Supplementary Figure 2. Complexity of phylogenetic tree, comparing WT DA to IR and BT WT.

ABSTRACT Background The European ALLTogether protocol for childhood acute lymphoblastic leukemia, initiated in Sweden 2019, introduced earlier asparaginase during induction, dexamethasone instead of prednisone for all patients, and omitted anthracyclines from low‐risk induction to reduce treatment‐related toxicity. Consolidation‐1 was based on the Induction 1B‐phase developed by the BFM‐group, replacing mercaptopurine, methotrexate, and asparaginase used in the previous NOPHO ALL2008 protocol, ALL2008. Following implementation, early treatment toxicity was considered unacceptably high, prompting a protocol amendment. We compared the prevalence of 14 predefined toxicities, the number of inpatient days, and weight changes during induction and consolidation‐1 between the two protocols. Methods We conducted a population‐based cohort study in Sweden, reviewing patient records from 117 children treated under ALLTogether protocol and 234 matched controls under ALL2008 protocol. Results The mean number of toxicities per patient was similar between the protocols (2.5 [290/117] vs. 2.3 [547/234]). ALLTogether cohort had significantly greater weight gain, with over 50% experiencing a > 10% increase ( p < 0.01). Hyperglycemia (OR 5.17, 95% CI 1.93–13.82) and osteonecrosis (3.4% vs. 0%, p = 0.012) were more common, while liver dysfunction (0.59, 0.38–0.93) was less frequent in the ALLTogether protocol. The number of inpatient days was similar across protocols, except for the initial hospitalization, which was longer in ALLTogether (median 11 vs. 7 days, p < 0.01). Conclusions The early introduction of asparaginase likely contributed to increased weight gain, hyperglycemia, and osteonecrosis. While overall toxicity burden remained similar between protocols, the shift in toxicity profile may explain the perception of increased early toxicity during treatment with the ALLTogether protocol. Trial Registration ClinicalTrials.gov identifier: NCT03911128; EudraCT numbers: 2018‐001795‐38 and 2008‐003235‐20

Supplementary Table 4. Segmental aberrations. All 20 Wilms tumor (WT) cases and their corresponding samples are listed. ‘Sample position’ refers to the individual sample and its position in a certain paraffin block (see Figure 1 for details.) For each genetic event the following information is given: the ploidy level (Ploidy), chromosome (chr), the genomic start and end positions, genetic events annotated as gain, loss, CNNI (copy neutral imbalance) or homozygous loss, the length of the aberration (bp), which chromosomal cytoband, the median log2 ratio, allelic composition and mutated cell fraction (MCF). IR= intermediate risk, BT= blastemal type, DA= diffuse anaplasia. Genomic positions accordning to hg19.

INTRODUCTION:Infection remains the most common treatment-related toxicity of childhood ALL, emphasizing the need to identify patients at risk and to tailor treatment strategies accordingly. AIMS:The primary aim was to compare infectious toxicity during early treatment for childhood ALL following the ALLTogether and NOPHO ALL-2008 (ALL-2008) protocols, and second, to identify risk factors for infectious toxicity. METHODS:A national retrospective matched cohort study was conducted, including 345 patients aged 1-17 years diagnosed with ALL and treated in Sweden according to the ALLTogether or ALL-2008 protocols. Nonparametric tests were used to compare infectious outcomes between protocols, and regression modeling was used to identify risk factors of the infectious outcomes. RESULTS:Treatment following ALL-2008 showed higher infectious toxicity during induction, whereas treatment following ALLTogether showed increased infectious toxicity during consolidation 1. Overall, treatment according to ALL-2008 was associated with a higher incidence of infections. Anthracycline use and young age (1-9 years) were associated with both higher infectious incidence and more severe infectious complications. Dexamethasone was associated with both lower incidence and lower severity of infectious complications as compared to prednisone. CONCLUSIONS:A notable shift in the timing of infectious toxicity was observed between the two treatment protocols. Risk factors for infectious toxicity during early treatment include anthracycline use and young age. Dexamethasone as an induction steroid was associated with lower infectious burden, although its effect is difficult to isolate from the simultaneous anthracycline effect. The findings suggest that treatment composition plays a central role in determining both the extent and timing of infectious complications.

