>11%; n=132) and low rate of PBBC (≤11%; n=85). Early PBBC and a high PBBC rate were associated with favorable BM responses (<5% leukemic cells) on D22 (86.7% vs. 68.4%, P<0.001; and 88.1% vs. 67.1%, P=0.001, respectively). No poor responders with ≥15% leukemic cells in BM on D22 were observed in the early PBBC group. Early PBBC and high PBBC rate were associated with higher flow-MRD negative rates on D22 (53.7% vs. 37.5%, P=0.027 and 51.9% vs. 38.4%, P=0.028). There was no significant association with RD or EFS in either group. Five-year OS was significantly higher in the early PBBC and high PBBC rate group (84.2% vs. 72.6%, P=0.02; and 86.5% vs. 73.7%, P=0.021, respectively). Conclusion: Early PBBC is a promising, non-invasive prognostic marker of treatment response and outcome in pediatric AML. Its simplicity and cost-effectiveness make it particularly advantageous for low-resource settings.

Introduction: MECOM-rearranged (r) acute myeloid leukemia (AML) is a distinct WHO-defined genetic subtype and includes cases with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) or 3q26.2 fusions with alternative partners. These rearrangements occur in <2% of adult AML cases and are even rarer and poorly characterized in pediatric AML. MECOM-r AML is associated with chemotherapy resistance and poor clinical outcomes. Management of children with this high-risk leukemia subtype remains insufficiently defined. This study thus aimed to characterize the clinical, cytogenetic, and molecular features of pediatric MECOM-r AML and to explore potential prognostic markers and therapeutic approaches. Methods: We conducted a retrospective international cohort study of pediatric and adolescent/young adult patients (0–21 years) newly diagnosed with MECOM-r AML between 1998 and 2022. Eligible cases harbored inv(3)/t(3;3) or alternative 3q26 rearrangements. Data were collected via 18 national and cooperative pediatric AML study groups. Patients with acute promyelocytic leukemia or myeloid leukemia of Down syndrome were excluded. Karyotypes were centrally reviewed. Descriptive statistics, Kaplan-Meier survival analysis, and multivariate logistic regression were used to assess clinical features and predictors of response and survival. Due to missing data, some analyses were limited to evaluable cases (EC). Results: Of 69 submitted cases, 10 were deemed ineligible as MECOM rearrangement was unconfirmed. Amongst the 59 patient study cohort, median age was 14.3 years (range 0.9–20.8) with equal sex distribution. Median diagnostic bone marrow blast percentage was 65% (range 6–95), although 12% (6/47 ECs) had <20% blasts and may have been classified as myelodysplastic syndrome (MDS) in prior years. Dysmegakaryopoiesis was observed in 61% (16/26 EC). Inv(3)was identified in54% (32/59 ECs), while others harbored rare fusions, most commonly from t(3;12)(q26;q22). Monosomy 7 was detected in 80% (45/56 EC) and FLT3-ITD in 26% (12/46 EC) with similar frequencies across subtypes. Six of 39 patients (15%) presented with diabetes insipidus at diagnosis, four with abnormal hypophyseal imaging. Nearly all patients (55/56 EC) received multi-agent AML chemotherapy. After two induction cycles, 68% of patients with response data (n=38) showed resistant disease. Overall, complete remission (CR) rates were low but similar between inv(3) and non-inv(3) cases (47% vs. 50%). In a multivariate logistic regression, monosomy 7 was significantly associated with resistant disease (OR 6.21, 95% CI 1.08–44.75, p=0.048), while inv(3) showed a non-significant trend toward poorer response (OR 2.60, 95% CI 0.54–14.95, p=0.25). Hematopoietic stem cell transplantation (HSCT) in first CR or without morphologic CR was performed in 67% (35/52) patients; 37% (13/35) survived (7 transplanted in CR, 3 in non-CR, 3 unknown), while 63% (22/35) died post-HSCT (13 due to relapse; toxicity 5; unknown cause 4). Of the 17 patients without primary HSCT, two survived, but both required HSCT subsequent to relapse. Only 29% (15/52 EC) of patients achieved long-term remission with 38% 5-year overall survival (OS). Kaplan-Meier analysis showed no significant survival difference between inv(3)and other MECOM-r subtypes (p=0.20). Cox regression analysis of the subset of 26 patients with evaluable data showed a non-significant increase in hazard of death for inv(3)(HR 2.04, 95% CI 0.72–5.77, p=0.18). Conclusion: This is the largest international cohort of pediatric MECOM-r AML to date and underscores the poor prognosis of this high-risk leukemia subtype also in children. Co-occurring monosomy 7 was a significant independent predictor of chemotherapy resistance. Our data prove that also pediatric MECOM-r AML shares features with MDS, including prominent dysplasia and monosomy 7. As updated in the WHO 2022 classification, blast count is no longer a defining criterion when specific genetic alterations are present. However, since MDS diagnosis was not an inclusion criterion, patients with <20% blasts were likely underrepresented, limiting insight into the full clinical spectrum of MECOM-driven myeloid malignancies. While HSCT remains the only chance for durable remission, OS of patients with MECOM-r AML remains poor. Ongoing molecular and functional analyses are needed to identify cooperating mutations and cellular vulnerabilities potentially amenable to new and more effective therapeutic approaches.

