Perturbations in dendritic cells (DCs) in B/T cell acute lymphoblastic leukemia (ALL), their cause(s) and consequence(s) on antileukemia immunity, and patient outcomes remain poorly explored. We find that maturation of DC1-6 subsets is disrupted in children and adults with ALL. Conventional DC1 and DC2 subpopulations are reduced at the expense of the progenitors and the DC4 fractions in ALL. The potential to mature, present antigens, and produce cytokines for initiating T cell surveillance appears to be impaired in every ALL DC subset. Such DC subsets are accordingly unable to induce T cell proliferation compared to DCs from healthy donors. MYC overexpression in ALL cells disrupts DC homeostasis and reduces the ability of DCs to induce T cell proliferation. Predominance of cells with transcriptional signatures typical of “stimulated DCs” predicts favorable clinical outcomes in B-ALL, while it is associated with unfavorable outcomes in T-ALL. Phenotyping DC subsets at ALL diagnosis could thus be valuable in informing treatment outcomes.

Pediatric solid tumors present a challenge for precision oncology as most tumors harbor low mutational burden, limiting the applicability of standard mutation-based liquid biopsy approaches for molecular monitoring. To overcome this, we developed a mutation-independent liquid biopsy framework based on Oxford Nanopore Technology sequencing, utilizing robust DNA methylation-based biomarkers for detection and surveillance of high-risk neuroblastoma (HR-NBL), a common pediatric solid tumor. Comparative analysis of tumor-derived DNA methylation profiles against a comprehensive atlas of normal human cell types identified 72 NBL-specific differentially methylated regions (termed meNBLs). Integration of 25 selected meNBLs into the methylation atlas enabled quantitative deconvolution of NBL-derived cfDNA, outperforming copy number and mutation-based estimates. NBL-specific blood plasma-derived cfDNA was detected at diagnosis but not in healthy controls, absent during remission, and markedly elevated at relapse, even before clinical detection of disease recurrence. These findings establish methylation-based deconvolution as a robust, mutation-independent approach for cfDNA monitoring in HR-NBL. ### Competing Interest Statement The authors have declared no competing interest. Cancer Biology Research Center (CBRC), Djerassi Oncology Center, Edmond J. Safra Center for Bioinformatics and Tel Aviv University Center for AI and Data Science (TAD), 0005670-2

INTRODUCTION:This article presents the "Stronger Than Yourself" program, developed within the integrative medicine service of the Oncology Department of Schneider Children's Medical Center of Israel. The program was designed to improve treatment adherence among pediatric oncology patients, with particular emphasis on oral medication intake. We describe the case of a 7.5-year-old girl diagnosed with acute lymphoblastic leukemia (ALL), who persistently refused to take her prescribed medication and struggled to cooperate with the medical team. Her condition required a tailored intervention to enable continuation of essential treatment. The "Stronger Than Yourself" program combines principles of empowerment and self-control, active parental guidance, and the use of mind-body modalities, such as touch, movement, guided imagery, and mindfulness. Through an individualized integrative intervention, her emotional and behavioral barriers were reduced, resulting in significant improvement in cooperation and full regular medication adherence. Since its launch in 2017, the program has supported more than 200 children, with the vast majority achieving independent and sustained medication intake. The model has been recognized by the professional community and presented internationally. This case demonstrates the potential of a focused integrative approach to address common barriers to medication adherence in pediatric oncology, to reduce distress, and strengthen coping in children and families. Further research is warranted to consolidate the model and extend its implementation in other pediatric care settings.