Supplementary Table 1. Overview of the study cohort and the clinical data. This table contains basic clinical information, including age, sex, whether tumors were uni- or bilateral, histology, stage, risk group, treatment to which the resection specimen analyzed was subjected to, the final outcome and the record of adverse events.

Supplementary Figure 3. Relationship between anaplasia, copy number aberrations, regression and mitotic rate.

Supplementary Figure 1 Geographic overview of histological and genetic features.

Supplementary Table 3. Table. Sample list with TP53 status. All 20 WT cases and their corresponding samples are listed. ‘Sample position’ refers to the individual sample and its position in a certain paraffin block (see Figure 1 for details.) IR= intermediate risk, BT= blastemal type, DA= diffuse anaplasia. Headings marked in grey show TP53 sequencing results; wt=wild type, mut=mutation, NA= not annotated (i.e. not sequenced), aa= amino acid, REF= reference nucleotide, ALT= alternative nucleotide, TP53 ref= number of wild type TP53 alleles, TP53 mut=number of TP53 mutated alleles. Genomic positions according to hg19. In all columns with grey headings (except column ‘TP53’), semicolons (;) denote when more than one unique TP53 mutation is present in the same tumor sample.

Supplementary Table 2. Representativeness of study participants. Details on how the patients with Wilms tumor in the present study compare to the general population of patients diagnosed with with Wilms tumor.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment. Neuroblastoma (NB) is a frequent childhood cancer that often becomes resistant to therapy. Here, the authors perform spatiotemporal genomic profiling of NBs before and after chemotherapy and find an evolutionary process characteristic of NBs growing resistant after first responding to treatment.