In most study protocols for children with acute myeloid leukemia (AML), treatment response is assessed with flow cytometry (FCM) after one and/or two courses of induction treatment, impacting risk stratification. In AML with RUNX1::RUNX1T1, which is the most common subtype of pediatric AML and generally associated with good prognosis, assessment of treatment response is complicated by two factors: 1) Reverse transcription quantitative PCR (RT-qPCR-MRD) often shows high levels of RUNX1::RUNX1T1 fusion transcripts indicating residual disease. 2) FCM-MRD sometimes detects cells with immature markers such as CD34 or CD117 and atypical immunophenotype, leading to an interpretation as MRD. However, we have observed that such cells lack the diagnostic immunophenotype and instead display basophilic or mast cell markers. In this study, we aimed to improve interpretation of FCM-MRD by characterization of AML with RUNX1::RUNX1T1 with focus on basophils and mast cells. Our study included 45 of 46 children with AML with RUNX1::RUNX1T1 in Sweden, Finland, Norway, Denmark, Hong Kong and Israel during 2013-2020, comprising 22 females and 23 males with median age of 10 years (range 4-17). They were treated according to the NOPHO-DBH AML2012 protocol. FCM-MRD analysis utilized antibodies allowing for identification of basophils as CD123+, HLA-DR-, CD33+ and mast cells as CD117++, HLA-DR-, CD33+. FCM-MRD was performed at diagnosis, day 22 after induction 1, before start of induction 2, and before start of consolidation. Results were compared with 70 children treated in Sweden with the same protocol during the same time period for AML without RUNX1::RUNX1T1. When analyzing children with AML with RUNX1::RUNX1T1 on day 22 after induction 1, both basophils (median 0.64%, range 0-40%) and mast cells (0.44%, 0-29%) were elevated compared with regenerating bone marrow (basophils 0.32%, 0.04-0.69%; mast cells 0.01%, 0.006-0.06%). In 80% of cases, the basophils displayed atypical immunophenotype with lower expression of CD123, CD38, CD11b and CD13 and higher CD34 and CD117, and in 38% mast cells exhibited lower CD117. Levels normalized before consolidation treatment (basophils 0.12%, 0-0.7%; mast cells 0.04%, 0-1.2%). To investigate the impact of these cells, we divided children into those with notably high basophils on day 22, defined as ≥2% (n=16), and those with low basophils, <2% (n=29). The group with high basophils day 22 had more atypical basophils and mast cells already at diagnosis, as well as high levels of mast cells day 22 (median 1.87% vs 0.27%, p=0.007). Leukemic origin of atypical basophils and mast cells at diagnosis was verified using FCM cell sorting and RT-qPCR of RUNX1::RUNX1T1. Children with high basophils did not differ from low basophil cases regarding age, white blood cell count, leukemia-associated immunophenotype, KIT or FLT3 mutation status, or treatment intensity. The clinical outcome was favorable, with only five relapses (11%), with no difference between children with high and low basophils. Similar findings were seen when children were divided into groups of high (≥0.9%, n=18) and low (<0.9%, n=27) mast cells day 22, partly overlapping with basophil groups. This suggests that both basophils and mast cells have leukemic origin but not relapse potential. A comparison with children with other AML (n=70) showed that elevated levels and atypical immunophenotype of basophils and mast cells day 22 was unique to AML with RUNX1::RUNX1T1. To understand the reason for this phenomenon, we investigated the gene expression profile of cases with AML with RUNX1::RUNX1T1 using the TARGET (children), Beat AML 1.0 and TCGA-LAML (both adults) datasets. In all three datasets, AML with RUNX1::RUNX1T1 showed an enrichment of the signature genes of the common myeloid progenitor with eosinophil/mast cell/basophil potential, namely CSF2RB (CD131), CLC, HDC, EPX and IL5RA, compared to other types of AML. This suggests that RUNX1:::RUNX1T1 leukemia can originate in this progenitor. In conclusion, during induction treatment of children with AML with RUNX1::RUNX1T1, basophils and mast cells are often increased. Since they are leukemia-related and often have atypical immature immunophenotype, they might cause concern. However, such cells do not seem to be associated with a worse prognosis but rather reflect an inherent feature of this leukemia and should not be interpreted as MRD.

