Whereas the molecular pathogenesis of childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) has been studied extensively, its 3D chromatin landscape – of vast importance for gene regulation – remains poorly explored. Here, we applied Micro-C, a high-resolution variant of Hi-C, to 35 primary pediatric BCP ALL cases, spanning all major genetic subtypes. We present a complete view of the chromatin interaction landscape in childhood ALL, with resolutions reaching up to 5 kb in individual samples and 1 kb in the aggregated dataset. Somatic genetic aberrations – including fusion genes, aneuploidy, and structural variants – were found to profoundly reshape the 3D genome organization, impacting chromatin compartmentalization (A/B), topologically associating domain (TAD) architecture, and regulatory element positioning. Notably, chromosomal gains were associated with weakened TAD boundaries and widespread gene dysregulation. In addition, our analysis identified over 25,000 chromatin loops anchored at regulatory elements—e.g., enhancer–promoter loops—regulating the expression of more than 10,000 protein-coding genes. Among these, we highlight regulatory loops that drive gene expression differences between BCP ALL subtypes in the absence of concurrent somatic genetic aberrations, including the known driver genes HOXA9, FLT3, TP53, CD44, IKZF1, ERG, and XBP1 . Taken together, our study gives unprecedented insights into chromatin organization and gene regulation in the leukemogenesis of BCP ALL. ### Competing Interest Statement The authors have declared no competing interest. Swedish Childhood Cancer Foundation, PR2020-0033, TJ2020-0024, PR2024-0058, PR2024-0002, PR2018-0023, PR2024-0033 Swedish Cancer Fund, 23 2694 PjF, 22 2062 Pj Governmental funding of clinical research within the National Health Service, ALFSKANE-623431 Swedish Research Council, 2020-01164, 2020-00997, 2024-02505 IngaBritt och Arne Lundbergs Forskningsstiftelse, LU2019-0100 Gunnar Nilsson Cancer Foundation Royal Physiographic Society of Lund

There is an ongoing discourse about patient and parent participation (PPP) in Clinical Ethics Support Services (CESS), and this paper focuses specifically on case-based CESS. Participation in CESS is increasing slowly in many contexts due to practical and moral complexity. To gain deeper understanding of PPP in CESS, we need to delve into stakeholders’ perspectives and the landscape in which they operate. The aim of the study was to explore perceptions regarding feasibility and moral appropriateness of PPP in CESS in pediatric oncology. Nordic healthcare personnel (n = 26) working as ethics support personnel in pediatric oncology (and/or pediatrics in general) participated in focus group interviews (n = 6). Data was analyzed with qualitative inductive content analysis. Despite engagement in CESS, most ethics support personnel had no former experience of PPP in CESS. The ethics support personnel expressed potential benefits with PPP in CESS, but these were overshadowed by fear of causing participant harm. The potential benefits and harms included to deepen understanding and trust, to catalyze confrontation and to create dilemmas of decision-making participation. Reported strategies to mitigate potential negative consequences and reduce risk of causing harm were at organizational, relational and individual levels. Despite seeing positive reasons for PPP in CESS, the ethics support personnel were mainly concerned about the potential participant harm and wanted to protect the child and the parent. This could be interpreted as a form of disguised paternalism. The perceived appropriateness of PPP in CESS in pediatric oncology seems to depend on the situation. Furthermore, in cases where it can be considered, there is no universal way of doing it. An important enabler may be to customize PPP in CESS on a case-by-case basis and to apply the identified strategies to reduce potential risk of causing harm. This study contributes to increased knowledge about PPP in CESS from the perspectives of ethics support personnel in pediatric oncology and informs us about what is needed to carefully foster PPP in CESS, both practically and morally.

To study the mechanisms of relapse in KMT2A-rearranged (KMT2A-r) acute lymphoblastic (ALL) and acute myeloid leukemia (AML), we performed whole-genome and exome sequencing of infants and children with relapsed ALL/AML (n = 36), and longitudinal deep-sequencing of 257 samples in 30 patients. Somatic alterations in drug-response genes, most commonly in TP53 and IKZF1 (64%), were highly enriched in early relapse ALL (79%, 9-36 months after diagnosis), but rare in very early relapse ALL (<9 months, 9%). A marked chemotherapy-exposure signature was detected for mutations in early relapse ALL but not in very early ALL or AML relapse, in line with different mechanisms of relapse. Longitudinal analyses could track residual leukemia cells, clonal drug responses, and the upcoming relapse. These results highlight that KMT2A-r ALL and AML evade therapy differently and provide insights into the mechanisms of relapse in this highly lethal form of pediatric acute leukemia.

