ABSTRACT:Acute myeloid leukemia (AML) is driven by diverse genetic abnormalities. We investigated clinical and molecular differences between clinically defined secondary AML following antecedent MDS, molecularly defined secondary type AML (st-AML), molecularly defined MDS/AML (st-MDS/AML; 10%-19% blasts) and other newly diagnosed AML (de novo AML). We also examined the prognostic value of molecular measurable residual disease (MRD) in st-AML. This retrospective cohort study included 2684 intensively treated patients with AML. Diagnostic (n = 2684) and complete remission (CR; n = 436) samples were sequenced using a 54-gene panel targeting frequently mutated genes in AML. Odds ratios were calculated to show the association between mutated genes and clinically defined sAML or de novo AML. Clinical outcomes of interest were overall survival (OS) and cumulative incidence of relapse (CIR). Not only the established mutations in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 but also ETV6 was significantly associated with clinically defined sAML, which defined the molecular signature for st-MDS/AML and st-AML. No OS differences were observed between st-MDS/AML and st-AML. Molecularly defined st-AML, now combined with st-MDS/AML, had worse OS compared with ELN2022 favorable- (5-year OS 39.9% vs 70.4%; P< .001) and intermediate-risk (5-year OS 39.9% vs 48.9%; P = .005) patients with AML. MRD based solely on secondary type mutations lacked predictive value, whereas MRD of non-DTA mutations in CR was associated with increased CIR in st-AML (subdistribution hazard ratio [SHR] 3.25; P< .001). Molecularly defined st-AML, including st-MDS/AML, defines a distinct AML category with a unique genetic, clinical and treatment response profile, in which next-generation sequencing (NGS)-based MRD holds markedly prognostic significance.

Therapy-related acute myeloid leukemia (t-AML) and AML with myelodysplasia-related changes (AML-MRC) are associated with poor outcomes. The liposomal formulation of cytarabine and daunorubicin (CPX-351) improved complete remission (CR) and CR with incomplete hematologic recovery (CRi) rates and overall survival (OS) compared with ‘standard’ induction (7+3) chemotherapy in a phase-III trial for patients aged 60-75 years. However, 7+3 dosing varies among trials and in clinical practice and it remains unknown whether CPX-351 is superior to 7+3 double-induction regimens including intermediate-dose cytarabine, as the one employed in the HOVON-SAKK-Nordic clinical trials. To address this question, we conducted a post-hoc analysis on t-AML/AML-MRC patients aged ≥60 years enrolled in three HOVON-SAKK-Nordic trials and defined a subset of patients that met the eligibility criteria of the CPX-351 trial and compared their outcomes with those of the CPX-351 arm using reconstructed survival data. CR/CRi rates were higher in the higher-intensity 7+3 cohort (67.8%) compared with CPX-351 (47.7%) with similar median OS between the two cohorts (10.1 months versus 8.9 months respectively, HR = 0.99; 95% CI 0.78-1.26, p=0.95). Thirty-day mortality (4.4% for higher-intensity 7+3 versus 5.9% for CPX-351) and adverse events, including febrile neutropenia (61% for higher-intensity 7+3 versus 68% for CPX-351), were comparable. The data suggest that obligatory double-induction may achieve outcomes similar to CPX-351 in these patients and provide a strong rationale for ongoing clinical trials comparing these regimens.

Novel therapies are needed for patients with multiple myeloma (MM) and extramedullary plasmacytomas. The prospective, Phase II EMN19 study assessed the efficacy and safety of daratumumab plus bortezomib, cyclophosphamide, and dexamethasone (DaraVCD) in 40 patients with newly diagnosed MM (NDMM; n = 29) or at first relapse (RMM; n = 11) and positron emission tomography or computed tomography (PET/CT)-confirmed extramedullary plasmacytomas (extraosseous [EMD] and/or paraosseous [PS]). DaraVCD was administered until disease progression or up to 3 years. The primary endpoint was hematological complete response (CR). Among patients, 22 (55.0%), 4 (10.0%), and 14 (35.0%) had EMD, EMD/PS, and PS plasmacytomas, respectively. Median patient age was 58.0 years, and 16 (40.0%), 12 (30.0%), and 10 (25.0%) patients were at International Staging System (ISS) Stages I, II, and III, respectively. Median circulating tumor cell (CTC) level was 0.002% (range, 0.000-0.353), significantly higher (P < 0.05) in patients with ISS Stage III and those with plasma cells > 60%. At a median follow-up of 30.0 months, all patients completed treatment (median duration: 19.8 months). The overall hematologic ≥CR rate was 47.5% (19/40; NDMM patients: 58.6% [17/29]; RMM patients: 18.2% [2/11]). Of patients with ≥CR, 80.0% (15/19) achieved minimal residual disease (MRD) negativity, and 68.4% (13/19) combined MRD negativity and complete metabolic response (CMR) on PET/CT. The overall median progression-free survival was 25.8 months, significantly longer in patients achieving hematologic ≥CR and/or CMR than others (not reached and 4.8 months, respectively; P < 0.001). DaraVCD showed encouraging efficacy in patients with MM and extramedullary plasmacytomas. Notably, this is the first report on CTC levels in EMD, and they were lower than previously reported NDMM thresholds.