Abstract BACKGROUND Whole genome sequencing (WGS) is the most informative singular molecular assay in cancer diagnosis. Recent evidence demonstrates that WGS can add diagnostic information and change the management of childhood cancer, and thus is being increasingly employed in clinical settings globally. However, it remains unknown whether WGS can accurately recapitulate existing multi-assay standard-of-care (SOC) genomic testing used in pediatric cancer diagnostics. In this study we evaluate the concordance between WGS and SOC findings from an unselected cohort of children across 8 centres from two healthcare systems (England and Sweden) that offer routine WGS. METHODS We compared WGS and SOC genomic test reports for children under 18 years presenting with new or relapsed cancer between January 2021 and November 2023 across 2 English centres; Cambridge University Hospital and Great Ormond Street Hospital, and 6 Swedish centres; Gothenburg, Karolinska, Linköping, Lund, Umeå and Uppsala. Only WGS findings reported to clinicians were evaluated, without re-analysis of genomes. Tests were described as ‘concordant’ where WGS and SOC were in complete concordance (positive or negative) for all SOC-detected variants. Discordance described occasions where SOC detected findings not identified by WGS. ‘Additional findings’ described cases where WGS provided disease-relevant findings above SOC testing. Only disease-relevant variants were considered in the analysis. RESULTS A cohort of 1032 patients with 1841 SOC molecular tests was included – 436 with haematological malignancies and 596 with solid tumor malignancies (528 from England, and 504 from Sweden). WGS recapitulated 99.3% of SOC tests performed, across all types of genomic alteration (1829/1841). Of the 12 instances of discordance, 3 related to poor WGS sample purity, 5 were gene fusions, 1 low variant allele frequency (0.02) internal tandem duplication, 2 single nucleotide variants and 1 copy-number aberration. WGS provided additional disease relevant findings in 19.7% of cases (203/1032). DISCUSSION Deployment of available SOC genomic testing for cancer diagnostics is highly variable across nations, individual centres and disease entities, and is usually dictated by test availability, cost and likely clinical yield, in a non-agnostic manner. For the first time we demonstrate, across two national systems, that WGS faithfully recapitulates the vast majority of SOC findings irrespective of mutation class, cancer type and variant calling algorithm. Sample quality and intra-tumoral heterogeneity likely account for the few discrepancies observed. Barriers to implementation of routine WGS as the only molecular diagnostic assay for pediatric cancer are cost, analytical expertise, and turnaround time (TAT). Our group is systematically studying the health economic benefits of WGS as a single assay to replace all SOC testing. Finally, an ongoing collaborative project aimed at reducing TAT to under 48 hours using novel technology has demonstrated feasibility in a small number of patients to date. Citation Format: Jonathan Kennedy, Sarah M. Leiter, Angus Hodder, Sheng-Yuan Kan, Jack Bartram, Giuseppe Barone, Michael Gattens, Matthew J. Murray, Sam Behjati, Patrick Tarpey, Matthew Cullen, Antony Ceraulo, Karin Langenberg, Jan Molenaar, Sandra Wessman, Frida Abel, Gustaf Ljungman, Geraldine Giraud, Hakon Anderson Blomstrand, Zdenek Rohan, Anna Staffas, Christina Orsmark-Pietras, Tatjana Pandzic, Irina Golovleva, Linda Fogelstrand, Jonas Abrahamsson, Ulrika Norèn-Nyström, Josefine Palle, Thoas Fioretos, Lucia Cavelier Franco, Gisela Barbany, Nadège Corradini, Gudrun Schleirmacher, Richard Rosenquist, David Gisselsson, Aditi Vedi. Whole genome sequencing can reproduce all standard-of-care diagnostics for childhood cancer: Results from two national systems [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B011.

