Figure S7: Drug-resilient 786-0 cells exhibit 1-2 whole genome duplications with high fidelity

Drug-resilient cells exhibit one to two whole-genome duplications with high fidelity. A, Copy numbers in untreated and treated surviving HCC1806 cells 5 DPT, as visualized with AneuFinder (reads per 10 Mb over total amount of reads) from scWGS each row representing a single nucleus. B, Ratio of DNA content within each cell in untreated and surviving HCC1806 cells 5 DPT. The heat maps show the normalized read depth (reads per 10 Mb bins over total amount of reads in the cell) of scWGS, where blue areas show a lower number of reads, and red areas show a higher number of reads. The blocks R1, R2, and R3 in the left represent replicates 1, 2, and 3, respectively. C, Copy number of chromosome X in untreated (CTL), surviving HCC1806 cells at 5 DPT and their progeny, as visualized with chromosomal FISH of cells in interphase.

Constitutional polymorphisms in ARID5B are associated with an increased risk of developing high hyperdiploid (HeH; 51-67 chromosomes) pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). Here, we investigated constitutional and somatic ARID5B variants in 1335 BCP ALL cases from five different cohorts, with a particular focus on HeH cases. In 353 HeH ALL that were heterozygous for risk alleles and trisomic for chromosome 10, where ARID5B is located, a significantly higher proportion of risk allele duplication was seen for the SNPs rs7090445 (p = 0.009), rs7089424 (p = 0.005), rs7073837 (p = 0.03), and rs10740055 (p = 0.04). Somatic ARID5B deletions were seen in 16/1335 cases (1.2%), being more common in HeH than in other genetic subtypes (2.2% vs. 0.4%; p = 0.002). The expression of ARID5B in HeH cases with genomic deletions was reduced, consistent with a functional role in leukemogenesis. Whole-genome sequencing and RNA-sequencing in HeH revealed additional somatic events involving ARID5B, resulting in a total frequency of 3.6% of HeH cases displaying a somatic ARID5B aberration. Overall, our results show that both constitutional and somatic events in ARID5B are involved in the leukemogenesis of pediatric BCP ALL, particularly in the HeH subtype.

Abstract Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole-genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF2α. We found altered chromatin accessibility, ablated expression of retinoblastoma protein (RB1), and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF2α in stress response by polyploidy suggests a novel avenue for tackling chemotherapy-induced resistance in cancer. Significance: In response to cisplatin treatment, some surviving cancer cells undergo whole-genome duplications without mitosis, which represents a mechanism of drug resistance. This study presents mechanistic data to implicate AP-1 and HIF2α signaling in the formation of this surviving cell phenotype. The results open a new avenue for targeting drug-resistant cells.

Protein changes validate the role of HIF2α and RB1 for cell survival. A, Protein level changes of HIF2α-interacting proteins, MCM7, HIC7, and NUR77 in HCC1806 and HCT116 cells when untreated (CTL), when surviving at 0 DPT, 5 DPT, and 10 DPT and as progeny; demonstrated by Western blot analysis. Actin was used as a loading control. Molecular weight markers in kDa are shown to the left. B, Representative images of protein level changes of RB1 and its phosphorylated sites (s790, s780, and s807) in HCC1806 and HCT116 cells when untreated (CTL), when surviving at 0 DPT, 5 DPT, 10 DPT and as progeny; as determined by Western blot analysis. C, Protein level changes of HIF2α and its targets SERPINB9, VEGF, and DEC1 in HCC1806 and HCT116 cells when untreated (CTL), as surviving at 0 DPT, 5 DPT, and 10 DPT and as progeny; as determined with Western blot analysis. D, Protein level changes of VHL and PHD1–3 in HCC1806 and HCT116 cells when untreated (CTL), when surviving at 0 DPT, 5 DPT, and 10 DPT, and as progeny; as determined with Western blot analysis. E, Number of HCC1806 and HCT116 cells surviving at 0 DPT and 10 DPT when treated with cisplatin only or cisplatin together with the HIF2α inhibitor Belzutifan. F, Number of LS174T and HCT116 colon cancer cells surviving cisplatin at 0 DPT and 10 DPT as “normal” and with k HIF2α KO from biological replicates (n = 3) and P-value (∗∗, P < 0.01; ∗, P < 0.05; significant relative to vehicle) by ANOVA test as indicated.