Dendritic cells (DCs) are a rare but critical component of anticancer defense. Perturbations in DCs in B/T-cell acute lymphoblastic leukemia (ALL), their cause(s) and consequence(s) on antileukemia immunity and patient outcome have not been thoroughly explored. Using high-dimensional flow cytometry and single-cell RNA sequencing (scRNA-seq), we profiled peripheral blood (PBMC) and bone marrow (BMMC) samples from 33 high-risk ALL patients and 35 tissue-matched healthy donors. We found significant defects in DC homeostasis that can predict clinical outcome at leukemia presentation. We found maturation of DC1-6 subsets to be disrupted in both children and adults with ALL. Within these subsets, conventional DC (cDC) subsets 1-3 showed greatest disruption with aberrant expression of subtype-specific markers. Computational lineage tracing using CellRANK identified ‘DC precursor-like’ cells within the peripheral DC4 fraction of healthy donors, which were aberrantly expanded in patients with ALL. DCs in ALL deviated from canonical maturation trajectories and had reduced expression of key transcription factors (IRF8 and IRF4), and cytokines receptors (e.g., CD116) required for their homeostatic development. Thus, the specialization of DCs into distinct functional lineages is impaired in ALL. Consistent with their perturbed functional specialization, antigen-primed ALL cDCs failed to induce T-cell proliferation when co-cultured with healthy donor pan-T cells, unlike healthy donor-derived DCs. Because elevated expressions of HLA-DR and CD11c on cDCs mark their mature stage with T-cell priming potential, we asked whether these markers are perturbed during primary human and murine leukemogenesis. In samples from patients and two preclinical ALL mouse models, CD11cHighHLA-DRHigh mature DCs were reduced at the expense of CD11cLowHLA-DRLow/- DCs. Interestingly, overexpression of MYC oncogene in ALL cells reduces the frequencies of CD11cHighHLA-DRHigh mature DCs and impairs their ability to induce T cell proliferation. Finally, we examined the prognostic significance of the defects in DCs in patients with ALL. In the ALL-patient cohort in which we identified defects in DCs, reduced frequencies of CD11cHighHLA-DRHigh DCs was associated with reduced overall survival (P<0.05). In an independent cohort of patients from the Children’s Oncology Group (COG) P9906 trial, children with increased frequencies of cells with a ‘dysfunctional DC’ transcriptome had inferior outcomes compared to their counterparts with ‘functional DC’ transcriptome (P<0.05). In conclusion, defective DC maturation impairs T-cell proliferation and subsequent surveillance and serves as an important predictor of poor patient outcome. Anil Kumar, Kory Hamane, Caroline Duault, Joao Rodrigues. Lima-Junior, Da Hae Jung, Hanjun Qin, Sheyla Salcido, Min Huang, Xinying Guo, Adeleh Taghi. Khani, Ashly Sanchez. Ortiz, Lucy Ghoda, Guido Marcucci, Norman J. Lacayo, Kathleen M. Sakamoto, Christian Hurtz, Martin Carroll, Sarah K. Tasian, Huimin Geng, Lingyun Ji, Saro Armenian, Shai Izraeli, Xiwei Wu, Holden T. Maecker, Srividya Swaminathan. Dendritic cell dysfunction predicts adverse prognosis in high-risk acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A046.

Germline GATA2 deficiency is a pleiotropic condition 1-8 characterized by numerous phenotypes, including monocytopenia, immunodeficiency, microbial susceptibilities, and high rates of myeloid malignancies 1,8. Accurate curation of germline GATA2 variants is critical for patient care and requires well-defined phenotypes associated with GATA2 deficiency. The many phenotypes attributed to the condition render a simple description of GATA2 deficiency difficult, complicating the development of GATA2 variant curation rules. Therefore, the Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) sought to define GATA2 deficiency based on a statistical comparison of phenotype data. To do so, the MM-VCEP systematically analyzed phenotype data and applied statistical comparisons to define the phenotypic features of GATA2 deficiency to inform germline variant curation. The MM-VCEP assembled an international cohort of 339 people with clinically diagnosed GATA2 deficiency from 16 centers in seven countries. The 73 phenotypes of these individuals were compared statistically to those of control participants from the UK Biobank (UKBB). We compared single phenotypes as well as combinations of two, three, and four phenotypes in people with clinically diagnosed GATA2 deficiency to UKBB controls. We defined GATA2 deficiency as any of the 2,903 combinations of two or three phenotypes with log10Odds Ratio ≥3 (OR ≥ 1000). This definition of GATA2 deficiency will inform gene-specific phenotypic criteria used in GATA2 variant curation guidelines that will facilitate standardized variant curation by clinical laboratories worldwide.