Background: The IGH locus is susceptible to translocations or insertions that contribute to B-cell precursor acute lymphoblastic leukemia (ALL) by ectopic or enhanced expression of a gene relocated to the IGH enhancer. The frequency of IGH rearrangements is relatively high in Down syndrome (DS) ALL. IGH rearrangements can be cryptic and might not be detected as a chimeric transcript, hence, their frequency, partner genes and prognostic value are largely unknown. Methods: We performed RNA-sequencing and IGH break-apart fluorescent in-situ hybridization (FISH) to determine the genetic and clinical characteristics of IGH rearrangements in 50 DS ALL patients. Results: We identified 10 patients with a chimeric IGH transcript and another 22 IGH-rearranged patients solely by FISH. The IGH rearrangement was clonal (>= 50 % of leukemic cells) in 11 cases and subclonal (10-50 % of cells) in 21 cases. Almost one-third of the subclonal IGH rearrangements co-occurred with known oncogenic driver aberration. The partner gene was identified in 16 cases and the most frequent partners were CEBPD (n = 6) and CRLF2 (n = 4). A trend towards a worse event-free survival was seen for DS ALL patients with a clonal IGH rearrangement (clonal: HR 3.34, p = 0.053; subclonal: HR 1.80, p = 0.31) compared with DS ALL patients without an IGH rearrangement. Conclusion: By combining RNA-sequencing and FISH, we identified IGH rearrangements in 64 % (n = 32) of DS ALL. A clonal IGH rearrangement (22 %) may point to an unfavorable outcome in DS ALL.

The transcription factor MECOM, located at 3q26, is essential for hematopoietic stem cells in healthy individuals. Enhancer translocations, due to 3q26 rearrangements, drive out-of-context MECOM expression in one of the most aggressive subtypes of acute myeloid leukemia (AML). Aberrantly expressed MECOM is essential for the survival and immature phenotype of these leukemia cells. Direct depletion of MECOM using an endogenous auxin-inducible degron immediately upregulates expression of CEBPA, which encodes a transcription factor required for neutrophil development and is frequently mutated in other AML subtypes. MECOM depletion is accompanied by a severe loss of CD34 and gain of mature myeloid cell surface marker CD15. MECOM exerts its inhibitory effect on differentiation by binding to the +42-kilobase CEBPA enhancer. This is partially dependent on the interaction between MECOM and its corepressor CTBP2. We demonstrate that CEBPA overexpression can bypass the MECOM-mediated block of differentiation. In addition, patients with AML with MECOM overexpression through enhancer hijacking show significantly reduced CEBPA levels. Our study directly connects 2 major players in normal and malignant hematopoiesis, MECOM and CEBPA, and unveils how MECOM maintains self-renewal by repressing CEBPA-induced differentiation.

Acute leukemia of ambiguous lineage (ALAL) is a rare, poor-prognosis acute leukemia subtype that cannot be assigned to a single hematopoietic lineage. Although ALAL patients are typically treated with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) regimens, optimal treatment choice is hindered by their lineage ambiguity. Therefore, we investigated the added value of transcriptomics for improving lineage assignment, currently based mainly on surface markers. First, we used an in-house pipeline to detect genetic lesions in RNA sequencing data (n = 30) with a sensitivity > 90% for small variants. Second, we compared ALAL gene expression profiles (GEPs) with representative AML (n = 145), B-ALL (n = 223), and T-ALL (n = 85) cases. In a principal component analysis (PCA), ALALs did not form a clear separate group, as most clustered with AML, B-ALL, or T-ALL. Accordingly, a machine learning classifier trained with GEPs of acute leukemias segregated 27/30 ALALs into myeloid-, B-, or T-lymphoid. These 27 cases harbored genetic abnormalities consistent with the classifier-assigned leukemia. Furthermore, deconvolution of ALAL GEPs revealed enrichment for signatures of normal hematopoietic cells corresponding to the leukemic type predicted by our algorithm. The classifier was also applied on an external ALAL cohort (n = 24), assigning 75% of the patients to a lineage matching their immunophenotypic and methylation profiles. In conclusion, integrative analysis of RNA sequencing data can accurately classify most ALAL cases as lineage-defined, while others show true transcriptional and epigenetic ambiguity driven by lesions like BCL11B. The pipeline and classifier developed here are valuable tools to improve ALAL diagnosis and guide therapeutic decisions.