Somatic copy number variations (CNVs), including abnormal chromosome numbers and structural changes leading to gain or loss of genetic material, play a crucial role in initiation and progression of cancer. CNVs are believed to cause gene dosage imbalances and modify cis-regulatory elements, leading to allelic expression imbalances in genes that influence cell division and thereby contribute to cancer development. However, the impact of CNVs on allelic gene expression in cancer remains unclear. Allele-specific expression (ASE) analysis, a potent method for investigating genome-wide allelic imbalance profiles in tumors, assesses the relative expression of two alleles using high-throughput sequencing data. However, many existing methods for gene-level ASE detection rely on only RNA sequencing data, which present challenges in interpreting the genetic mechanisms underlying ASE in cancer. To address this issue, we developed a robust framework that integrates allele-specific copy number calls into ASE calling algorithms by leveraging paired genome and transcriptome data from the same sample. This integration enhances the interpretability of the genetic mechanisms driving ASE, thereby facilitating the identification of driver events triggered by CNVs in cancer. In this study, we utilized BASE to conduct a comprehensive analysis of ASE in high hyperdiploid acute lymphoblastic leukemia (HeH ALL), a prevalent childhood malignancy characterized by gains of chromosomes X, 4, 6, 10, 14, 17, 18, and 21. Our analysis unveiled the comprehensive ASE landscape in HeH ALL. Through a multi-perspective examination of HeH ASEs, we offer a systematic understanding of how CNVs impact ASE in HeH, providing valuable insights to guide ASE studies in cancer.

Surviving polyploid cells demonstrate an overall reduction of chromatin openness while AP-1 motifs were enriched. A, Visualization of accessible regions in surviving HCC1806 cells at 0 DPT; as quantified by ATAC-seq. B, Visualization of more (green) or less (red) accessible regions in surviving HCC1806 cells at 0 DPT; as quantified by ATAC-seq. C, Visualization of DNA motifs for AP-1 family members in HCC1806 surviving at 0 DPT, as quantified by ATAC-seq. D, Openness of region for EPAS1 in HCC1806 cells surviving at 0 DPT, as visualized with genome browser tracks. E, Protein level changes in HCC1806 and HCT116 cells of the AP-1 members FOS, FOSL1, JUN, and ATF-3 in untreated (CTL), surviving cells at 0 DPT, 5 DPT, and 10 DPT, and as progeny. F, Number of HCC1806 and HCT116 cells surviving at 0 DPT and 10 DPT when treated with cisplatin alone and cisplatin together with the FOS/AP-1 inhibitor T-5224 from biological replicates (n = 3) and P-value (∗∗, P < 0.01; ∗, P < 0.05, significant relative to vehicle) by ANOVA test as indicated.