Background: Central nervous system disease (CNS3) in pediatric acute myeloid leukemia (pAML) is reported in 6% to 29% of cases. However, its impact on event-free survival (EFS) and overall survival (OS) remains uncertain. This study evaluates the effect of CNS involvement at diagnosis on relapse and survival in patients treated on the NOPHO-DBH AML2012 protocol. Methods: Data from 931 pediatric AML patients in the NOPHO-DBH AML2012 protocol were analyzed, comparing outcomes, relapse rates, and survival between those with and without CNS disease. CNS-directed therapy included intensified intrathecal chemotherapy without irradiation. Results: Of 922 patients with available CNS status, 10.9% had CNS3 at diagnosis. CNS3 patients were younger (median age 3.5 vs. 8 years,P=0.001) with higher white blood cell counts (56.1x109/L vs 18.7x109/L,P<0.001) and higher frequency of other extramedullary disease (30.4% vs 11.3%,P<0.001) and inv(16)(P<0.001). EFS5y was 71.5% for CNS-positive patients vs. 62.7% for CNS-negative (p=0.14), and OS5y was 81.4% vs. 79.3% (p=0.54). Patients with CNS disease had a lower cumulative incidence of relapse (15.6% vs 26.5%,P=0.023), and CNS relapse occurred in 0.9% and 0.7% of patients with and without CNS disease. Conclusion: CNS disease at diagnosis in pAML does not adversely affect survival or treatment response.

PURPOSE Measurable residual disease (MRD) by using flow cytometry after induction therapy is strongly prognostic in pediatric AML, and hematopoietic stem-cell transplant (hSCT) may counteract a poor response. We designed a phase III study with intensified response-guided induction and MRD-based risk stratification and treated poor induction response with hSCT. The efficacy of liposomal daunorubicin (DNX) in induction was compared with mitoxantrone. METHODS The study planned to randomly assign 300 patients, but the production of DNX ceased in 2017. One hundred ninety-four patients were randomly assigned to mitoxantrone or experimental DNX in induction 1. Ninety-three non–randomly assigned patients served as an observation cohort. Primary end point was fraction of patients with MRD <0.1% on day 22 after induction 1. Patients with MRD ≥15% after induction 1 or ≥0.1% after induction 2 or FLT3-ITD with NPM1 wildtype were stratified to high-risk therapy, including hSCT. RESULTS Outcome for all 287 children was good with 5-year event-free survival (EFS5y) 66.7% (CI, 61.4 to 72.4) and 5-year overall survival (OS5y) 79.6% (CI, 75.0 to 84.4). Overall, 75% were stratified to standard-risk and 19% to high-risk. There was no difference in the proportion of patients with MRD <0.1% on day 22 after induction 1 (34% mitoxantrone, etoposide, araC [MEC], 30% DNX, P = .65), but the proportion increased to 61% for MEC versus 47% for DNX ( P = .061) at the last evaluation before induction 2. EFS5y was significantly lower, 56.6% (CI, 46.7 to 66.5) versus 71.9% (CI, 63.0 to 80.9), and cumulative incidence of relapse (CIR) was higher, 35.1% (CI, 25.7 to 44.7) versus 18.8% (CI, 11.6 to 27.2) for DNX. The inferior outcome for DNX was only in standard-risk patients with EFS5y 55.3% (CI, 45.1 to 67.7) versus 79.9% (CI, 71.1 to 89.9), CIR 39.5% (CI, 28.4 to 50.3) versus 18.7% (CI, 10.5 to 28.7), and OS5y 76.2% (CI, 67.2 to 86.4) versus 88.6% (CI, 81.4 to 96.3). As-treated analyses, including the observation cohort, supported these results. For all high-risk patients, 85% received hSCT, and EFS5y was 77.7 (CI, 67.3 to 89.7) and OS5y was 83.0 (CI, 73.5 to 93.8). CONCLUSION The intensification of induction therapy with risk stratification on the basis of response to induction and hSCT for high-risk patients led to improved outcomes. Mitoxantrone had a superior anti-leukemic effect than liposomal daunorubicin.