Most patients with acute myeloid leukemia (AML) may obtain remission upon induction chemotherapy, but relapse is frequent and associated with poor survival. Previous prognostic models for outcomes after relapse lacked analysis of comprehensive molecular data. A validated prognostic model integrating clinical, cytogenetic, and molecular variables may support treatment decisions. We studied 943 patients with AML who relapsed after intensive induction treatment in a development cohort (HOVON-SAKK). A random survival forest algorithm was used to evaluate the association of clinical parameters, cytogenetic abnormalities, and molecular variables at diagnosis with overall survival (OS). Relapsing patients (n = 377) who were enrolled in the NCRI-AML18 trial were used for validation. In the development cohort, the median age at relapse was 58 years, and patients were classified as 2022 European LeukemiaNet favorable (22%), intermediate (31%), and adverse risk (48%). One-third underwent allogeneic transplantation in the first complete remission. Variable selection yielded 9 variables associated with 1-year OS, including relapse-free interval, age, white blood cell count, mutated TP53, FLT3 internal tandem duplication, core-binding factor abnormalities, t(v;11q23)/KMT2A rearrangement, and complex/monosomal karyotype, which were assigned points according to their estimated hazard ratios. Three prognostic groups were defined with distinct 1-year OS in both development (favorable, 51% ± 3%; intermediate, 29% ± 3%; and poor, 14% ± 2%, respectively) and validation cohorts (51% ± 4%, 26% ± 5%, and 14% ± 3%, respectively). Validation confirmed the improved accuracy in predicting outcomes for patients with AML in first relapse. The revised AML relapse model improved on previous prognostic models for outcomes after first relapse. It provides stratification that might support tailoring second line treatment.

PURPOSE AML is a genetically heterogeneous disease, particularly in older patients. In patients older than 60 years, survival rates are variable after the most important curative approach, intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (allo-HCT). Thus, there is an urgent need in clinical practice for a prognostic model to identify older patients with AML who benefit from curative treatment. METHODS We studied 1,910 intensively treated patients older than 60 years with AML and high-risk myelodysplastic syndrome (HR-MDS) from two cohorts (NCRI-AML18 and HOVON-SAKK). The median patient age was 67 years. Using a random survival forest, clinical, molecular, and cytogenetic variables were evaluated in an AML development cohort (n = 1,204) for association with overall survival (OS). Relative weights of selected variables determined the prognostic model, which was validated in AML (n = 491) and HR-MDS cohorts (n = 215). RESULTS The complete cohort had a high frequency of poor-risk features, including 2022 European LeukemiaNet adverse-risk (57.3%), mutated TP53 (14.4%), and myelodysplasia-related genetic features (65.1%). Nine variables were used to construct four groups with highly distinct 4-year OS in the (1) AML development, (2) AML validation, and (3) HR-MDS test cohorts ([1] favorable: 54% ± 4%, intermediate: 38% ± 2%, poor: 21% ± 2%, very poor: 4% ± 1%; [2] 54% ± 9%, 43% ± 4%, 27% ± 4%, 4% ± 3%; and [3] 54% ± 10%, 33% ± 6%, 14% ± 5%, 0% ± 3%, respectively). This new AML60+ classification improves current prognostic classifications. Importantly, patients within the AML60+ intermediate- and very poor-risk group significantly benefited from allo-HCT, whereas the poor-risk patients showed an indication, albeit nonsignificant, for improved outcome after allo-HCT. CONCLUSION The new AML60+ classification provides prognostic information for intensively treated patients 60 years and older with AML and HR-MDS and identifies patients who benefit from intensive chemotherapy and allo-HCT.