Extensive genetic and epigenetic variegation has been demonstrated in many malignancies. Importantly, their interplay has the potential to contribute to disease progression and treatment resistance. To shed light on the complex relationships between these different sources of intra-tumour heterogeneity, we explored their relative contributions to the evolutionary dynamics of Acute Lymphoblastic Leukaemia (ALL) in children with Down syndrome, which has particularly poor prognosis. We quantified the tumour propagating potential of genetically distinct sub-clones using serial transplantation assays and SNP-arrays. While most leukaemias were characterized by a single dominant subclone, others were highly heterogeneous. Importantly, we provide clear and direct evidence that genotypes and phenotypes with functional relevance to leukemic progression and treatment resistance can co-segregate within the disease. Hence, individual genetic lesions can be restricted to well-defined cell immunophenotypes, corresponding to different stages of the leukemic differentiation hierarchy and varied proliferation potentials. As a result of this difference in fitness, which can be accurately quantified via competitive transplantation assays, matching diagnostic, post-treatment, and relapse leukaemias can be dominated by different genotypes, including pre-leukemic clones persisting throughout the disease progression and treatment. Intriguingly, plasticity also appears to be a temporally defined property that can segregate with genotype. These results suggest that Down Syndrome ALL should be viewed as a complex matrix of cells exhibiting genetic and epigenetic heterogeneity that foster extensive clonal evolution and competition. Therapeutic intervention reshapes this ‘eco-system’ and may provide the right conditions for the preferential expansion of selected compartments and subsequently relapse.

Childhood cancers are life-threatening diseases that affect the whole family. During the treatment moral situations might arise. Research on how parents perceive moral challenges in childhood cancer care is sparse. The aim was to explore parents’ main concern, and how they deal with their main concern, when facing moral challenges in childhood cancer care. Data collection included focus group interviews with parents. The data analysis was carried out according to a classical theory and revealed the core category “Sheltering in chaos”. The strategies to handle the main concern included “To bring the child through a life-saving trajectory” by “Balancing control” and “Deliberating of suffering. The consequences included feelings of being “Torn between roles”. “Familiarity” emerged as a facilitating factor to handle moral challenges. The results indicate that parents are torn between different roles, and that the care experience is improved through familiarity with the healthcare professionals. The following findings may inspire new ways of offering moral support to families in childhood cancer care.