The survival rate for childhood acute lymphoblastic leukaemia (ALL) has improved over time, due to uniform treatment with risk-adapted therapy developed through international collaboration. The international ALLTogether treatment protocol (EUDRACT 2018–001795-38; ClinicalTrials.gov: NCT03911128) replaced the NOPHO ALL2008 (ALL2008; EUDRACT NCT03911128) protocol in Sweden in 2019. ALLTogether introduced a high dose of intravenous asparaginase during early induction combined with dexamethasone. Both asparaginase and glucocorticoids lead to a transient coagulopathy. Asparagine depletion reduces liver synthesis both pro- and anticoagulants, most importantly fibrinogen and antithrombin,1 leading to coagulopathy, whereas dexamethasone induces hypercoagulability.2, 3 After the initiation of ALLTogether, we encountered patients with bleeding tendency during early treatment regardless of sufficient platelet levels. Further examination revealed activated partial thromboplastin time (aPTT) without clot formation and immeasurable low fibrinogen (<0.3 g/L). Replacement of fibrinogen corrected the bleeding tendency and aPTT. To address concerns on increased risk of bleeding, we collected antithrombin and fibrinogen values and clinical data on thromboses and bleeding events during early treatment, and compared the results between the two protocols. This national study was approved by the Swedish Ethical Review Authority (2021–03146). We identified all children (n = 120) between the age of 1 and 18 years at diagnosis and treated in the six Swedish childhood cancer centres according to ALLTogether protocols between August 2019 (initiation of the pilot in Sweden) and March 2021 (the first substantial amendment) from the Swedish Childhood Cancer Registry. Patients with Down syndrome (n = 2) or transferred to high-risk block therapy during consolidation 1 (n = 1) were excluded. We selected controls matched by NCI-risk group treated with the ALL2008 protocol (n = 185). Patient characteristics between groups were balanced (Table 1). Data on coagulation parameters, thromboses and bleeding events during induction and consolidation 1 were collected from medical charts. As clinical practice in measuring fibrinogen and antithrombin values during treatment varied between the paediatric oncology centres, we could not compare the values at specific timepoints and recorded only the lowest measured abnormal values. Otherwise, fibrinogen and antithrombin were classified as normal or below reference value. Fibrinogen values were available for 76/117 (65%) of the ALLTogether and 99/185 (53%) of the ALL2008 patients. During ALLTogether induction, 46% (29/63) had fibrinogen levels below the reference (<2.0 g/L), comparable to 54% (45/84) during ALL2008 (p = 0.295). Hypofibrinogenemia was more pronounced during ALLTogether induction, 86% (25/29) had fibrinogen values <1.0 g/L, and 35% (10/29) had fibrinogen values <0.5 g/L, p < 0.001 (Table 1). During consolidation 1, 58% (28/48) of the ALLTogether patients had fibrinogen levels below the reference, compared with 75% (51/68) in the ALL2008 group (p = 0.058). Extremely low fibrinogen values (<0.5 g/L) during consolidation 1 were uncommon (1/28 and 1/51, respectively). Antithrombin was measured in 56/117 (48%) in the ALLTogether and 86/185 (46%) in the ALL2008 groups. More patients treated according to ALLTogether (65%; 28/43) than ALL2008 (10%; 7/72) had antithrombin values under the reference (<0.85 IU/mL) during induction (p < 0.001), but the level of reduced antithrombin activity did not differ between the protocols. No differences between the groups were seen under consolidation 1 (Table 1). As the initial reason to compare the two protocols was the unexpectedly low fibrinogen levels, we collected both major (>20 g/L decrease in haemoglobin level due to the bleed and/or bleeding event involving critical organs such as the central nervous system) and non-major (defined as bleeding events leading to intervention by healthcare professionals, e.g. transfusions, hospital admission or other measures that would otherwise not have been taken) bleeding events. There were no significant differences in bleeding events between the protocols: 15/117 (13%) of the ALLTogether patients had 23 bleedings compared with 34/185 (18%) patients with 47 bleeding events in ALL2008 (p = 0.202). Both groups had two major bleedings: one epidural bleeding and one secondary to cerebral sinus venous thrombosis (CSVT; ALLTogether) and one haemopneumothorax and one intestinal bleeding (ALL2008). We then compared thrombosis during early treatment: 6% (7/117) of ALLTogether patients had a thrombotic event, defined as thrombosis leading to anticoagulant treatment (three CSVTs, two upper and two lower venous system). One CSVT was associated with secondary bleeding. By comparison, 4/182 (2%) had a thrombosis (three CSVTs and one lower venous system) in the ALL2008 group. In line with previous reports,3-5 combining asparaginase with dexamethasone led to a pronounced hypofibrinogenemia during induction in ALLTogether. Despite low fibrinogen during ALLTogether, the number of bleeding events did not increase. This is likely due to simultaneous decrease in anticoagulant proteins maintaining the delicate haemostatic balance. Non-major bleeds, expected but seldom reported side effects during ALL treatment, were common and led to outpatient visits, hospital admissions or transfusions. Our cohort was not powered to compare the incidence of thrombotic events between the two protocols, but we would expect the timing of thromboses to be earlier during ALLTogether than during ALL2008. Study limitations included the relatively low number of patients, retrospective approach, and differences in routines and the laboratory methods used for measuring coagulation parameters between centres and protocols. Many patients lacked fibrinogen and antithrombin values, especially during ALL2008 induction, before the start of asparaginase treatment. Due to these limitations, the results should be interpreted with caution. Study strengths were the national, population-based design and detailed patient chart review. In conclusion, our study showed no significant differences in the frequency of thrombosis or bleeding events between treatment protocols despite significantly decreased fibrinogen levels during ALLTogether. Yet, haemostatic reserve capacity during ALLTogether may be even more reduced than during ALL2008 and should be considered with clinical bleeding tendency and prior to surgery. As some patients had immeasurable fibrinogen levels, we encourage to consider the increased potential predisposition to bleeding in ALLTogether, particularly during induction, to optimise haemostasis in high-risk scenarios when managing these patients. Future studies should try to clarify whether novel methods including global haemostasis assays can help to identify children at risk of bleeding and those who may benefit from thromboprophylaxis. SR, AH, JA, MB, MH, ASH and HV contributed to the design and implementation of the study. JF, JJ, ID, LH, AV, AW and OZ collected data from paediatric cancer centres. JF, AH and SR performed data analysis and interpretation. JF wrote the original draft. All authors reviewed, edited and approved the original draft. We thank the Ellen Bachrach Memorial Fund (AH) and the Swedish Childhood Cancer Fund (MH, SR) for financial support. The authors have no conflict of interest to report.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Supplementary Materials and Methods, Supplementary Figures S1-S4, Supplementary Tables S1-S4