Background: Chromosome 21 is affected in ∼60% of paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients and includes somatic and constitutional gains, intrachromosomal amplification of chromosome 21 (iAMP21), and the translocation t(12;21) resulting in the ETV6::RUNX1 gene fusion. Methods: Since these numeric and structural chromosome 21 alterations are not targetable, we studied the type and frequency of yet-proven targetable events co-occurring with chromosome 21 alterations. Results: Among 307 primary paediatric BCP-ALL cases, JAK/STAT pathway lesions were most frequent in patients with constitutional gain of chromosome 21 (Down syndrome ALL; 35/71, 49%) and iAMP21 (9/22, 41%). RAS pathway lesions were most frequent in high hyperdiploidy (62/108, 57%) and FLT3 lesions were most frequent in iAMP21 (7/22, 32%). Virtually all cases expressed CD19 and CD22 at the cell surface. Positivity for CD20 surface expression ranged from 67% in iAMP21 (8/12) to 20% in ETV6::RUNX1 (26/129). Conclusion: Activated JAK/STAT, RAS or FLT3 signalling, and CD marker surface expression may provide targetable treatment options for the majority of chromosome 21-altered BCP-ALL cases.

Inotuzumab ozogamicin (InO) is a CD22-directed antibody conjugated with calicheamicin. The phase IB of the ITCC-059 trial tested InO combined with chemotherapy in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Relapsed /refractory CD22+ BCP-ALL pediatric patients were enrolled. The primary objective was to establish the recommended phase II dose (RP2D). Secondary objectives included preliminary efficacy and tolerability. InO was combined with 1.5 mg/m2 of vincristine (days 3, 10, 17, 24), 20 mg/m2 of dexamethasone (2 5-day blocks, then amended), and intrathecal therapy. A rolling-6 design was used testing InO from 0.8 to 1.8 mg/m2/cycle. Between May 2020 and April 2022, 30 patients were treated, and 29 were evaluable for dose limiting toxicities (DLT). At 1.1 mg/m2/cycle, two of four patients had DLT (liver toxicity). InO was de-escalated to 0.8 mg/m2/cycle (N=6) without DLT while awaiting a protocol amendment to reduce dexamethasone dose to 10 mg/m2. Post amendment, InO was re-escalated to 1.1 mg/m2/cycle (N=6, 1 DLT), then to 1.4 mg/m2/ cycle (N=3, no DLT), and finally to 1.8 mg/m2/cycle (N=7, 1 DLT). Three additional patients were treated in an expansion cohort. The pooled response rate was 80% (24/30; 95% confidence interval [CI]: 61.4-92.3) and, among responders, 66.7% achieved minimal residual disease negativity. The RP2D of InO combined with vincristine, dexamethasone and intrathecal therapy was declared at 1.8 mg/m2/cycle (1.5 mg/m2/cycle after remission) in a fractioned schedule. This combination showed a response rate similar to the single agent cohorts of this trial, with liver toxicity issues at the initial higher dexamethasone dose (clinicaltrials gov. Identifier: NTR5736).