Allopurinol can be used in maintenance therapy (MT) for pediatric acute lymphoblastic leukemia (ALL) to mitigate hepatic toxicity in patients with skewed 6-mercaptopurine metabolism. Allopurinol increases the erythrocyte levels of thioguanine nucleotides (e-TGN), which is the proposed main mediator of the antileukemic effect and decreases methyl mercaptopurine (e-MeMP) levels, associated with hepatotoxicity. We investigated the effects of allopurinol in thiopurine methyltransferase (TPMT) wild-type patients without previous clinical signs of skewed 6-mercaptopurine metabolism. Fifty-one patients from Sweden and Finland were enrolled in this prospective before-after trial during ALL MT. Mean e-TGN increased from 280 nmol/mmol hemoglobin (Hb) after 12 weeks of standard MT to 440 after 12 weeks of MT with addition of allopurinol 50 mg/ m2 (P<0.001). Mean e-MeMP decreased simultaneously from 9,481 nmol/mmol Hb to 2,791 (P<0.001) and mean alanine aminotransferase declined by almost 50%. Primary endpoint, defined as e-TGN >200 nmol/mmol Hb, was reached for 91% of the patients after 12 weeks of allopurinol (week 25) compared to 67% before (week 13) (P<0.001). This level was chosen as the median e-TGN in a previous NOPHO ALL-2008 study was just below 200 nmol/mmol Hb. During weeks on allopurinol a slightly higher proportion of the patients had a white blood cell count within target 1.5-3.0×109/L. Allopurinol did not increase severe adverse events and no life-threatening events were reported. In conclusion, allopurinol add-on treatment is safe and leads to increased e-TGN and reduced e-MeMP also in ALL-patients without previous signs of skewed thiopurine metabolism and is a promising approach to increase antileukemic effect and reduce toxicity.

Hyperleukocytosis in pediatric acute myeloid leukemia (AML) is associated with severe complications and an inferior outcome. We report results on patients with hyperleukocytosis included in the NOPHO-DBH AML 2012 study. We recommended immediate initiation of full-dose chemotherapy (etoposide monotherapy for 5 days as part of the first course), avoiding leukapheresis and prephase chemotherapy. Of 714 patients included in the NOPHO-DBH AML 2012 study, 122 (17.1%) had hyperleukocytosis, and 111 were treated according to the recommendations with etoposide upfront without preceding leukapheresis or prephase chemotherapy. The first dose was applied the same day as the AML diagnosis or the day after in 94%. Etoposide was administered via peripheral veins in 37% of patients without major complications. After initiation of etoposide the white blood cell counts on days 2-5 were 69%, 36%, 17% and 8%, respectively, of the pre-treatment level. On day 3, 81% of patients had a white blood cell count <100 x109/L. Five-year event-free and overall survival rates for all patients with hyperleukocytosis were 52.9% (95% confidence interval [95% CI]: 44.4-63.0) and 74.1% (95% CI: 66.4-82.6), compared to 64.9% (95% CI: 60.9-69.1) and 78.9% (95% CI: 75.4-82.4) for patients without hyperleukocytosis (P<0.001 for event-free survival, P=0.1 overall survival). Six-week early mortality was 4.1% for all patients with hyperleukocytosis (2.7% for the 111 patients treated with etoposide upfront). We conclude that management of hyperleukocytosis in pediatric AML with immediate etoposide monotherapy without leukapheresis or prephase chemotherapy is feasible, safe and effective. The reduction in white blood cell count during the first days is comparable to the reported results of leukapheresis, and outcomes seem at least equivalent to therapies including leukapheresis. Based on our results, we advocate abandoning leukapheresis for hyperleukocytosis in pediatric AML. Instead, it is crucial to start induction chemotherapy as early as possible.