Abstract A streamlined, sensitive, and universal molecular method for MRD detection in pediatric AML remains an unmet clinical need. Here, we propose a novel approach based on tracking somatic non‐coding passenger variants. Using whole‐genome sequencing (WGS) of diagnostic bone marrow DNA, we identify patient‐specific somatic passenger variants, which we term “leukemia‐specific passenger variants” (LSPVs). Single‐cell DNA proteogenomic analyses demonstrate that LSPVs are specific and robust markers of leukemic cells, present across the entire leukemic cell population. Moreover, we show that LSPVs are accurate markers of disease burden, stable from diagnosis to relapse. Importantly, LSPVs can be detected in all patients, suggesting the universal applicability of this approach. Leveraging these findings, we developed DAISY‐MRD, a streamlined WGS‐based MRD method that does not rely on the presence of specific trackable genetic aberrations and does not require tailored assay design, making it simple, efficient, and feasible in clinical settings.

PURPOSE:Liquid biopsy monitoring in pediatric solid tumors is limited by low mutational burden and lack of trackable genomic drivers. We sought to develop a mutation-agnostic, methylation-based liquid biopsy framework enabling universal molecular surveillance of high-risk neuroblastoma. EXPERIMENTAL DESIGN:Using whole-genome Oxford Nanopore Technologies sequencing of high-risk neuroblastoma tumors, we compared tumor-derived methylation profiles to a comprehensive atlas of normal human cell types and identified 72 neuroblastoma-specific differentially methylated regions (meNBLs) that were reliably detectable in cfDNA. Marker robustness and specificity were validated using independent neuroblastoma methylation datasets and assessed against methylation profiles from other cancer types. We established neuroblastoma as a distinct methylation entity within the reference atlas by integrating a panel of 25 meNBLs, enabling quantitative estimation of tumor-derived cfDNA. Assay performance was evaluated across diagnostic, remission, relapse, and healthy control samples and compared with mutation- and copy number-based approaches. RESULTS:Neuroblastoma-derived cfDNA was consistently detected at diagnosis and relapse, but was absent in healthy controls and during confirmed remission. Methylation-based deconvolution demonstrated high specificity, with no detectable background signal in controls, and improved performance relative to copy number-based tumor fraction estimation. Longitudinal profiling enabled early molecular detection of relapse and reliable disease monitoring. CONCLUSIONS:We establish a robust, mutation-independent methylation-based liquid biopsy strategy for neuroblastoma that enables accurate, quantitative disease monitoring across all high-risk patients including those lacking trackable genomic alterations. This approach supports clinical translation of methylation-based cfDNA deconvolution as a broadly applicable platform for pediatric precision oncology.

Pediatric acute lymphoblastic leukemia (pALL) is the most common childhood malignancy, yet its etiology remains incompletely understood. However, over the course of three waves of germline genetic research, several non-environmental causes have been identified. Beginning with trisomy 21, seven overt cancer predisposition syndromes (CPSs)—characterized by broad clinical phenotypes that include an elevated risk of pALL—were first described. More recently, newly described CPSs conferring high risk of pALL are increasingly covert, with six exhibiting only minimal or no non-cancer features. These 13 CPSs now represent the principal known hereditary causes of pALL, and human pangenomic data indicates a strong negative selection against mutations in the genes associated with these conditions. Collectively they affect approximately 1 in 450 newborns, of which just a minority will develop the disease. As evidenced by tailored leukemia care protocols for children with trisomy 21, there is growing recognition that CPSs warrant specialized diagnostic, therapeutic, and long-term management strategies. In this review, we investigate the evidence that the 12 other CPSs associated with high risk of pALL may also see benefits from specialized care — even if these needs are often incompletely mapped or addressed in the clinic. Given the rarity of each syndrome, collaborative international research and shared data initiatives will be crucial for advancing knowledge and improving outcomes for these patients.