Introduction: Multiple myeloma (MM) with extramedullary disease (EMD) is an aggressive disease that requires innovative treatment strategies. Methods: The ongoing multinational, open-label phase 2 EMN19 trial (NCT04166565) enrolled patients (pts) with MM and EMD either as newly diagnosed MM (NDMM) or at first relapse (RMM). Pts received Daratumab (Dara) in combination with bortezomib (V), Cyclophosphamide (C), and Dexamethasone (D) (DaraVCD), either until disease progression or for a maximum of 36 months, along with autologous stem cell transplantation. Response to treatment was assessed using both the International Myeloma Working Group (IMWG) and the Impetus criteria for evaluating the EMD responses. Complete metabolic response (CMR) was defined as: Deauville scores 1, 2 or 3, with absence of FDG-avid bone marrow lesion(s), irrespective of a persistent mass on CT, or complete disappearance of EMD. Partial metabolic response as: Deauville score of 4 or 5 with decreased uptake compared to baseline and absence of structural progression development on CT or 50% reduction in EMD size. MRD assessment was performed locally at the time of CR or at an earlier time point, as per clinical practice. Pts without MRD assessment were included in the denominator when estimating the MRD(-) rate. Pts who had discontinued treatment by Cycle 4 were considered as non-responders (both in terms of hematologic and EMD response). Progression-Free Survival (PFS) was analyzed by the Kaplan-Meier method. Cox regression analysis with Firth's correction was used for the estimation of the hazard ratios (HRs) and 95% confidence intervals (CIs). Results: A cohort of 40 pts (73% NDMM; 28% RMM) was enrolled. The mean age was 58 years, with 22 (55%) of the pts being male. At baseline, 33 (83%) of the pts were in stage ≤II of the International Staging System and 6 (15%) were classified as high-risk according to FISH (t(4;14), t(14;16) and/or del17p). Among the pts, 22 (55%) had extramedullary plasmacytomas (PCTs), 14 (35%) had paraosseous involvement, and 4 (10%) had both. The median number of PCTs per pt was 2. At the cut-off date with a median follow-up (FU) of 23 months, 17 (43%) pts were still on treatment, while 23 (58%) had discontinued treatment mainly due to disease progression (13, 33%), inadequate response at the end of cycle 3 (5, 13%) or death (3, 8%). In the overall population, deepest responses were as follows: MRD(-) was achieved by 15 (38%) pts, ≥Complete response (CR) by 18 (45%) pts, ≥VGPR by 30 (75%) pts with an overall response rate (ORR) of 80% (32 pts). The median (range) time to MRD(-) was 19 months (4-33); 13 of the 15 MRD(-) pts were NDMM. Median PFS (24-months PFS rate) was 26 months (56%) in NDMM and 15 months (36%) in RMM. Overall, 28/40 (70%) pts had a concurrent hematologic and EMD response of partial response (PR) or better; the best hematologic responses achieved among those pts were: MRD(-) (15 pts), ≥CR (16 pts), VGPR (10 pts) and PR (2 pts). Additionally, 4/40 pts had a response of ≥PR (1 stringent CR, 1 CR and 2VGPR) without achieving an EMD response, while 8/40 pts had discontinued treatment by cycle 4 and did not have a hematologic or EMD response. The combinations of hematologic and EMD responses for 28 pts with concurrent hematologic and EMD response are shown in Figure 1. As seen in Figure 2, pts with concurrent hematologic CR (hemCR) and CMR had the best outcomes in terms of PFS, while having only one type of response (hematologic or EMD) was associated with inferior outcomes compared to having a response in and outside of marrow (p<0.001 overall and in NDMM). Overall, the respective HRs (95% CI) were: no response vs both types: 20.5 (3.5-119.8), p<0.001; one type vs both types: 8.3 (1.2-58.2), p=0.034. The median (range) time to first concurrent hemCR and CMR was 6 months (3-15); for pts achieving only one type of response (hemCR or CMR) the median (range) time to first response was 5 months (1-11). Conclusions: After a median FU of 23 months of DaraVCD treatment, 33% of the pts have achieved both deep hematological responses with MRD(-) and CMR resulting in a median PFS of 20 months. However, having only one type of response (hemCR or CMR) is associated with an inferior outcome. These results, in terms of response, are comparable to those found in the LYRA study in which NDMM EMD pts are treated using a similar DaraVCD protocol. Even in this high-risk population of unmet need, long-term deep responses can be achieved with DaraVCD in and outside of marrow.