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

AbstractPurpose:While patients with intermediate-risk (IR) Wilms tumors now have an overall survival (OS) rate of almost 90%, those affected by high-stage tumors with diffuse anaplasia have an OS of only around 50%. We here identify key events in the pathogenesis of diffuse anaplasia by mapping cancer cell evolution over anatomic space in Wilms tumors.Experimental Design:We spatially mapped subclonal landscapes in a retrospective cohort of 20 Wilms tumors using high-resolution copy-number profiling and TP53 mutation analysis followed by clonal deconvolution and phylogenetic reconstruction. Tumor whole-mount sections (WMS) were utilized to characterize the distribution of subclones across anatomically distinct tumor compartments.Results:Compared with non-diffuse anaplasia Wilms tumors, tumors with diffuse anaplasia showed a significantly higher number of genetically distinct tumor cell subpopulations and more complex phylogenetic trees, including high levels of phylogenetic species richness, divergence, and irregularity. All regions with classical anaplasia showed TP53 alterations. TP53 mutations were frequently followed by saltatory evolution and parallel loss of the remaining wild-type (WT) allele in different regions. Morphologic features of anaplasia increased with copy-number aberration (CNA) burden and regressive features. Compartments demarcated by fibrous septae or necrosis/regression were frequently (73%) associated with the emergence of new clonal CNAs, although clonal sweeps were rare within these compartments.Conclusions:Wilms tumors with diffuse anaplasia display significantly more complex phylogenies compared with non-diffuse anaplasia Wilms tumors, including features of saltatory and parallel evolution. The subclonal landscape of individual tumors was constrained by anatomic compartments, which should be considered when sampling tissue for precision diagnostics.

Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.

Our current knowledge of the different immune cells in neuroblastoma is based on in vitro and in vivo studies mainly focusing on a single cell type. Different studies have conveyed conflicting results and despite the use of anti-GD2 immunotherapy in the clinic, a comprehensive immune cell overview at the single-cell level is still missing and understanding the complete composition of immune cells neuroblastoma will be crucial for the development of novel immunotherapeutics against the disease. In this study, we performed single-cell RNA-sequencing on human neuroblastoma coupled with multiplex immunohistochemistry and survival analysis to provide a complete cellular and molecular immune cell landscape of human neuroblastoma. Further, we contrasted the neuroblastoma data with single-cell RNA-sequencing data from normal fetal adrenal gland to characterize cell-state changes from normal tissue to neuroblastoma. Our analysis revealed 27 immune cell subtypes including distinct subpopulations of myeloid, NK, B and T cells that were associated with a survival benefit. Multiple subtypes of B and NK cells, not seen in neuroblastoma before, were identified. We propose the presence of tertiary lymphoid structures and showed that Active NK cells correlated with improved survival. In contrast to adult cancers, we detected no difference in cytotoxicity and exhaustion score for cytotoxic T cells, nor Treg activity. However, we demonstrated an increase in inflammatory monocyte signature score from normal to tumor derived myeloid cells. Finally, we performed receptor-ligand interaction analysis between tumor, stroma and immune cells, where we highlight several interactions that we suggest for future studies of how to exploit immune cells as a therapeutic option in neuroblastoma. Our findings significantly broaden the understanding of the immune composition in neuroblastoma and provides a resource for the development of novel immunotherapeutics. Citation Format: Bronte Manouk Verhoeven, Shenglin Mei, Thale K. Olsen, Karin K. Gustafsson, Anders Valind, Axel Lindström, David G. Nord, Shahrzad S. Fard, Catharina Hagerling, Peter V. Kharchenko, Per Kogner, John I. Johnsen, Ninib Baryawno. The immune cell atlas of human neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6215.

Supplementary Data from Branching Copy-Number Evolution and Parallel Immune Profiles across the Regional Tumor Space of Resected Pancreatic Cancer