BACKGROUND:With improving childhood cancer cure rates, there is a growing focus on the long-term health and well-being of survivors. School reintegration is crucial for their rehabilitation and psychosocial adaptation, yet little is known about school violence and bullying faced by cancer survivors upon their return. AIMS:This study aimed to investigate the prevalence and forms of school bullying against childhood acute lymphoblastic leukemia (ALL) survivors, identifying risk and protective factors. METHODS:ALL survivors aged 6-18 years were assessed through interviews and questionnaires. A paired comparison group of healthy students was recruited from their classrooms. RESULTS:The study included 70 students from grades 6-12 (mean age 14.9 ± 1.82 years), comprising 35 ALL survivors and a paired comparison group. ALL survivors endured more physical (M = 1.64, SD = 1.00) and relational bullying (M = 1.50, SD = 1.04) than their healthy classmates (M = 0.90, SD = 0.58; M = 0.90, SD = 0.51 respectively), while facing similar verbal bullying levels (M = 1.71, SD = 0.99 vs. M = 1.38, SD = 0.69). However, survivors reported less cyberbullying (M = 0.45, SD = 0.36) and severe bullying (M = 0.41, SD = 0.05) than their healthy classmates (M = 1.49, SD = 0.48; M = 0.66, SD = 0.53 respectively). Survivors who returned to a different school cohort post-remission faced higher bullying risks than those who rejoined their original cohort (-0.79 < β < -0.92; p < 0.001). Physical disfigurements following cancer treatment were a risk factor for victimization only among those who returned to their original cohort. CONCLUSIONS:The findings highlight the unique challenges faced by childhood leukemia survivors in school reintegration after cancer therapy, emphasizing the significant effect of the school cohort environment on their bullying experiences.

PURPOSE:Modern ALL therapy aims to reduce toxicity, while maintaining and improving the current high cure rates. Acute and late sequelae of anthracyclines are of major concern. The AIEOP-BFM ALL 2009 trial aimed to clarify the need for anthracyclines in low-risk patients. PATIENTS AND METHODS:After 2 weeks of induction therapy, which included two daunorubicin (DNR) doses once weekly (30 mg/m2 each) as part of a 4-drug therapy, patients age 1-17 years with newly diagnosed non-high-risk B-ALL either positive for ETV6::RUNX1 or with rapid treatment response, as assessed by induction day-15 evaluation, were randomly assigned to receive either two additional doses of DNR (control arm [CA]) or no further DNR during induction (experimental arm [EA]). Patients treated as randomly assigned were included in the primary analysis on noninferiority in event-free survival (EFS). Adverse reactions of special interest (ARSI) were analyzed in the as-treated population. RESULTS:Of 6,136 patients enrolled in AIEOP-BFM ALL 2009, 2,514 patients (41.0%) were eligible for this random assignment, with 82.7% actually randomly assigned (EA: n = 1,040 and CA: n = 1,039). The 5-year EFS was 92.5% (SE 0.8%) in CA and 92.2% (SE 0.9%) in EA. Accordingly, cumulative incidence of relapse was 5.8% (SE 0.7%) and 5.7% (SE 0.7%), and overall survival was 97.6% (SE 0.5%) and 97.4% (SE 0.5%) in CA and EA, respectively. Life-threatening and fatal ARSI were similar in the two arms, but there was a three times lower incidence of invasive fungal infections in the EA (0.5% v 1.5%, P = .02). CONCLUSION:A reduced DNR dose during induction did not compromise the outcome of patients with favorable prognostic factors but did diminish infectious toxicity indicated by the lower rate of invasive fungal infections.

