Single-cell genomics details the maturation block in BCP-ALL and identifies therapeutic vulnerabilities in DUX4-r cases.

B cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy, driven by multiple genetic alterations that cause 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, even in subtypes with a favorable outcome. Here, we used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs, belonging to the BCR::ABL1-positive, ETV6::RUNX1-positive, high hyperdiploid, and recently discovered DUX4-rearranged (DUX4-r) subtypes. Projection of the ALL cells along the normal hematopoietic differentiation axis revealed a diversity in the maturation pattern 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-r ALL cells also displayed transcriptional signatures resembling mature B cells. Focusing on the DUX4-r subtype, we found that the blast population displayed 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 in vivo. Given the multilineage priming of the DUX4-r blasts with aberrant expression of the myeloid marker CD371 (CLL-1), we generated chimeric antigen receptor T cells, which effectively eliminated DUX4-r ALL cells in vivo. These results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-r subtype with implications for the understanding of ALL biology and new therapeutic strategies.

Biologic and clinical analysis of childhood gamma delta T-ALL identifies LMO2/STAG2 rearrangements as extremely high-risk.

Acute lymphoblastic leukemia expressing the gamma delta T cell receptor (yo T-ALL) is a poorly understood disease. We studied 200 children with yo T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. yo T-ALL diagnosed in children under three years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by Poly(ADP-ribose) polymerase (PARP) inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric yo T-ALL.

Immune cell populations and induced immune responses at admission in patients hospitalized with vaccine breakthrough SARS-CoV-2 infections.

Background: Vaccine breakthrough SARS-CoV-2 infections are common and of clinical and public health concern. However, little is known about the immunological characteristics of patients hospitalized due to these infections. We aimed to investigate and compare immune cell subpopulations and induced immune responses in vaccinated and non-vaccinated patients hospitalized with severe COVID-19. Methods: A nested case-control study on adults (≥ 18 years) who received at least two doses of a mRNA-COVID-19 vaccine and were hospitalized with SARS-CoV-2 breakthrough infections and severe COVID-19 between January 7, 2021, and February 1, 2022, were eligible for inclusion. Age- and sex-matched non-vaccinated controls were identified. Immunophenotyping was performed using a custom-designed 10-color flow cytometry prefabricated freeze-dried antibody panel (DuraClone, Beckman Coulter (BC), Brea, Calif). TruCulture (Myriad RBM, Austin, USA) was used to assess induced immune response in whole blood, revealing different critical signaling pathways as a proxy for immune function. All samples were obtained within 48 hours of admission. Results: In total, 20 hospitalized patients with severe COVID-19 and a breakthrough SARS-CoV-2 infection were included, ten vaccinated and ten non-vaccinated patients. Vaccinated patients had lower concentrations of CD19 B cells (p = 0.035), naïve CD4 T cells (p = 0.015), a higher proportion of γδ1 T cells (p = 0.019), and higher unstimulated immune cell release of IL-10 (p = 0.015). Conclusion: We observed immunological differences between vaccinated and non-vaccinated patients hospitalized due to severe COVID-19 that indicate that vaccinated patients had lower B cell concentrations, lower concentrations of CD4 naïve T cells, a skewed gamma-delta V1/V2 ratio, and an exaggerated IL-10 response at admission. These results could indicate a suboptimal immune response involved in SARS-CoV-2 breakthrough infections that cause severe COVID-19 in vaccinated adults. However, the sample size was small, and further research is needed to confirm these results.

Widespread alterations in systemic immune profile are linked to lung function heterogeneity and airway microbes in cystic fibrosis.

BACKGROUND: Excessive inflammation and recurrent airway infections characterize people with cystic fibrosis (pwCF), a disease with highly heterogeneous clinical outcomes. How the overall immune response is affected in pwCF, its relationships with the lung microbiome, and the source of clinical heterogeneity have not been fully elucidated. METHODS: Peripheral blood and sputum samples were collected from 28 pwCF and an age-matched control group. Systemic immune cell subsets and surface markers were quantified using multiparameter flow cytometry. Lung microbiome composition was reconstructed using metatranscriptomics on sputum samples, and microbial taxa were correlated to circulating immune cells and surface markers expression. RESULTS: In pwCF, we found a specific systemic immune profile characterized by widespread hyperactivation and altered frequencies of several subsets. These included substantial changes in B-cell subsets, enrichment of CD35+/CD49d+ neutrophils, and reduction in dendritic cells. Activation markers and checkpoint molecule expression levels differed from healthy subjects. CTLA-4 expression was increased in Tregs and, together with impaired B-cell subsets, correlated with patients' lung function. Concentrations and frequencies of key immune cells and marker expression correlated with the relative abundance of commensal and pathogenic bacteria in the lungs. CONCLUSION: The CF-specific immune signature, involving hyperactivation, immune dysregulation with alteration in Treg homeostasis, and impaired B-cell function, is a potential source of lung function heterogeneity. The activity of specific microbes contributes to disrupting the balance of the immune response. Our data provide a unique foundation for identifying novel markers and immunomodulatory targets to develop the future of cystic fibrosis treatment and management.