Introduction Despite high rates of complete remission (CR) after intensive induction chemotherapy in newly diagnosed AML, relapse is frequent and associated with poor outcome with a median overall survival (OS) of 3-5 months. While cytogenetic and molecular markers are associated with outcome in newly diagnosed patients, the prognostic value in the relapsed setting is unclear, even though it may facilitate therapeutic decision making. Genetic data was only limitedly available when earlier models for relapsed patients were developed, such as reported by HOVON-SAKK (Breems, JCO 2005) and GOELAMS (Chevallier, Leukemia, 2011). Therefore, a comprehensive analysis of clinical, cytogenetic and molecular variables in a prognostic model for outcome after relapse is needed. We set out to evaluate the impact of these variables on outcome after first relapse in the current therapeutic era, and develop a new prognostic model and compare that model with earlier models. Methods A total of 946 (44%) relapsing patients out of 2127 newly diagnosed AML patients who participated in prospective HOVON-SAKK trials of intensive induction chemotherapy between 2000-2018 were included in this analysis. A random survival forest machine learning algorithm was used to identify top markers impacting 1 year survival after relapse among 45 candidate predictors, including 7 clinical parameters, and 9 cytogenetic and 29 molecular abnormalities present at diagnosis. Cox regression with backward selection was performed to obtain hazard ratios (HRs) from relevant predictors identified by the random survival forest algorithm. A clinical prognostic tool was developed with points assigned to each marker based on the rounded logarithm of HR. Four groups were defined based on quartiles of the individual predictions. Prognostic accuracy of risk models was assessed using Harrel's C-index. Results The median age of relapsed patients was 58 years (range: 18-81), with 56% being male. Adverse risk AML according to ELN2022 risk was found in 48% of patients, whereas favorable and intermediate risk were classified in 22% and 31%, respectively. Prior allogeneic stem cell transplantation (SCT) in first CR was performed in 33% of patients, while 14% received autologous SCT. The median time between CR and relapse was 14 months (range: 0-124). The median follow-up time after relapse for patients alive was 49 months (range: 0-124). The random survival forest identified 16 parameters that were associated with 1 year OS based on variable importance, including: age at relapse ≥60 years, relapse-free interval ≤1 year, white blood cell count ≥10 x 10e9/L at diagnosis, prior allogeneic SCT, absence of t(8;21)/inv16, and presence of complex or monosomal karyotype (CK/MK), -5 or del(5q), -7, t(v;11q23), chromosome 17 alterations (-17, del(17p) or abn(17p)), FLT3-ITD, and mutations found in either TP53 with VAF ≥10%, DNMT3A, EZH2, NPM1, or NRAS. From these parameters, nine remained significant with Cox regression: age (HR 1.48, p<0.01), relapse free interval ≤1 year (HR 1.74, p<0.01), white blood cell count ≥10 x 10e9/L at diagnosis (HR 1.23, p=0.02), prior allogeneic SCT (HR 1.58, p<0.01), CK/MK (HR 1.53, p<0.01), no t(8;21)/inv(16) (HR 2.34, p<0.01), t(v;11q23) (HR 1.51, p=0.02), mutated TP53 with VAF ≥10% (HR 1.70, p<0.01), FLT3-ITD (HR 1.32, p<0.01). Next, weights were assigned to these variables based on the multivariate HRs (Figure 1A). Scores were calculated for individual patients and four groups were defined. OS at 1 year estimated 54±3% in the favorable risk group (≤5 points), 35±3% in the intermediate group (6-7 points), 18±3% in the high risk group (8-9 points), and 8±2% in the very high risk group (≥10 points) (Figure 1B). The prognostic accuracy for 1 year OS as measured by the C-index was 0.70. In contrast, in this cohort C-indices for the HOVON-SAKK and GOELAMS models were 0.65 and 0.64, respectively. In addition, comparing the earlier models with the new model, 50% of patients in the adverse group of the HOVON-SAKK model were classified to the favorable (14%) and intermediate group (36%) in the new model. In the GOELAMS model, 42% of intermediate risk patients were considered high (10%) or very high risk (32%) in the new model. Conclusion Analysis of clinical, cytogenetic and molecular variables identified nine variables for a new prognostic model which was associated with distinct OS at 1 year after first AML relapse and improved prognostic accuracy.

10017 Background: Bosutinib is a tyrosine kinase inhibitor (TKI), approved for adults with Philadelphia Chromosome (Ph+) CML; at the standard initial dose of 400 mg/day in ND patients, and 500 mg/day in resistant/intolerant (R/I) patients, administered orally once daily (QD) with food. Compared to the TKIs already approved in pediatrics, bosutinib has a different tolerability profile, and preclinical data suggest that longitudinal growth is potentially less impaired. Study NCT04258943 is an international, open-label, phase I/II trial, sponsored by the Erasmus Medical Center and the Children's Oncology Group, and funded by Pfizer Inc. The phase II arm enrolled ND patients, as well as R/I ones. The latter are not included in this analysis. Methods: ND patients aged 1-18 years with chronic phase CML, without evidence for organ toxicities, were enrolled. Main exclusion criteria included known T315I or V299L BCR-ABL1 mutations, and use of proton pump inhibitors and CYP3A inducers/inhibitors. The primary objectives of the phase II part of the study were to assess the safety and pharmacokinetics (PK) of bosutinib at the recommended phase II dose, which is body surface area adjusted, and consists of 300 mg/m 2 QD for ND patients (max 500 mg/day), as determined in the phase I part of the study. Based on regulatory authorities requirements, at least 35 patients have to be enrolled in phase II, with a total of 50-60 patients in phase I and II combined. This allows for pooled AE rates to be estimated with a maximum standard error of 0.071 and 0.065, respectively. Results: On 20/12/2022, 25 ND patients were screened, and 24 were enrolled: 15 males, median age 13 years (range: 5-17). The median follow up was 14 (range: 0.9-31) months. The most common non-hematological adverse events (AEs) were diarrhea (n = 16, Grade (Gr) ≤ 2 = 13), abdominal pain (n = 10, Gr ≤ 2 = 10), and nausea/vomiting (n = 8/8, Gr ≤ 2 = 7/8). Most common hematological AEs included platelet count decrease (n = 11, Gr ≤ 2 = 5), and anemia (n = 10, Gr ≤ 2 = 4). At 6 and 12 months, the cumulative incidence of Major Cytogenetic Response (MCyR) was 85% (95%CI 49%-96%) and 92.5% (95%CI 39%-99%), while for Complete CyR it was 77% (47%-91%) and 88.3% (95%CI 55%-98%), and for Major Molecular Response it was 23% (95%CI 7%-46%) and 30% (95%CI 10%-54%), respectively. Three patients permanently discontinued bosutinib due to intolerance, four due to unsatisfactory response per investigator’s judgment, 17 were still on treatment at time of dataset lock. Conclusions: Bosutinib was well tolerated, despite common grade 1-2 gastrointestinal AEs. The preliminary efficacy seems comparable to published data from other second generation TKIs in children and to ND adult patients treated with bosutinib. Clinical trial information: NCT04258943 .