Oxford Nanopore Technology (ONT)-based methylation sequencing is emerging as a powerful approach for the rapid and accurate classification of brain tumors, an essential component of precision oncology. However, its broader clinical adoption has been limited by reliance on fresh-frozen (FF) tissue, whereas the vast majority of clinical specimens are formalin-fixed paraffin-embedded (FFPE). In this study, we address this limitation by evaluating the effects of FFPE processing on DNA methylation profiles and introducing a validated protocol for ONT-based classification using DNA extracted directly from pathology-marked regions on stained FFPE slides. This approach enables the targeted selection of tumor-rich areas following histological assessment, thereby improving DNA input quality and tumor content. We demonstrate that even small, low-input samples (≥25 ng) can be successfully classified using this method, with high concordance to final integrated neuropathological diagnoses. Our results show that, despite modest methylation loss associated with formalin fixation, classification performance remains robust. Notably, we identify a correlation between methylation degradation and fixation time, supporting a recommendation to limit formalin exposure to ≤3-4 days when possible. By enabling accurate methylation-based tumor classification from routinely processed, stained FFPE tissue, our protocol integrates seamlessly into existing clinical workflows. This expands the accessibility of ONT-based diagnostics and supports informed, timely treatment decisions-even in cases with minimal tissue availability or urgent clinical need.

To improve the outcome of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients, the AIEOP-BFM ALL 2009 trial modified T-ALL stratification and treatment based on AIEOP-BFM ALL 2000 and other pediatric ALL groups' results. This report aims to describe the outcome of T-ALL patients in trial AIEOP-BFM ALL 2009 and evaluate prognostic features defined within the end of induction (EOI) therapy, for future protocols stratification and interventions. From 06/2010 to 02/2017, 872 T-ALL patients, aged 1-17, were enrolled. High risk (HR) criteria were prednisone poor response (PPR), Day 15 flow cytometry minimal residual disease (MRD) ≥ 10%, no complete remission at EOI, or polymerase chain reaction (PCR)-MRD ≥ 5 × 10-4 at end of consolidation (EOC). Three Cox regression models on event-free survival (EFS) evaluated prognostic factors. Overall, 5-year EFS and survival were 79.9% ± 1.4% and 84.9% ± 1.2% with cumulative incidence of relapse (CIR) and death of 13.0% ± 1.2% and 5.9% ± 0.8%. Five-year EFS and CIR were 86.8% ± 1.6% and 8.7% ± 1.3% in non-HR patients (n = 470); 71.9% ± 2.3% and 18.0% ± 1.9% in HR patients (n = 402). High PCR-MRD levels at EOI and EOC were prognostic in all models, with EOC-MRD ≥ 5 × 10-3 related to a hazard ratio of 6.22 (P < 0.001). When a model considered factors identified at EOI only, central nervous system (CNS)3 (hazard ratio = 2.3, P < 0.001), PPR (hazard ratio = 1.74, P = 0.02), and high EOI-MRD (hazard ratio 4.71 for ≥5 × 10-2 vs. negative, P < 0.001) significantly impacted EFS. Results of T-ALL patients in AIEOP-BFM ALL 2009 were favorable. While EOC-MRD remained the strongest prognostic predictor, PPR, CNS3 disease, and EOI-MRD showed relevant prognostic value, with CNS3 and EOI-MRD ≥ 5 × 10-2 being candidate criteria for early stratification and intervention modifications.