Improved Innate Immune Function in Patients with Chronic Lymphocytic Leukemia Treated with Targeted Therapy in Clinical Trials.

PURPOSE: Patients with chronic lymphocytic leukemia (CLL) have increased risk of severe infections. Although adaptive immune dysfunction is well described, clinical tools for identifying patients at risk are lacking, warranting investigation of additional immune components. In contrast to chemotherapy, targeted agents could spare or even improve innate immune function. Therefore, we investigated innate immune phenotypes and function in patients with CLL before and during targeted treatment. EXPERIMENTAL DESIGN: Baseline and consecutive blood samples were collected from patients with CLL treated with acalabrutinib (n = 17) or ibrutinib+venetoclax (n = 18) in clinical trials. Innate immune function was assessed by TruCulture, a whole-blood ligand-stimulation assay quantifying cytokine release in response to standardized stimuli. Innate immune phenotypes were characterized by flow cytometry. As a proxy for infections, we mapped antimicrobial use before and during treatment. RESULTS: At baseline, patients with CLL displayed impaired stimulated cytokine responses to the endotoxin lipopolysaccharide (LPS) along with deactivated monocytes, enrichment of myeloid-derived suppressor cells and metamyelocytes, and elevated (unstimulated) proinflammatory cytokines. Two/three cycles of acalabrutinib or ibrutinib normalized LPS-stimulated responses, in parallel with decreased duration of infections. Innate immune profiles and elevated proinflammatory cytokines further normalized during longer-term acalabrutinib or ibrutinib+venetoclax, paralleled by decreased infection frequency. CONCLUSIONS: Innate immune impairment and infection susceptibility in patients with CLL were restored in parallel during targeted therapy. Thus, targeted treatment may reduce the risk of infections in CLL, as currently under investigation in the PreVent-ACaLL phase 2 trial of acalabrutinib+venetoclax for high-risk CLL (NCT03868722).

Multimodal classification of molecular subtypes in pediatric acute lymphoblastic leukemia.

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Biologic and clinical features of childhood gamma delta T-ALL: identification of STAG2/LMO2 γδ T-ALL as an extremely high risk leukemia in the very young.

PURPOSE: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease. METHODS: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts. RESULTS: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping resulting in deregulation of gene expression associated with T-cell differentiation. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine. CONCLUSION: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL. SUPPORT: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

Integrins and the Metastasis-like Dissemination of Acute Lymphoblastic Leukemia to the Central Nervous System.

Acute lymphoblastic leukemia (ALL) disseminates with high prevalence to the central nervous system (CNS) in a process resembling aspects of the CNS surveillance of normal immune cells as well as aspects of brain metastasis from solid cancers. Importantly, inside the CNS, the ALL blasts are typically confined within the cerebrospinal fluid (CSF)-filled cavities of the subarachnoid space, which they use as a sanctuary protected from both chemotherapy and immune cells. At present, high cumulative doses of intrathecal chemotherapy are administered to patients, but this is associated with neurotoxicity and CNS relapse still occurs. Thus, it is imperative to identify markers and novel therapy targets specific to CNS ALL. Integrins represent a family of adhesion molecules involved in cell-cell and cell-matrix interactions, implicated in the adhesion and migration of metastatic cancer cells, normal immune cells, and leukemic blasts. The ability of integrins to also facilitate cell-adhesion mediated drug resistance, combined with recent discoveries of integrin-dependent routes of leukemic cells into the CNS, have sparked a renewed interest in integrins as markers and therapeutic targets in CNS leukemia. Here, we review the roles of integrins in CNS surveillance by normal lymphocytes, dissemination to the CNS by ALL cells, and brain metastasis from solid cancers. Furthermore, we discuss whether ALL dissemination to the CNS abides by known hallmarks of metastasis, and the potential roles of integrins in this context.

Erratum: Author Correction: Early stimulated immune responses predict clinical disease severity in hospitalized COVID-19 patients.

[This corrects the article DOI: 10.1038/s43856-022-00178-5.].

Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia.

Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3 D835H or NRAS G13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

Early stimulated immune responses predict clinical disease severity in hospitalized COVID-19 patients.

Background: The immune pathogenesis underlying the diverse clinical course of COVID-19 is poorly understood. Currently, there is an unmet need in daily clinical practice for early biomarkers and improved risk stratification tools to help identify and monitor COVID-19 patients at risk of severe disease. Methods: We performed longitudinal assessment of stimulated immune responses in 30 patients hospitalized with COVID-19. We used the TruCulture whole-blood ligand-stimulation assay applying standardized stimuli to activate distinct immune pathways, allowing quantification of cytokine responses. We further characterized immune cell subsets by flow cytometry and used this deep immunophenotyping data to map the course of clinical disease within and between patients. Results: Here we demonstrate impairments in innate immune response pathways at time of COVID-19 hospitalization that are associated with the development of severe disease. We show that these impairments are transient in those discharged from hospital, as illustrated by functional and cellular immune reconstitution. Specifically, we identify lower levels of LPS-stimulated IL-1ß, and R848-stimulated IL-12 and IL-17A, at hospital admission to be significantly associated with increasing COVID-19 disease severity during hospitalization. Furthermore, we propose a stimulated immune response signature for predicting risk of developing severe or critical COVID-19 disease at time of hospitalization, to validate in larger cohorts. Conclusions: We identify early impairments in innate immune responses that are associated with subsequent COVID-19 disease severity. Our findings provide basis for early identification of patients at risk of severe disease which may have significant implications for the early management of patients hospitalized with COVID-19.

Added Value of Reanalysis of Whole Exome- and Whole Genome Sequencing Data From Patients Suspected of Primary Immune Deficiency Using an Extended Gene Panel and Structural Variation Calling.

Background: Knowledge of the genetic variation underlying Primary Immune Deficiency (PID) is increasing. Reanalysis of genome-wide sequencing data from undiagnosed patients with suspected PID may improve the diagnostic rate. Methods: We included patients monitored at the Department of Infectious Diseases or the Child and Adolescent Department, Rigshospitalet, Denmark, for a suspected PID, who had been analysed previously using a targeted PID gene panel (457 PID-related genes) on whole exome- (WES) or whole genome sequencing (WGS) data. A literature review was performed to extend the PID gene panel used for reanalysis of single nucleotide variation (SNV) and small indels. Structural variant (SV) calling was added on WGS data. Results: Genetic data from 94 patients (86 adults) including 36 WES and 58 WGS was reanalysed a median of 23 months after the initial analysis. The extended gene panel included 208 additional PID-related genes. Genetic reanalysis led to a small increase in the proportion of patients with new suspicious PID related variants of uncertain significance (VUS). The proportion of patients with a causal genetic diagnosis was constant. In total, five patients (5%, including three WES and two WGS) had a new suspicious PID VUS identified due to reanalysis. Among these, two patients had a variant added due to the expansion of the PID gene panel, and three patients had a variant reclassified to a VUS in a gene included in the initial PID gene panel. The total proportion of patients with PID related VUS, likely pathogenic, and pathogenic variants increased from 43 (46%) to 47 (50%), as one patient had a VUS detected in both initial- and reanalysis. In addition, we detected new suspicious SNVs and SVs of uncertain significance in PID candidate genes with unknown inheritance and/or as heterozygous variants in genes with autosomal recessive inheritance in 8 patients. Conclusion: These data indicate a possible diagnostic gain of reassessing WES/WGS data from patients with suspected PID. Reasons for the possible gain included improved knowledge of genotype-phenotype correlation, expanding the gene panel, and adding SV analyses. Future studies of genotype-phenotype correlations may provide additional knowledge on the impact of the new suspicious VUSs.

Flow Cytometric Detection of Malignant Blasts in Cerebrospinal Fluid: A Biomarker of Central Nervous System Involvement in Childhood Acute Lymphoblastic Leukemia.

Despite the excellent prognosis for children and adolescents with acute lymphoblastic lymphoma (ALL), the involvement of the central nervous system (CNS) represents a major therapeutic challenge. Patients who develop CNS relapse have a very poor prognosis, and since current methods cannot reliably identify patients with CNS involvement or patients at high risk of CNS relapse, all children with ALL receive CNS-directed treatment. The current golden standard for detecting CNS involvement is the assessment of cytomorphology on cytospin slides of cerebrospinal fluid (CSF). This technique is inadequate due to low sensitivity and reproducibility. Flow cytometric analysis of CSF represent a novel, highly specific and sensitive technique for the detection of leukemic cells in the CNS. In prospective studies, CSF flow cytometry demonstrated two to three times higher rates of CNS involvement at diagnosis of childhood ALL than conventional cytospin, and especially demonstrated superior sensitivity in detecting low-level CNS disease. CNS involvement determined via flow cytometry has been linked to a higher risk of CNS relapse and poor outcomes in several studies. In this review, we discuss the central analytical concepts of CSF flow cytometry and summarize the current evidence supporting the use of flow cytometric detection of malignant blasts as a biomarker of CNS involvement in childhood ALL.