Acute myeloid leukemia (AML) with t(7;12)(q36;p13) is a recurrent translocation in AML in infants or very young children and was recently included in the World Health Organization (WHO) Classification of Hematolymphoid Tumors. AML with t(7;12) is reported to involve MNX1 and ETV6 signaling; however, the mechanism of leukemogenesis is not well understood, and the presence of MNX1::ETV6 fusion transcripts has only been confirmed in approximately 50% of cases. In contrast, high expression of MNX1 has been seen in all investigated cases. In this study, we investigated the clinical as well as biological characteristics of 12 pediatric AML with t(7;12) and performed whole transcriptome (WTS) and whole genome sequencing (WGS) on six of these. There was no significant difference in event-free survival or overall survival of these t(7;12) AML patients compared with other AML in the same age group. Interestingly, WTS identified several fusion transcripts involving ETV6 but not together with MNX1. WGS identified the genomic breakpoints and revealed that a common fusion partner on chromosome 7 was NOM1. Principal component analysis (PCA) of the WTS data showed that all t(7;12) AML cases cluster together, separate from all other pediatric AML subtypes; all cases had high expression of MNX1, MNX1-AS1, and MNX1-AS2. Hence, t(7;12) AML, despite expressing different fusion transcripts and with varying translocation breakpoints, constitutes a phenotypically homogenous subgroup. This underlines that the leukemia-driving event most likely is ectopic expression of MNX1 and that this therefore should be the defining Classifying criteria of this type of AML.

Background: In children with acute myeloid leukemia (AML), treatment intensification based on measurable residual disease (MRD) detected by multiparameter flow cytometry (MFC) shows promising results. However, a substantial proportion of patients with no detectable MRD by MFC relapse, indicating that more sensitive methods for MRD analysis are needed. Aims: Our aim was to investigate the feasibility of targeted deep sequencing for analysis of MRD during treatment of children with AML. We assessed the sensitivity of targeted deep sequencing as compared to MFC and characterized the genetic and protein signature on the single cell level to understand the nature of residual mutated cells. Methods: Bone marrow samples were collected from children with AML. Leukemia-specific mutations for tracking were identified in diagnostic samples by whole exome sequencing of sorted leukemic cells. Samples from day 22 and before start of second induction and first consolidation courses were assessed with patient-tailored targeted deep sequencing with a limit of detection of 0.02% variant allele frequency. Results were compared to parallel MRD analyses with 8 color MFC with a limit of detection of 0.1% cells, and when applicable RT-qPCR. Single cell multi-omics analysis was performed using the Tapestri platform with 45 primer-barcoded antibodies for cell surface proteins and customized mutation-specific primers for DNA, followed by sequencing. Results: In diagnostic samples, at least one leukemia-specific mutation suitable for tracking was detected in 25/27 patients; most often both clonal and subclonal mutations (average 12 mutations per case). For these children (of which four eventually relapsed), targeted deep sequencing assays to track mutations in samples during treatment were designed for clonal mutations in any gene, and subclonal mutations in cancer-associated genes (in total 74 mutations). When compared to MFC results, targeted deep sequencing was more sensitive; 50% of analyzed samples were positive for at least one mutation with targeted deep sequencing but negative with MFC, 44% were positive with both methods, 6% negative with both methods and none negative with targeted deep sequencing and positive with MFC. In 8 patients assessed for MRD also by RT-qPCR of leukemic transcript, targeted deep sequencing and RT-qPCR showed similar performance. Mutations were detected at the end of induction in patients that eventually relapsed, but also in some patients not experiencing relapse. Therefore, we aimed to characterize residual mutated cells. We used single cell multi-omics analysis on 15 samples from 7 patients (6 diagnostic, 8 during treatment and one relapse) to enable tracking of mutated residual cells and describing protein expression and copy number aberrations. Protein expression signatures were verified using a normal sample with MFC performed in parallel. Our analysis provided detection of MRD on a single cell level, as well as phenotypic characterization of cells harboring leukemia-specific mutations. The detected patterns were patient-specific and included subclonality, as indicated by whole exome and targeted deep sequencing, and the presence of a preleukemic clone in stem/progenitor cells. Summary/Conclusion: Targeted deep sequencing enables a more sensitive detection of MRD than MFC and is applicable in most children with AML. Residual mutated cells during treatment can be relapse-causing but also pre-leukemic. Further studies are needed to address the prognostic value of highly sensitive molecular MRD in childhood AML. Keywords: OMICS, Quantitative molecular analysis, Acute myeloid leukemia, MRD