PAX5 is genetically altered in over 30% of B-ALL cases, with point mutations representing the second most common type of alteration. These point mutations are highly enriched in the DNA binding (paired) domain and have been recognized as founder events in PAX5 P80R and PAX5alt B-ALL subtypes, which account for over 12% of B-ALL. However, the molecular features and underlying mechanisms of these mutations in B-ALL remain largely undefined, limiting the development of targeted therapies and resulting in poor outcomes, especially in adult patients. We systematically investigated the functional roles and oncogenic mechanisms of PAX5 paired domain mutations. Integrated CRISPR screening and genomic profiling revealed that the paired domain is critical for PAX5 function. Disruptions in this region significantly reduced cell viability in B-ALL cell lines, and analysis of 2,955 B-ALL samples showed that recurrent PAX5 missense mutations are highly enriched in this domain, further underscoring its functional importance. Functional analysis of knock-in mice with PAX5 mutations revealed that R38H and R140L severely impair DNA binding and block B-cell differentiation, while P34L and P80R retain binding ability but alter binding specificity and gene regulation. Mice carrying homozygous (P34L, R38H, P80R, R140L) or heterozygous (R38H, P80R) mutations developed spontaneous B-ALL within one year. To model co-occurring mutations observed in patients, Pax5 R38H/P34L and R38H/R140L mice were generated. These compound mutations exhibited greater leukemogenic potential than single mutants. Transcriptomic analysis of leukemia samples showed consistent up-regulation of metabolic pathways and Myc target genes. Notably, biallelic PAX5 alterations and additional genetic lesions in signaling pathways were detected in all PAX5R38H/+ and PAX5P80R/+ leukemias, faithfully recapitulating the genetic alteration features of patient B-ALL driven by PAX5 mutations. Among Pax5-mutant mouse strains, Pax5P80R/+ mice develop B-ALL with short latency and complete penetrance, making them an ideal model to study the mutational evolution and leukemogenesis of B-ALL. To characterize the stepwise leukemogenesis process, we performed serial bone marrow aspirations in the same Pax5P80R/+ mice. In all 17 mice analyzed, we identified early preleukemic clones with only Pax5 WT deletions, followed by the emergence of leukemic clones harboring Jak1/3 mutations. Single-cell RNA/BCR-seq revealed that Pax5 WT allele deletions relieve metabolic constraints, thereby promoting clonal expansion of preleukemic B cells blocked at the pre-B stage. These changes coincide with downregulation of metabolic checkpoint genes and upregulation of glycolytic enzymes, promoting genetic instability and facilitating malignant transformation. Clinically, PAX5 P80R patients have better outcomes than PAX5alt cases. Using our mouse models, we observed that leukemic cells driven by the PAX5 P80R mutation exhibit greater sensitivity to dexamethasone compared to the ones carrying other PAX5 mutations in PAX5alt subtype, potentially due to their heightened dependence on glucose uptake and glycolysis, suggesting that metabolic rewiring driven by PAX5 mutants may influence therapeutic response. Interestingly, we found that an aberrant isoform of MEGF10 (aMEGF10) is highly and specifically expressed in PAX5 P80R B-ALL. This N-terminal truncated isoform encodes a protein that retains the intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). Using CUT&Tag, we showed that PAX5 P80R directly binds to the aMEGF10 promoter. By engineering human CD34⁺ cells, we established a humanized PAX5 P80R leukemia model that recapitulates the gene expression profile of patient samples, including robust activation of aMEGF10. Functionally, aMEGF10 binds to and activates SYK, thereby promoting leukemic cell proliferation. Importantly, this oncogenic effect depends on intact ITAM motifs and SYK signaling, revealing a novel targetable vulnerability in PAX5 P80R leukemias. Overall, the study establishes that PAX5 DNA binding domain mutations contribute to leukemogenesis through stepwise loss of PAX5 WT alleles and acquisition of signaling pathway mutations, involving impaired differentiation, metabolic deregulation, and novel oncogene activation. These findings not only deepen the understanding of PAX5-driven leukemias but also highlight aMEGF10–SYK signaling as a promising therapeutic target.