Does minimal central nervous system involvement in childhood acute lymphoblastic leukemia increase the risk for central nervous system toxicity?

Central nervous system (CNS) involvement in childhood acute lymphoblastic leukemia (ALL) implicates enhanced intrathecal chemotherapy, which is related to CNS toxicity. Whether CNS involvement alone contributes to CNS toxicity remains unclear. We studied the occurrence of all CNS toxicities, seizures, and posterior reversible encephalopathy syndrome (PRES) in children with ALL without enhanced intrathecal chemotherapy with CNS involvement (n = 64) or without CNS involvement (n = 256) by flow cytometry. CNS involvement increased the risk for all CNS toxicities, seizures, and PRES in univariate analysis and, after adjusting for induction therapy, for seizures (hazard ratio [HR] = 3.33; 95% confidence interval [CI]: 1.26-8.82; p = 0.016) and PRES (HR = 4.85; 95% CI: 1.71-13.75; p = 0.003).

Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro-a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.

High CD34 surface expression in BCP-ALL predicts poor induction therapy response and is associated with altered expression of genes related to cell migration and adhesion.

Minimal residual disease (MRD) constitutes the most important prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Flow cytometry is widely used in MRD assessment, yet little is known regarding the effect of different immunophenotypic subsets on outcome. In this study of 200 BCP-ALL patients, we found that a CD34-positive, CD38 dim-positive, nTdT dim-positive immunophenotype on the leukemic blasts was associated with poor induction therapy response and predicted an MRD level at the end of induction therapy (EOI) of ≥ 0.001. CD34 expression was strongly and positively associated with EOI MRD, whereas CD34-negative patients had a low relapse risk. Further, CD34 expression increased from diagnosis to relapse. CD34 is a stemness-associated cell-surface molecule, possibly involved in cell adhesion/migration or survival. Accordingly, genes associated with stemness were overrepresented among the most upregulated genes in CD34-positive leukemias, and protein-protein interaction networks showed an overrepresentation of genes associated with cell migration, cell adhesion, and negative regulation of apoptosis. The present work is the first to demonstrate a CD34-negative immunophenotype as a good prognostic factor in ALL, whereas high CD34 expression is associated with poor therapy response and an altered gene expression profile reminiscent of migrating cancer stem-like cells.

Characteristics of white blood cell count in acute lymphoblastic leukemia: A COST LEGEND phenotype-genotype study.

BACKGROUND: White blood cell count (WBC) as a measure of extramedullary leukemic cell survival is a well-known prognostic factor in acute lymphoblastic leukemia (ALL), but its biology, including impact of host genome variants, is poorly understood. METHODS: We included patients treated with the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL-2008 protocol (N = 2347, 72% were genotyped by Illumina Omni2.5exome-8-Bead chip) aged 1-45 years, diagnosed with B-cell precursor (BCP-) or T-cell ALL (T-ALL) to investigate the variation in WBC. Spline functions of WBC were fitted correcting for association with age across ALL subgroups of immunophenotypes and karyotypes. The residuals between spline WBC and actual WBC were used to identify WBC-associated germline genetic variants in a genome-wide association study (GWAS) while adjusting for age and ALL subtype associations. RESULTS: We observed an overall inverse correlation between age and WBC, which was stronger for the selected patient subgroups of immunophenotype and karyotypes (ρBCP-ALL  = -.17, ρT-ALL  = -.19; p < 3 × 10-4 ). Spline functions fitted to age, immunophenotype, and karyotype explained WBC variation better than age alone (ρ = .43, p << 2 × 10-6 ). However, when the spline-adjusted WBC residuals were used as phenotype, no GWAS significant associations were found. Based on available annotation, the top 50 genetic variants suggested effects on signal transduction, translation initiation, cell development, and proliferation. CONCLUSION: These results indicate that host genome variants do not strongly influence WBC across ALL subsets, and future studies of why some patients are more prone to hyperleukocytosis should be performed within specific ALL subsets that apply more complex analyses to capture potential germline variant interactions and impact on WBC.