Introduction Allopurinol can be used in maintenance therapy (MT) for acute lymphoblastic leukemia (ALL) to mitigate hepatic toxicity in patients with skewed 6-mercaptopurine (6MP) metabolism. These patients have high erythrocyte levels of methylated mercaptopurine metabolites (e-MeMP) associated with liver toxicity, including transaminitis and hypoglycemia, and low erythrocyte levels of thioguanine nucleotides (e-TGN), the key intermediate metabolites mediating the antileukemic effect. Retrospective studies and case reports show that the unfavorable metabolite ratio (high e-MeMP/TGN) can be modified by adding allopurinol leading to lower MeMP and higher TGN. In a recent publication https://doi.org/10.3324/haematol.2023.284390 we showed that allopurinol leads to a similar metabolic shift also in unselected pediatric ALL-patients, without previous severe liver toxicity or other signs of skewed 6MP metabolism. We have now analysed the levels of DNA incorporated TGN (DNA-TG) in our cohort since more recent studies, e.g. https://doi.org/10.1016/s1470-2045(17)30154-7, suggests that relapse-free survival is better correlated to DNA-TG than to e-TGN. Methods Pediatric ALL patients on NOPHO ALL-2008 non-high risk protocols with thiopurine methyltransferase wild-type were studied in a prospective before-after trial, NCT03022747. 6MP metabolites were measured, in total 9 blood samples per patient, during 12 weeks of standard MT, followed by 12 weeks of MT with addition of allopurinol 50 mg/m2 and finally 4 weeks of MT without allopurinol. Mean DNA-TG for each patient was calculated for all study phases separately. DNA-TG/e-TGN ratio was calculated as there was a concern that a decrease in e-MeMP might reduce the DNA incorporation of TGN, since e-MeMP is known to inhibit de novo purine synthesis. The 6MP dose was reduced by 50% when allopurinol was initiated to prevent excessive myelosuppression. DNA-TG was quantified using the same method as in the study cited above, with 1−2 µg DNA purified from whole blood and thioguanine measured with ultra-performance liquid chromatography tandem mass spectrometry. Results 51 patients from Sweden and Finland, age 0-15 (median 4) years, were included, of whom 48 completed the study. In paired analysis DNA-TG was 393 fmol/mg DNA higher (1248 vs 855) (p<0.001), e-TGN 193 nmol/mmol Hb higher (457 vs 264) (p<0.001) and e-MeMP 6324 nmol/mmol Hb lower (2614 vs 8938) (p<0.001) when allopurinol was added, Mean DNA-TG/e-TGN ratio did not change significantly, 3.15 on allopurinol compared to 3.51 before, (p=0.10). Four weeks after allopurinol was discontinued, mean DNA-TG decreased 564 fmol/mg DNA (p<0.001), e-TGN decreased 271 nmol/mmol Hb (p<0.001) whereas e-MeMP showed a non-significant increase of 777 nmol/mmol Hb (p=0.17). Alanine aminotransferase decreased by 39% a few weeks after the e-MeMP-levels decreased. Mean absolute neutrophil count (ANC) was lower on allopurinol, 1.35 x 109/L, compared to 1.72 before (p<0.001). Allopurinol did not increase the number of severe adverse events (SAE) nor cause any life-threatening episodes. In accordance with study protocol the mean given dose 6MP was halved during the weeks on allopurinol, 187 mg/m2/week compared to 379 (p<0.001) for the weeks before allopurinol. Conclusions Addition of allopurinol to standard MT with 6MP and methotrexate leads to substantially increased DNA-TG levels. DNA-TG/e-TGN ratio was unchanged indicating that DNA incorporation of TGN was not significantly affected during allopurinol treatment. The 46 % rise in DNA-TG, together with our previously published data with higher proportion of ANC levels within target, lower ALT and no increase in SAE, indicate that addition of allopurinol could be an effective strategy to optimize ALL MT.

B cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. It is initiated by multiple genetic alterations, causing a maturation arrest and accumulation of abnormal progenitor B cells. Current treatment protocols with chemotherapy have led to favorable outcomes, but are associated with significant toxicity and risk of side effects, highlighting the necessity for highly effective, less toxic, targeted drugs that also show efficacy in children experiencing relapse. We have used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs, including the BCR::ABL1-positive, ETV6::RUNX1-positive, high hyperdiploid, and recently discovered DUX4-rearranged subtypes. In total, 188,546 single cells derived from 23 BCP-ALLs and 9 normal bone marrow samples were profiled using the 10X Genomics platform. Cellranger multi and atac count (10X Genomics) were used to generate read count matrices. Further analysis was performed using Seurat and Signac. The in-house developed program SingleCellProjections was used to create a normal bone marrow reference graph onto which the BCP-ALL data was projected. Projection of the ALL cells along the normal B cell differentiation axis, revealed a diversity in the maturation block between the different BCP-ALL subtypes. Whereas the BCR::ABL1-, ETV6::RUNX1-positive and high hyperdiploidy cells mainly showed similarities to normal pro-B cells, the DUX4-rearranged ALL cells also displayed a transcriptional signature resembling mature B cells. In addition, the blast population in DUX4-rearranged ALLs showed multilineage priming toward non-hematopoietic cells, myeloid and T cell lineages, but also an activation of PI3K/AKT signaling that sensitized the cells to PI3K inhibition. Finally, we show that chimeric antigen receptor T cell therapy targeting the upregulated myeloid receptor CD371 (CLL-1), effectively eliminates DUX4-rearranged ALL cells. Our results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-rearranged subtype with important implications for the understanding of ALL biology and new therapeutic strategies.