Patients with multiple myeloma (MM) demonstrate variable outcomes with treatment. With increasing treatment options, predictive factors for response and outcome are relevant to inform treatment choices. Immunomodulating agents (IMiDs) represent the cornerstone of MM treatment and act through binding to Cereblon (CRBN), affecting downstream targets of this E3 ubiquitin ligase. We hypothesized differential expression of effector or target proteins from the CRBN pathway to predict outcome in patients treated with IMiDs. Bone marrow (BM) biopsies were obtained from 148 newly diagnosed, transplant non-eligible patients with MM. Per HOVON-87/NMSG-18 trial protocol, these patients were treated with thalidomide or lenalidomide combined with melphalan and prednisone followed by thalidomide/lenalidomide maintenance (i.e. MPT-T or MPR-R). Immunohistochemistry was performed for CRBN, its neosubstrates Ikaros and Aiolos and the downstream targets interferon regulatory factor 4 (IRF-4) and cellular myelocytomatosis oncogene (c-MYC). Patients with response of VGPR or better have higher nuclear CRBN expression compared to patients with PR or worse (≥VGPR: median CRBN H-score=185 (interquartile range (IQR), 147-211) vs ≤PR median CRBN H-score=159 (IQR 129-193); p=0.02). Higher nuclear CRBN expression was associated with a longer progression-free survival (PFS) and overall survival (OS). For PFS a hazard ratio (HR) of 0.53 was found (95% confidence interval (CI) =0.37-0.77; p<0.001); for OS: HR = 0.59 (95% CI=0.38-0.90; p=0.02). The association between CRBN and OS varied with IRF-4 levels. In patients with IRF-4 levels above the median, a hazard ratio of 0.22 was found (95% CI=0.10-0.49; p=0.0002); in contrast, patients with IRF-4 levels below the median, had a hazard ratio of 0.82 (95% CI=0.44-1.53; p=0.5). For Ikaros, Aiolos and c-MYC no correlation with survival was found, either alone or in combination with CRBN. In conclusion, higher expression of nuclear CRBN was associated with a superior PFS and OS upon MPT or MPR treatment. Levels of nuclear CRBN protein, possibly in combination with IRF-4, may represent a biomarker for predicting treatment outcome in patients treated with IMiDs.

A common finding in pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) is that chromosome 21 is never lost and an extra chromosome 21 is often gained. This implies an important role for chromosome 21 in the pathobiology of BCPALL, emphasized by the increased risk of BCPALL in children with Down syndrome. However, model systems of chromosome 21 gain are lacking. We therefore developed a BCPALL cell line (Nalm-6, DUX4-rearranged) with an additional chromosome 21 by means of microcell-mediated chromosome transfer. FISH, PCR, multiplex ligation-dependent probe amplification, and whole exome sequencing showed that an additional chromosome 21 was successfully transferred to the recipient cells. Transcription of some but not all genes on chromosome 21 was increased, indicating tight transcriptional regulation. Nalm-6 cells with an additional chromosome 21 proliferated slightly slower compared with parental Nalm-6 and sensitivity to induction chemotherapeutics was mildly increased. The extra copy of chromosome 21 did not confer sensitivity to targeted signaling inhibitors. In conclusion, a BCPALL cell line with an additional human chromosome 21 was developed, validated, and subjected to functional studies, which showed a minor but potentially relevant effect in vitro. This cell line offers the possibility to study further the role of chromosome 21 in ALL.