Drug-resilient cells triple in size and mass for up to 10 days posttreatment. A, Our treatment protocol entailed that seeded cells were treated with cisplatin (T = −3 days) for 72 hours (T = 0 days posttreatment; DPT) and, following filtration, studied for 10 DPT. After a subsequent time interval (between 2–12 weeks depending on cell line), surviving cells gave rise to progeny. B, Cells from four cancer cell lines stained with Giemsa when untreated (CTL) and treated at timepoint 0 DPT, 5 DPT, and 10 DPT, and progeny from these cells at 28 DPT (HCC1806, 786-0, and HCT116 cells), and 49 DPT (U1890 cells; n = 3 biological replicates). Scale bar, 20 µm. C, Detailed view of nuclei of untreated HCC1806 cells and when surviving 5 DPT, using TEM. Scale bar, 2 µm. D, Size of untreated (CTL) cells, surviving cells at 0 DPT, 5 DPT, 10 DPT, and progeny at 28 DTP (HCC1806, 786-0), 21 DTP (HCT116), and 49 DTP (U1890). Sizes acquired by imaging of adherent cells and analyzed in ImageJ. Cell size average from biological triplicates (n = 3) and P-value (∗∗, P < 0.01; ∗, P < 0.05; NS, not significant) by ANOVA test as indicated. E, Mass of untreated (CTL) cells, surviving cells at 10 DPT, and progeny at 28 DTP (HCC1806, 786-0), 21 DTP (HCT116), and 49 DTP (U1890). Cell mass average from biological triplicates (n = 3) and P-value (∗∗, P < 0.01; ∗, P < 0.05; NS, not significant) by ANOVA test as indicated. F, Cell diameter distributions and frequency of 10,000 sorted HCC1806 cells in control (CTL) and treated cells at 0 DPT, 5 DPT, and 10 DPT (biological replicates n = 3). G, Representative image of proliferating clones of progeny 28 DTP (HCC1806, 786-0, HCT116, and U1890). Cells are stained with 0.5% crystal violet solution. H, Distribution of surviving cells that died or regained proliferative capacity 2 months after treatment of HCC1806, 786-0, HCT116, and U1890 cells. Treated and filtered cells (n = 96) at 0 DPT were transferred to individual wells. Average of the number of wells with dead cells, large cells, and proliferating progeny cells from biological replicates (n = 3) and P value (∗∗, P < 0.01; ∗, P < 0.05, significant relative to vehicle) by ANOVA test as indicated.

Whilst the molecular pathogenesis of childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) has been studied extensively, its 3D chromatin landscape remains poorly explored. Genome-wide chromosome conformation capture methods have provided the tools to investigate the different units of chromatin organization, such as transcriptionally active (A) and inactive (B) compartments, topologically associating domains (TADs), and fine-scale chromatin loops and enhancer-promoter interactions. The aim of this study is to elucidate the chromatin architecture and topological gene regulation in childhood BCP ALL. To date, 29 primary patient samples were included, comprising the high hyperdiploid (HeH) (n=11), ETV6:: RUNX1-positive (n=8), BCR:: ABL1-positive (n=2), TCF3:: PBX1-positive (n=2), DUX4-rearranged (n=2), intrachromosomal amplification of chromosome 21 (iAMP21) (n=1), KMT2A-rearranged (n=1), near-haploid (n=1) and near-triploid (n=1) genetic subtypes. Leukemic blast cells obtained at diagnosis were analyzed using Micro-C, a high-resolution variation of Hi-C (average number of total reads = 1.4 billion, highest resolution = 5 kb) combined with pair-end sequencing. Chromatin contact heatmaps were generated for each case using Juicer and Cooler. A/B compartments were identified using FanC at 500 kb resolution and visualized in the software IGV, while TAD calling was performed by Juicer, Domaincaller and Insulation Score. Differential chromatin interaction loop calling was made using Pareidolia and Mustache, and structural variant (SV) calling was carried out by EagleC. Preliminary principal component analysis of the 29 cases, based on the first two eigenvectors of the contact matrix (500 kb resolution), showed that HeH, ETV6:: RUNX1-positive, TCF3:: PBX1-positive and DUX4-rearranged cases each clustered based on their chromatin 3D organization. Furthermore, the A/B compartments of 11 HeH and 8 ETV6:: RUNX1-positive cases were analyzed at 500 kb resolution and compartment shifts among the two subtypes were annotated. A total of 390 shifts were detected, where activating shifts (from B to A compartment) happened more often in HeH (263 shifts) than in ETV6:: RUNX1-positive cases (127 shifts). Analysis of TAD boundary strength at 25 kb resolution revealed that HeH cases displayed significantly weaker boundaries compared to ETV6:: RUNX1-positive cases. TAD boundary strength showed no bias towards the frequently gained or non-gained chromosomes in HeH ALL. By merging individual heatmaps of all HeH and ETV6:: RUNX1-positive cases using Cooler, we created subtype-specific profiles and compared the intensity of chromatin interactions between the two genetic subtypes. Chromatin interaction intensity analysis was then combined with previously published RNA-sequencing data to identify transcriptional dysregulation events that could be associated with chromatin interaction changes. Preliminary results show that there was a chromatin loop missing close to the well-known leukemia-related gene IKZF1 in HeH compared to ETV6:: RUNX1-positive cases; this gene also showed lower expression in the RNA-sequencing data. FLT3 was associated with weakened chromatin interactions and down-regulated in ETV6:: RUNX1-positive cases compared to HeH, in agreement with its known high expression in HeH. Finally, we performed screening of SVs using EagleC and Micro-C heatmaps in HeH and ETV6:: RUNX1-positive samples. Out of the 19 included cases, previous whole-genome sequencing (WGS) data were available for 16. We detected 75 SVs, of which 50 were intrachromosomal rearrangements and 25 were translocations. Micro-C heatmaps allowed visual detection of SVs smaller than 1 Mb and permitted identification of the type of SVs. WGS detected 61% of the SVs found in the HeH samples with Micro-C and 51% of those in the ETV6:: RUNX1-positive cases. In summary, we present the first high-resolution genome-wide map of chromatin 3D organization in pediatric ALL. Our results indicate that different subtypes of childhood BCP ALL have distinct 3D chromatin landscapes and that abnormal chromatin architectures affect the regulation of leukemia-related genes.