Children with hematological malignancies, are often hospitalized in pediatric intensive care units (PICU) for sepsis with high mortality and re-admission rates. Risk factors for both are inconsistent. We reviewed data of 190 admissions of children with hematological malignancies to PICU for sepsis. Survival rate (SR) was 85%. Mortality risk factors were: non-complete remission (p < 0.01) and status post-stem cell transplantation (p = 0.02), and best predictors were inotropic drugs (p < 0.01), and Pediatric logistic organ dysfunction-2 (p < 0.01) scores. Patients with viremia had the lowest SR (50%, 0.001). One-quarter of the children were re-admitted due to sepsis, and risk factors were: High-risk (HR) hematological malignancy (p < 0.01) and lack of central venous line (CVL) removal (p < 0.01). Sepsis remains a major cause of death in children with hematological malignancies, and re-admissions are common. Our findings support the recommendation of removing CVL during sepsis and highlight those at the highest risk for sepsis to consider individualized anti-infectious prophylaxis.

Chimeric Antigen Receptor (CAR) T-cells can eliminate leukemia and lymphoma within the central nervous system (CNS). Following a case with CD19+ relapse in the CNS in the presence of persisting Tisagenlecleucel, we generated several models to study CAR T-cells in the CNS. In both immunocompetent and humanized leukemia-bearing mice, CNS-residing CAR T-cells were shown to have inferior ex-vivo killing capacities compared to CAR T-cells harvested from the spleen or bone marrow of receptive mice. In a mechanistic work-up, we identified two contributing factors: First, CAR T-cells cultured in cerebrospinal fluid showed impaired cytotoxic and proliferative function compared to those in serum or other media. Second, using a unique in vitro human blood-brain barrier (BBB) model composed of endothelial cells and brain pericytes, we demonstrated that CAR T-cells that crossed the BBB exhibited antigen-independent activation and diminished cytolytic capacity. This led to increased activation-induced cell death of the BBB-migrating CAR T-cells and was confirmed to be independent of the CAR target and the costimulatory domain. Our findings add possible mechanisms of CAR T-cell resistance in the CNS, relevant for CNS hematologic and non-hematologic tumors. ### Competing Interest Statement The authors have declared no competing interest. Israel Cancer Association, https://ror.org/03yxd7304, 20211196 Dotan center for hematologic malignancie

ABSTRACT:Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome that complicates hematologic malignancies. The Optimized HLH Inflammatory (OHI) index, based solely on the combined elevation of soluble CD25 (sCD25; >3900 U/mL) and ferritin (1000 ng/mL) levels, predicts mortality more effectively than the conventional HLH criteria. This study aimed to determine whether mortality in OHI-positive (OHI+) patients is primarily caused by lymphoma or inflammation-related causes. In a multicenter, retrospective study of patients with newly diagnosed lymphoma with sCD25 and ferritin measurements available, patients were classified as OHI+ or OHI negative (OHI-). The 1-year and 3-year overall survival (OS), event-free survival, and causes of death were analyzed, along with predicted vs observed mortality based on other lymphoma-relevant prognostic indexes. Among 135 patients with lymphoma (70 OHI- and 65 OHI+), OHI+ patients had significantly lower OS at 1 year (33% vs 81%; P < .0001) with a median survival of 190 days. OHI+ patients had a 13-fold increased mortality risk (odds ratio, 13.3; 95% confidence interval, 6.0-28.5) despite similar predicted OS based on conventional indexes (72% vs 65%; P = .46). Mortality causes differed significantly. A total of 58% of OHI+ patients died from multiorgan failure, whereas only 6% died from progressive lymphoma. In contrast, 43% of the OHI- patients died from lymphoma progression. The incorporation of etoposide into lymphoma-directed treatment was associated with improved OS in OHI+ T-cell lymphomas (P = .007). These findings underscore the clinical significance of the OHI index as a prognostic tool in lymphoma, to elucidate the mechanisms of mortality, and to identify a high-risk subgroup for which tailored treatments may lead to improved outcomes.