Inotuzumab Ozogamicin is a CD22-directed antibody conjugated to calicheamicin, approved in adults with relapsed or refractory (R/R) B cell acute lymphoblastic leukemia (BCP-ALL). Patients aged 1–18 years, with R/R CD22 + BCP-ALL were treated at the RP2D of 1.8 mg/m 2 . Using a single-stage design, with an overall response rate (ORR) ≤ 30% defined as not promissing and ORR > 55% as expected, 25 patients needed to be recruited to achieve 80% power at 0.05 significance level. Thirty-two patients were enrolled, 28 were treated, 27 were evaluable for response. The estimated ORR was 81.5% (95%CI: 61.9–93.7%), and 81.8% (18/22) of the responding subjects were minimal residual disease (MRD) negative. The study met its primary endpoint. Median follow up of survivors was 16 months (IQR: 14.49–20.07). One year Event Free Survival was 36.7% (95% CI: 22.2–60.4%), and Overall Survival was 55.1% (95% CI: 39.1−77.7%). Eighteen patients received consolidation (with HSCT and/or CAR T-cells therapy). Sinusoidal obstructive syndrome (SOS) occurred in seven patients. MRD negativity seemed correlated to calicheamicin sensitivity in vitro, but not to CD22 surface expression, saturation, or internalization. InO was effective in this population. The most relevant risk was the occurrence of SOS, particularly when InO treatment was followed by HSCT.

Substantial heterogeneity within mutant TP53 acute myeloid leukemia (AML) and myelodysplastic syndrome with excess of blast (MDS-EB) precludes the exact assessment of prognostic impact for individual patients. We performed in-depth clinical and molecular analysis of mutant TP53 AML and MDS-EB to dissect the molecular characteristics in detail and determine its impact on survival. We performed next-generation sequencing on 2200 AML/MDS-EB specimens and assessed the TP53 mutant allelic status (mono- or bi-allelic), the number of TP53 mutations, mutant TP53 clone size, concurrent mutations, cytogenetics, and mutant TP53 molecular minimal residual disease and studied the associations of these characteristics with overall survival. TP53 mutations were detected in 230 (10.5%) patients with AML/MDS-EB with a median variant allele frequency of 47%. Bi-allelic mutant TP53 status was observed in 174 (76%) patients. Multiple TP53 mutations were found in 49 (21%) patients. Concurrent mutations were detected in 113 (49%) patients. No significant difference in any of the aforementioned molecular characteristics of mutant TP53 was detected between AML and MDS-EB. Patients with mutant TP53 have a poor outcome (2-year overall survival, 12.8%); however, no survival difference between AML and MDS-EB was observed. Importantly, none of the molecular characteristics were significantly associated with survival in mutant TP53 AML/MDS-EB. In most patients, TP53 mutations remained detectable in complete remission by deep sequencing (73%). Detection of residual mutant TP53 was not associated with survival. Mutant TP53 AML and MDS-EB do not differ with respect to molecular characteristics and survival. Therefore, mutant TP53 AML/MDS-EB should be considered a distinct molecular disease entity.

Background In addition to treatment response, cytogenetic and molecular aberrations are the most important prognostic factors in children with de novo acute myeloid leukemia (AML). However, little is known about cytogenetics at the time of relapse. Methods This international study analyzed the prognostic value of cytogenetic profiles and karyotypic changes in pediatric relapsed AML in relation to the probability of event-free (pEFS) and overall survival (pOS). For this purpose, cytogenetic reports from all patients registered on the Relapsed AML 2001/01 Study were reviewed and classified. Results Cytogenetic information at relapse was available for 403 (71%) of 569 registered patients. Frequently detected aberrations at relapse were t(8;21)(q22;q22) (n = 60) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) (n = 24), both associated with relatively good outcome (4-year pOS 59% and 71%, respectively). Monosomy 7/7q-, t(9;11)(p22;q23), t(10;11)(p12;q23), and complex karyotypes were associated with poor outcomes (4-year pOS 17%, 19%, 22%, and 22%, respectively). Of 261 (65%) patients for whom cytogenetic data were reliable at both diagnosis and relapse, pEFS was inferior for patients with karyotypic instability (n = 128, 49%), but pOS was similar. Unstable karyotypes with both gain and loss of aberrations were associated with inferior outcome. Early treatment response, time to relapse, and cytogenetic profile at time of relapse were the most important prognostic factors, both outweighing karytoypic instability per se. Conclusion The cytogenetic subgroup at relapse is an independent risk factor for (event-free) survival. Cytogenetic assessment at the time of relapse is of high importance and may contribute to improved risk-adapted treatment for children with relapsed AML.