High hyperdiploid acute lymphoblastic leukemia (HeH ALL), one of the most common childhood malignancies, is driven by nonrandom aneuploidy (abnormal chromosome numbers) mainly comprising chromosomal gains. In this study, we investigate how aneuploidy in HeH ALL arises. Single cell whole genome sequencing of 2847 cells from nine primary cases and one normal bone marrow reveals that HeH ALL generally display low chromosomal heterogeneity, indicating that they are not characterized by chromosomal instability and showing that aneuploidy-driven malignancies are not necessarily chromosomally heterogeneous. Furthermore, most chromosomal gains are present in all leukemic cells, suggesting that they arose early during leukemogenesis. Copy number data from 577 primary cases reveals selective pressures that were used for in silico modeling of aneuploidy development. This shows that the aneuploidy in HeH ALL likely arises by an initial tripolar mitosis in a diploid cell followed by clonal evolution, in line with a punctuated evolution model.

Background: Amplification of 1q (amp(1q); =4 1q copies) has repeatedly been reported to predict a worse outcome in multiple myeloma (MM), whereas the impact of gain of 1q (gain(1q); three 1q copies) is less clear.Methods: We investigated survival of MM in relation to amp(1q) and gain(1q) by retro-spectively analysing 346 consecutively newly diagnosed MM (NDMM) patients. Of these, 62 (18%) had amp(1q), 97 (28%) gain(1q) and 187 (54%) a normal number of 1q copies (no1q).Results: The patients with amp(1q) had a shorter median progression-free survival than those with gain(1q) or no(1q) (13.1 months, 95% confidence interval [CI] 8.2- 18.1 months vs. 36.1 months, 95% CI 23.1-49.1 months vs. 25.4 months, 95% CI 19.8-31.1 months, p = .005). The 3-year overall survival (OS) was 56% for amp(1q), 76% for gain(1q) and 80% for no1q (p = .003). In the multivariate analysis, the pres-ence of amp(1q) was independently associated with a shorter OS (hazard ratio 1.99, 95% CI 1.03-3.82, p = .039), whereas gain(1q) had no negative effect on survival.Conclusion: Our results thus suggest that amp(1q) should be considered a high-risk abnormality in NDMM and that new treatment strategies should be explored to miti-gate its negative effect on survival.