Background: Dendritic cells (DCs) are key regulators of long-term anti-leukemia immunity. The functional status of DCs and mechanisms by which they are perturbed in B/T- cell acute lymphoblastic leukemia (ALL) are unknown. We delineated the defects in DC homeostasis in patients with high-risk ALL and assessed the cause and prognostic significance of these defects. Approach: Using high-dimensional flow cytometry and single cell RNA-sequencing (scRNA-seq) respectively, we profiled proteins and mRNA in single myeloid cells including DC subsets in mononuclear cell samples from peripheral blood (PBMC) and bone marrow (BMMC) of 33 high-risk ALL patients and 35 tissue-matched healthy donor (HD) controls. We used co-cultures of DCs with immune effector cells (T or NK) and primary mouse models of B- and T-ALL to determine the cause and consequence of DC dysfunction in ALL. We correlated DC dysfunction with clinical outcome. Results: Monocytes and DCs exhibit functional similarities and phenotypic plasticity. In the non-malignant immune cell fraction, we found significant reduction in CD14+ total monocytes in PBMC and BMMC of ALL patients compared to HD controls. Classical monocytes (CD14HighCD16-) were significantly reduced at the expense of other monocytes subsets (p<0.0001). Frequencies of CD14+CD209+ monocyte-derived DCs, which can impair T-cell surveillance, were aberrantly increased in ALL patients (p<0.001). In non-monocytic, non-B, non-T, HLA-DR+ fraction of ALL patients, CD123LowCD11c- population aberrantly emerged at the expense of CD11c+ conventional DCs (cDC) and CD123HighCD11c- plasmacytoid DCs (pDCs) (p<0.001 PB, p<0.001 BM for all DC subsets). High-dimensional flow cytometry and scRNA-seq found CD141HighCD1c- (cDC1) and CD141Low/-CD1c+ (cDC2-3) to be reduced at the expense of CD141Low/-CD1c- (cDC4-5) in ALL patient PBMC and BMMC compared to healthy counterparts. We identified DC progenitors (DCP), cDC1-5, and pDCs (DC6) in healthy PBMCs, but this distinction was lost in ALL. cDC1-3 showed greatest disruption, with subtype-specific markers being expressed aberrantly in ALL. Computational lineage tracing showed that ALL DCs do not follow the healthy differentiation trajectory; this could be potentially due to below threshold expression levels of transcription factors IRF8 and IRF4, which are essential for the terminal differentiation of DC subsets. Consistent with this, cytokines (e.g., GM-CSF) and receptors (e.g., CD116 and CD117) required for DC maturation and differentiation were significantly reduced in ALL microenvironments compared to healthy controls. Thus, functional specialization of DC subsets is impaired in ALL. Antigen-primed DCs derived from ALL patients, when co-cultured with healthy donor-derived pan-T cells, were unable to induce T-cell proliferation, unlike their healthy donor-derived DC counterpart. Thus, perturbed DC homeostasis in ALL impairs the ability of DCs to induce T-cell mediated immunity. Increased HLA-DR and CD11c mark mature DCs with T-cell priming potential. Consistent with the inability of DCs in ALL patients to induce T-cell proliferation, we found a reduction in CD11cHighHLA-DRHigh and a concomitant increase in CD11cLowHLA-DRLow/-DC fraction in patients with ALL and in two transgenic mouse models that develop high-risk disease. MYC overexpression in leukemia cells drove the reduction in frequencies of CD11cHighHLA-DRHigh DCs. ALL patients with lower than median frequencies of CD11cHighHLA-DRHighDCs had poor overall survival. Finally, using transcriptomic profiles of less mature/dysfunctional and mature/functional DCs, we used CIBERSORT to estimate the relative fractions of dysfunctional and functional DCs in patients with B-ALL enrolled in the Children's Oncology Group (COG) P9906 trial. Higher proportion of dysfunctional DCs at diagnosis predicted poor clinical outcomes in children with ALL independent of known indicators of poor prognosis including central nervous system involvement of leukemia cells and high white blood cell count at diagnosis (P<0.05). Conclusion: Oncogenic signaling from leukemic cells impairs DC differentiation and function in ALL. The subsequent loss in DC-mediated T-cell priming disrupts anti-leukemic immune surveillance. Level of DC dysfunction in patients with ALL is a reliable predictor of patient prognosis and could inform treatment strategies.