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 Background Childhood cancers affect about 350 children every year in Sweden and are life-threatening diseases. During the treatment period, situations arise that can become morally challenging for the child. When knowing children’s values and morally challenging situations in childhood cancer care, targeted ethics support could be developed and used in care. Aim To explore children’s values and moral dilemmas ​​when undergoing cancer treatment. Methods This is a qualitative study based on empirical data. The data collection was conducted through three focus group interviews and six individual interviews with children between 10 and 18 years (n = 16). A content analysis methodology was used to generate themes. Children who were/have been treated for cancer at three childhood cancer centres in Sweden were invited to participate. The study was approved by the Swedish Ethical Review Authority. The children’s participation was based on voluntariness and consent/assent. Findings During the analysis, five themes of values emerged: Personal relationships, Bodily ease and identity, Feeling in control and being involved, Positive distractions and Right care that is needed. Their moral dilemmas were thematized into: Should I consider others or not? Should I rest or not? and Should I refuse treatment or not? Conclusion Children undergoing cancer treatment want to have personal relationships with healthcare professionals. Their moral dilemmas were about questioning their own physical and psychological well-being against their expectations, the values of others and the treatment required. Further research is needed to understand how to deal with moral dilemmas in children undergoing cancer treatment.

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.

Activating FLT3 and RAS mutations commonly occur in leukemia with KMT2A-gene rearrangements (KMT2A-r). However, how these mutations cooperate with the KMT2A-r to remodel the epigenetic landscape is unknown. Using a retroviral acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3, we show that FLT3 ITD , FLT3 N676K , and NRAS G12D remodeled the chromatin accessibility landscape and associated transcriptional networks. Although the activating mutations shared a common core of chromatin changes, each mutation exhibits unique profiles with most opened peaks associating with enhancers in intronic or intergenic regions. Specifically, FLT3 N676K and NRAS G12D rewired similar chromatin and transcriptional networks, distinct from those mediated by FLT3 ITD . Motif analysis uncovered a role for the AP-1 family of transcription factors in KMT2A::MLLT3 leukemia with FLT3 N676K and NRAS G12D , whereas Runx1 and Stat5a/Stat5b were active in the presence of FLT3 ITD . Furthermore, transcriptional programs linked to immune cell regulation were activated in KMT2A-r AML expressing NRAS G12D or FLT3 N676K , and the expression of NKG2D-ligands on KMT2A-r cells rendered them sensitive to CAR T cell-mediated killing. Human KMT2A-r AML cells could be pharmacologically sensitized to NKG2D-CAR T cells by treatment with the histone deacetylase inhibitor LBH589 (panobinostat) which caused upregulation of NKG2D-ligand levels. Co-treatment with LBH589 and NKG2D-CAR T cells enabled robust AML cell killing, and the strongest effect was observed for cells expressing NRAS G12D . Finally, the results were validated and extended to acute leukemia in infancy. Combined, activating mutations induced mutation-specific changes in the epigenetic landscape, leading to changes in transcriptional programs orchestrated by specific transcription factor networks.

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.