Ulrika Norén-Nyström1,*, Mette K. Andersen2, Gisela Barbany3, Vaidas Dirse4, Martine Eilert-Olsen5, Marie Engvall6, Arja Harila-Saari7, Mats Heyman8,9, Randi Hovland10, Satu Häikiö11, Jón J. Jónsson12,13, Ritva Karhu14, Eigil Kjeldsen15, Anna Norberg16, Birgitte S. Preiss17, Kati Pulkkinen18, Petter Quist-Paulsen19, Hannele Räsänen20, Kjeld Schmiegelow21, Anne Seitsonen22, Helene Sjögren23, Pille Tammur24, Bertil Johansson25,26,*

Cytogenetic analysis provides important information on the genetic mechanisms of cancer. The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (Mitelman DB) is the largest catalog of acquired chromosome aberrations, presently comprising >70 000 cases across multiple cancer types. Although this resource has enabled the identification of chromosome abnormalities leading to specific cancers and cancer mechanisms, a large-scale, systematic analysis of these aberrations and their downstream implications has been difficult due to the lack of a standard, automated mapping from aberrations to genomic coordinates. We previously introduced CytoConverter as a tool that automates such conversions. CytoConverter has now been updated with improved interpretation of karyotypes and has been integrated with the Mitelman DB, providing a comprehensive mapping of the 70 000+ cases to genomic coordinates, as well as visualization of the frequencies of chromosomal gains and losses. Importantly, all CytoConverter-generated genomic coordinates are publicly available in Google BigQuery, a cloud-based data warehouse, facilitating data exploration and integration with other datasets hosted by the Institute for Systems Biology Cancer Gateway in the Cloud (ISB-CGC) Resource. We demonstrate the use of BigQuery for integrative analysis of Mitelman DB with other cancer datasets, including a comparison of the frequency of imbalances identified in Mitelman DB cases with those found in The Cancer Genome Atlas (TCGA) copy number datasets. This solution provides opportunities to leverage the power of cloud computing for low-cost, scalable, and integrated analysis of chromosome aberrations and gene fusions in cancer.

Introduction The suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.Methods For this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.Results Both the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.Discussion The filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6 :: RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6 :: RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6 :: RUNX1 cases (n = 26) were IKZF1 -deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values ( P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6 :: RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the high hyperdiploid (HeH) subtype accounting for approximately 25% of B-cell precursor (BCP) ALL cases. It has been shown that germline variants in the ARID5B gene in chromosome band 10q21.2 are associated with increased risk of BCP ALL, in particular HeH ALL, and that the risk alleles result in lower expression of ARID5B in hematopoietic cells. ARID5B codes for a protein involved in regulating gene expression and chromatin remodeling. We have previously reported somatic deletions in the ARID5B locus in two cases of HeH ALL, but the overall frequency of acquired copy number changes and rearrangements involving ARID5B in BCP ALL remains unknown. Here, we have investigated constitutional and somatic ARID5B variants in pediatric BCP ALL, with a particular focus on HeH cases. METHODS Constitutional variants We studied four known risk single nucleotide polymorphisms (SNPs) in the ARID5B locus (rs7090445, rs7089424, rs7073837 and rs10740055) in HeH ALL cases heterozygous for the risk SNP and with trisomy 10. These were investigated in three different cohorts, including a total of 92 cases informative for rs7090445, 92 cases for rs7089424, 119 cases for rs7073837 and 66 cases for rs10740055. The genotype and relative allele frequencies were ascertained from SNP array or whole genome sequencing (WGS) data. One-sided binomial tests were applied to investigate whether the risk allele was more often in the duplicated chromosome than the non-risk allele. Somatic variants For somatic variants, we ascertained copy number status based on SNP array and/or WGS analysis in the ARID5B region in a total of 466 pediatric HeH ALL cases and structural rearrangements based on WGS in 77 cases. We also studied, using SNP array analysis, somatic copy number variants in a separate cohort consisting of 590 non-HeH BCP ALL cases. To compare the proportions of deletions in the HeH cohort and in the other genetic subtypes, we used Fisher's Exact two-sided test. RESULTS Constitutional variants All four risk SNPs showed a significantly higher proportion of risk allele duplication (one-sided binominal test); rs7090445 ( P=0.009), rs7089424 ( P=0.005), rs7073837 ( P=0.03) and rs10740055 ( P=0.04). This validates that there is a clonal selection for HeH blast cells with gain of the chromosome 10 homologue carrying the risk allele as opposed to gain of the homologue that carries the non-risk allele. Somatic variants Somatic deletions targeting ARID5B were found in 9/466 cases (1.9%) of HeH cases. The deletions covered different parts of ARID5B, with no minimally deleted region, suggesting that the functional outcome was downregulation of ARID5B expression. In the cohort with non-HeH BCP ALL (other genetic subtypes), 4/590 somatic deletions were found (0.68%). There was no statistically significant difference between the frequency of copy number aberrations targeting ARID5B between HeH and the other genetic subtypes of ALL (P=0.09). WGS analysis of the HeH cases revealed one translocation and one missense mutation in 2/77 cases (2.6%). The translocation involved the PAN3 and ARID5B genes and was also present in RNA sequencing data from this case. CONCLUSIONS We show that our previous finding that HeH ALL constitutionally heterozygous for ARID5B risk alleles and with an acquired trisomy 10 more commonly duplicates the chromosome 10 homologue carrying the risk allele holds true in a much larger cohort. Furthermore, somatic deletions involving ARID5B are recurrent in pediatric BCP ALL.