Supplementary Figure Legend from Appearance of the Novel Activating F1174S ALK Mutation in Neuroblastoma Correlates with Aggressive Tumor Progression and Unresponsiveness to Therapy

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 × 109 /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.

Risk of treatment‐related life‐threatening toxicity is high in childhood acute myeloid leukaemia (AML), and access to intensive care units (ICU) is crucial. We explored the ICU admission rate and outcome after intensive care in childhood AML in Sweden.

BACKGROUND:Robot-assisted nephroureterectomy (RANU) is the primary treatment for upper tract urothelial carcinoma (UTUC) at our hospital for patients with clinical stage less than T2, and for patients with invasive tumours, but unfit for major surgery.OBJECTIVE:To assess peri-operative conditions and outcomes of RANU at our unit, and to evaluate the safety of the procedure.METHODS:The medical records of all 166 patients undergoing RANU for suspected UTUC and followed for more than three months in a large university hospital in Sweden were reviewed retrospectively. After the exclusion of twenty patients because of previous cystectomy, simultaneous surgical procedure, or other tumour types than UTUC in the pathological report, 146 patients remained for the analyses. The primary endpoint was complication rate according to Clavien-Dindo at 90 days. Secondary endpoints were perioperative bleeding, violation of oncological surgical principles, hospital stay, and re-admission within 90 days.RESULTS:The median age was 75 [(Inter Quartile Range) IQR 70-80] years and 57% of the patients had an ASA score above 2. According to Clavien-Dindo, one patient had a grade 3 complication, and no patient had a grade 4-5 complication. The median blood loss was 50 (IQR 20-100) ml and the median hospital stay was 6 (IQR 5-7) days. Twelve patients were re-admitted to the hospital within 90 days (eight with urinary tract infection/haematuria, one with hematoma, and three with other diseases).CONCLUSION:Robot-assisted nephroureterectomy is a safe procedure for patients with upper tract urothelial carcinoma, with a low risk of major surgical complications.

Risk of treatment-related life-threatening toxicity is high in childhood acute myeloid leukaemia (AML), and access to intensive care units (ICU) is crucial. We explored the ICU admission rate and outcome after intensive care in childhood AML in Sweden. Patients diagnosed between 2008 and 2016 were identified from the Swedish Childhood Cancer Registry (SCCR), a national quality registry. Data from SCCR was cross-referenced with clinical questionnaire data from paediatric oncology centers and the Swedish Intensive Care Registry (SIR), another national quality registry. According to combined data, 46% of the children (58/126) were admitted to ICU, 17% (21/126) within 1 month from diagnosis. Overall, ICU mortality per admission was 12% and 6% during first-line treatment. There was a discrepancy between admission rate from the clinical questionnaires and SCCR (29%; 36/126 children) and SIR (44%; 55/126) All deaths during first-line treatment occurred at or after ICU care. Although admission rate under AML treatment was high, the treatment-related mortality under first-line treatment was low. No child died under first-line treatment without admission to ICU, suggesting good availability. The discrepancy between the two registries, SCCR and SIR, highlights the need for future validation of registry data.

Cisplatin-based combination chemotherapy (CHT) improves survival in patients with muscle-invasive (MIBC) and metastatic urothelial cancer (mUC). Here we map the cellular landscape in MIBC and mUC and explore its associations to outcome.