The Src homology 2B protein 3, encoded by the SH2B3 gene, is involved in growth factor/cytokine receptor signalling and plays a critical role in haematopoiesis.1 Certain germline single nucleotide polymorphisms in SH2B3 have been associated with an increased risk of cardiovascular and autoimmune disorders, myeloproliferative neoplasms (MPN), and B-cell precursor acute lymphoblastic leukaemia (BCP ALL), whereas acquired SH2B3 abnormalities have been reported in, for example, BCR::ABL1-like BCP ALL, MPN, and Down syndrome-related acute megakaryoblastic leukaemia.2-6 Considering that the latter is characterised by a constitutional trisomy 21, it is noteworthy that somatic SH2B3 changes are enriched in BCP ALLs with acquired chromosome 21 gain (chr21gain), such as intrachromosomal amplification of chromosome 21 and trisomy/tetrasomy 21.7, 8 Interestingly, Sinclair et al.8 reported a high frequency of homozygous SH2B3 aberrations in BCP ALL cases with chr21gain and uniparental isodisomies (UPIDs) involving the long arm of chromosome 12 (UPID12q), including the SH2B3 locus at 12q24.12. We ascertained whether UPIDs of the whole chromosome 12 (UPID12) or of chromosome arm 12p (UPID12p) also are particularly common in BCP ALL with chr21gain. Furthermore, we investigated if these UPIDs, in particular UPID12 that results in homozygosity of the SH2B3 alleles, also are associated with SH2B3 abnormalities. Informed consent was obtained according to the Declaration of Helsinki and the study was approved by the Research Ethics Committee of Lund University (Dnr 2011/289). A total of 345 paediatric/adolescent (<18 years) BCP ALLs diagnosed between January 1992 and May 2022 were analysed by single nucleotide polymorphism arrays (SNP-A), as detailed in the Supporting Information.9 The cell lines NALM-1610 and MHH-CALL-211 with duplicated near-haploid (NH) karyotypes, tetrasomy 21, and UPID12 (among several other UPIDs), were also included in the analyses. Of the 345 BCP ALLs, 142 (41%) had various types of chr21gain, 20 (14%) of which harboured UPID12, UPID12p, or UPID12q (Table 1). None of the 203 BCP ALLs without chr21gain had any of these UPIDs (p < 0.0001; Yates's chi-squared test). Thus, not only UPID12q, as previously reported,8 but also other types of UPID involving chromosome 12 are clearly enriched in, perhaps even exclusive for, BCP ALLs with chr21gain. The UPIDs were shown to be acquired in all 10 cases where constitutional DNA was available for comparison. With regard to UPID12, this was not unexpected considering that this abnormality is extremely rare in phenotypically normal individuals.12 Furthermore, the segments involved in the UPID12p and UPID12q were terminal in most instances, strongly indicating that they were acquired.13 Based on the cytogenetic features of the few cases with interstitial UPID12p, it was also most likely acquired, as discussed below. We hence conclude that UPIDs involving chromosome 12 in BCP ALL should, a priori, be considered acquired. Of the 10 UPID12-positive cases (including the two cell lines; Table 1), six were NH with duplicated clones and four were high hyperdiploid (HeH; proved by SNP-A analyses not to represent duplicated NH clones). This is in agreement with our previous findings showing that whole-chromosome UPIDs are almost exclusively found in massively aneuploid BCP ALLs.13 There were no SH2B3 deletions or pathogenic sequence variants in any of the UPID12-positive cases (details on SH2B3 sequencing are provided in the Supporting Information). This clearly shows that homozygous SH2B3 aberrations are not a consequence of UPID12. In fact, based on whole-exome-sequencing of BCP ALL, we have previously reported that only 3% of UPID-associated homozygous gene variants are associated with whole chromosome UPIDs—the vast majority (97%) are located within segmental UPIDs.14 Thus, homozygous gene mutations are generally not the functionally important outcome of whole chromosome UPIDs. Furthermore, considering that both HeH and duplicated NH clones invariably have chr21gain (https://mitelmandatabase.isb-cgc.org/), the strong association between UPID12 and chr21gain found herein is unsurprising and does not, as such, provide any evidence that these two genetic features cooperate in the leukaemogenic process. In fact, whole-chromosome UPIDs, including UPID12, may merely be a consequence of the mechanisms underlying aneuploidies, without necessarily having any pathogenic impact.14 None of the 10 cases with UPID12p had any homozygous SH2B3 abnormalities (Table 1). This was not to be expected considering that these UPIDs did not involve the SH2B3 locus at 12q; in fact, the sequencing of SH2B3 was mainly performed to exclude the presence of mutations in this gene. Among the cases with UPID12p, two were near-triploid HeH with three copies of chromosome 12. One of the HeH cases had a del(12p) corresponding to the UPID12p segment (Table 1). Thus, the 12p deletion occurred in the non-duplicated chromosome 12, changing the allelic ratio from 2:1 to 2:0 in the deleted segment. The other HeH case had a uniparental trisomy of 12p, which most likely occurred through a near-centromeric recombination event after the occurrence of trisomy 12. The remaining eight UPID12p-positive cases carried the ETV6::RUNX1 fusion and all of them had a del(12p) together with an additional der(21)t(12;21). If the 12p deletion occurred in the non-translocated chromosome 12, the +der(21)t(12;21) would result in duplication of the same 12p, resulting in UPID12p. Thus, finding UPID12p in BCP ALLs with ETV6::RUNX1 constitutes indirect evidence for the presence of both del(12p) and +der(21)t(12;21). This obviously also explains the strong association between UPID12p and chr21gain. Only two (0.6%) of the 345 BCP ALLs had UPID12q and both were classified as B-other harbouring either tetrasomy 21 or isochromosomes of 21q (Table 1). None of them had deletions involving SH2B3 but one carried a homozygous SH2B3 frameshift mutation (Table 1). In the study by Sinclair et al.,8 the SH2B3 gene was either homozygously deleted or mutated in five of seven cases with UPID12q. Thus, including the two cases presented herein, two-thirds of UPID12q-positive BCP ALLs have now been shown to carry homozygous SH2B3 abnormalities. Why UPID12q is only present in cases with chr21gain and whether/how these two changes cooperate in the leukaemogenic process remains to be elucidated. We conclude that UPIDs involving chromosome 12 are specific for BCP ALL with chr21gain, that UPID12 typically occurs in NH or HeH and UPID12p in t(12;21), and that only UPID12q is associated with the presence of homozygous SH2B3 abnormalities. Kristina B. Lundin-Ström and Bertil Johansson designed the study, performed analyses, analysed the data, and wrote the manuscript; Andrea Biloglav performed analyses and analysed the data; Anders Castor supplied patient samples and clinical data; Linda Olsson-Arvidsson supplied cytogenetic data. This study was supported by grants from The Swedish Cancer Society (20 0792 PjF), the Swedish Childhood Cancer Foundation (PR2021-0005), the Swedish Research Council (2020-01164), and Governmental Funding of Clinical Research within the National Health Service. The authors declare no conflict of interest. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The genomic landscape and mechanisms driving relapse in KMT2A-rearranged ( KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 36 KMT2A-r ALL (n=19) and AML (n=17) patients of which 25 relapsed and 11 remained in remission. Twenty diagnose-relapse-germline trios and 5 multiple relapse samples were analyzed by whole genome (WGS) and whole exome sequencing (WES) and 30 patients longitudinally by using patient-specific mutations identified by WGS/WES, including the KMT2A-r (average coverage 3300X). The mutational burden increased from diagnosis to relapse and relapse evolved through branching evolution. Relapse was seeded by multiple diagnostic clones in 56%, by a single sweeping clone detected at diagnosis in 22%, and by a single sweeping clone not detected at diagnosis in 22%. Notably, the evolutionary patterns correlated to relapse time, where multiple diagnostic clones seeding relapse were connected to an earlier relapse with all very early relapse ALL (3/3, relapse <9 months from diagnosis) and half of the early AML relapse showing this pattern (2/4, relapse <1 year in complete remission, CR1). By contrast, later relapse was connected to a sweeping clone at relapse with 67% of early relapse ALL (>9 months from diagnosis) and 40% of late relapse AML (>1 year in CR1) showing this pattern. Pathway analysis showed that cell cycle genes, glucocorticoid signalling, purine metabolism, mismatch repair, and B-cell differentiation, were enriched in early relapse ALL (83%, 5/6) and included TP53, CREBBP, NT5C2 PMS2, PRPS2, NR3C1, IKZF1, with none of the very early relapse infant ALL harboring such alterations (n=4). Further, TP53 and IKZF1alterationsco-occurred (n=4/4). These results were validated in public data sets of 98 KMT2A-r ALL infants (n=84) and children (n=14) at diagnosis and relapse (n=24) and showed that 50% of early relapse ALL, and none of the 8 very early relapse ALL, had such alterations. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the PMS2 and NR3C1 deletions. In AML, TP53 and CCND3 alterations were maintained, and gain of WT1 was seen in late relapse AML. Signalling mutations were the most common type of mutations at diagnosis (64%) and relapse (56%) and the frequency was similar in patients that remained in remission and in those that relapsed (55% versus 60%). One infant ALL and four AML patients had multiple relapses, allowing us to study how the genetic landscape evolved across consecutive relapses. This showed a stepwise replacement of clones during treatment in agreement with a fitter clone that evolves under chemotherapeutic selective pressure. Longitudinal analysis allowed sensitive detection of residual leukemia cells and showed that the relapse clone could be detected at diagnosis in 64% of patients. Further, infants with >10% of molecularly detectable leukemia cells after induction therapy, had a high risk of a very early relapse. Ultra-deep sequencing allowed detection of the relapse clone up to 4 months before relapse. In 11 of the 30 patients (3 remission and 8 relapse), low-frequency KMT2A-fusion positive leukemic cells were found at remission outside of the MRD time points. Our longitudinal data also provided unique insights into clonal response to treatment by showing that 1) a change in therapy can favour the eradication of one clone and expansion of another, 2) a clone that initially was the most sensitive clone to therapy, was the one that eventually caused relapse, and 3), a diagnostic clone can be undetectable for a long time before expanding to cause relapse, suggesting that molecular monitoring with personal mutations is a powerful tool to follow response to therapy. These results provide new biological insights into the relapse mechanisms in KMT2A-r leukemia. The data shows different clonal evolution patters depending on when in time the patient relapsed, with very early relapse ALL being seeded by multiple diagnostic subclones and a paucity of acquired genetic alterations at relapse. By contrast, early relapse ALL was characterized by a single diagnostic clone seeding relapse by a clonal sweep along with acquired mutations in chemoresistance-associated genes. To validate and extend these findings, we are currently analyzing 11 additional infant relapse samples with WGS.

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.

The genomic landscape, evolutionary trajectories and the mechanisms driving relapse in KMT2A-rearranged (KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 30 KMT2A-r ALL (n=16) and AML (n=14) patients of which 19 relapsed. Eleven diagnose-relapse-germline trios were analyzed by whole genome (WGS) and whole exome sequencing (WES), and all patients by ultra-deep resequencing (average 3400X) of patient-specific mutations identified by WGS/WES on the diagnostic (n=30) and relapse (n=11) samples, at diagnosis (n=30), during treatment (n=203) and at relapse (n=30, 1-4 bone marrow (BM) relapses in 17 patients). The mutational burden increased from diagnosis to relapse in the diagnose-relapse-germline trios. The combined analysis of WGS/WES and ultra-deep data showed that the leukemias typically evolved through branching evolution, and that a selective sweep occurred at first relapse in 76% of patient. Further, additional subclones were often detected at relapse and in 69% the relapse clone was detected already at diagnosis as a subclone. Eight pathways were recurrently altered including signalling, cell cycle, epigenetic, B-cell development, transcription factor, glucocorticoid receptor signalling, purine metabolism and cohesin. Signalling mutations were the most common type of mutations at diagnosis (50%) and at relapse (41%) and the diagnostic frequency was similar in patients that remained in complete remission (CR) and in those that relapsed (55% versus 47%). Signalling mutations were often subclonal (60%) and 30% had more than one mutation and mutations in signalling genes were usually gained or maintained at relapse in ALL but mainly lost or reduced in size in AML. Alterations in cell cycle genes were enriched at relapse and either gained (60%) or maintained (27%), and included TP53 in ALL and AML, CDKN2A/B in ALL and CCND3 in AML. The TP53 alterations were typically biallelic at relapse. Only 2/30 patients had diagnostic TP53 mutations and both relapsed. Further, relapse-specific mutations in genes involved in glucocorticoid signalling and purine metabolism (CREBBP, NT5C2,PRPS2, NR3C1) were detected in all ALL patients with early relapse (n=4, during maintenance therapy). These relapse samples also had co-occurring IKZF1 deletions with 3 acquired at relapse and 1 maintained, as well as acquired TP53 mutations (3/4 patients). By contrast, infant ALL with a very early relapse (n=4, before maintenance therapy) lacked cell cycle and chemoresistance-associated mutations at relapse. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2 or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the NR3C1 deletion. In AML, gain of WT1 mutations was seen in patients with late relapse (after >1 year in CR1). Longitudinal sequencing on the 30 patients showed that at day 15, ALL patients that remained in CR (n=5) had higher levels of molecularly detectable leukemia cells as compared to those that relapsed (n=8), however at day 29, the reverse was seen with relapse patients having higher levels (n.s). Further, infants with very early relapse (n=3) had a higher leukemic burden at both time points as compared to those with early relapse (n=5). Patients with very early relapse typically reached CR later and the relapse clone could be detected at diagnosis in all 4 patients compared to 2/4 with early relapse. In 7 patients, 4 that relapsed and 3 that remained in CR, low-frequency mutations and KMT2A-fusion positive leukemic clones (DNA-level) were found at remission. Moreover, 4 patients had molecularly detectable clones across all investigated time points until relapse (6-13 time points), 2 had persistent disease but 2 entered CR. Finally, the relapse clone could be detected 46-126 days before relapse in 6 patients. Combined, early relapse in infant and childhood KMT2A-r ALL was characterized by relapse-specific TP53, IKZF1, CREBBP, NR3C1, NTC52 or PRPS2 alterations. By contrast, very early relapse KMT2A-r infant ALL likely arises from pre-existing chemo resistant cells with a paucity of genetic alterations in relapse and chemoresistance-associated genes at relapse. Further, longitudinal molecular tracking provided biological insight into clonal response to treatment and captured the complexity of the temporal evolution in very early and early relapse patients.