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and recent cure rates approach 90% on first line therapy. IKZF1 deletions have been reported as an unfavorable prognostic factor and are rare in ETV6::RUNX1 and high hyperdiploid (HeH) ALL, the two cytogenetic subgroups with the most favorable prognosis. Due to its rarity, the prognostic effect of IKZF1deletions within this subset of patients remains unclear. Therefore, we assessed the prognostic impact of IKZF1deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients. We performed this retrospective analysis on data of children and adolescents of 1-18 years with B-cell precursor ALL diagnosed in 1991-2016 and treated on one of 16 trials from 9 study groups, of which 6 were minimal residual disease (MRD) guided. Cytogenetic, fluorescence in-situ hybridization, and RT-PCR analyses of pre-treatment bone marrow samples to determine ploidy and fusion gene status were performed locally. Trials using MRD for risk stratification applied either PCR or flow cytometry analyses. The IKZF1 status was determined by multiplex ligation-dependent probe amplification by each individual study group. 3% of ETV6::RUNX1 (n=26) cases had an IKZF1 deletion; this affected survival adversely overall (5-year event free survival [EFS]: 79% vs. 92%, p = 0.02) (Table 1). However, in minimal residual disease (MRD) guided protocols, IKZF1 did not affect outcome in ETV6::RUNX1 cases ( 5-year EFS: 100% vs. 93%, p = 0.34) (Table 1). 9% of HeH cases had an IKZF1 deletion (n=85); this adversely affected survival overall (5-year EFS: 76% vs. 89%, p = 0.006), and in MRD-guided protocols (73% vs. 88%, p = 0.004) (Table 1). Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (Hazard ratio of EFS [95% CI]: 2.00 [1.20-3.30]) (Table 1). As the prognostic effect of IKZF1 deletions has been shown to vary by MRD risk group, we examined the prognostic effect of IKZF1deletions in HeH cases with detectable MRD <5% at end of induction in more detail. Of these cases, 10% carried an IKZF1deletion (n=34) which negatively affected outcome (5-year EFS: 78% vs. 92%, p = 0.01) Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in this intermediate MRD HeH ALL (Hazard ratio of EFS [95% CI]: 2.90 [1.20-6.80]). However, cases with an IKZF1 deletion had significantly higher MRD values than their wild-type counterparts (p = 0.03, Figure 1). In conclusion, our analysis of a large composite cohort consisting of 16 trials shows that IKZF1deletions do not affect survival in ETV6::RUNX1 ALL when treated on MRD-guided protocols. In contrast, our data shows that in HeH ALL IKZF1deletions are associated with lower survival rates, higher relapse rates, and higher MRD values. Future results of current trials such as the ALLTogether will likely reveal whether risk stratification predominantly reliant on MRD is adequate for HeH patients or whether stratification by copy number alteration profile, including IKZF1 status, or by other methods would be more suitable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal