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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., Competing Interests: Declaration of competing interest We declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Overweight and obesity are linked to increased hospitalization and mortality in COVID-19 patients. This study aimed to characterize induced immune responses and deep immune cell profiles stratified by BMI in hospitalized COVID-19 patients., Methods and Results: This observational multicenter cohort pilot study included 122 adult patients with PCR-confirmed COVID-19 in Denmark, stratified by BMI (normal weight, overweight, obese). Inflammation was assessed using TruCulture® and immune cell profiles by flow cytometry with a customized antibody panel (DuraClone®). Patients with obesity had a more pro-inflammatory phenotype with increased TNF-α, IL-8, IL-17, and IL-10 levels post-T cell stimulation, and altered B cell profiles. Patients with obesity showed higher concentrations of naïve, transitional, and non-isotype switched memory B cells, and plasmablasts compared to normal weight patients and healthy controls., Conclusions: Obesity in hospitalized COVID-19 patients may correlate with elevated pro-inflammatory cytokines, anti-inflammatory IL-10, and increased B cell subset activation, highlighting the need for further studies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: CN received research funding and/or consultancy fees outside this study from Abbvie, Janssen, AstraZeneca, Takeda, Octapharma, CSL Behring, Beigene and Genmab. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sejdic, Hartling, Holler, Klingen Gjærde, Lindegaard, Dungu, Gnesin, Møller, Teglgaard, Niemann, Brooks, Jørgensen, Franck, Fischer, Marquart, Harboe and Ostrowski.)

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)., (©2024 American Association for Cancer Research.)

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., (© 2023. The Author(s).)

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.

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.

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., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

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)., (© 2022 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

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., Competing Interests: TK has participated in advisory boards for Gilead Sciences, GlaxoSmithKline/ViiV and MSD. TK has received fees for teaching from Takeda and CSL Behring, and a research grant from Gilead Sciences. MH has participated in advisory boards for AstraZeneca, Gilead Sciences, GlaxoSmithKline/ViiV, MSD, Roche, and Sobi. MH has received fees for teaching from Gilead Sciences and GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mørup, Nazaryan-Petersen, Gabrielaite, Reekie, Marquart, Hartling, Marvig, Katzenstein, Masmas, Lundgren, Murray, Helleberg and Borgwardt.)

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.

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., (© 2022 Duncan et al.)

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 (ρ BCP-ALL  = -.17, ρ T-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., (© 2022 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

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., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness., Objective: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level., Methods: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated., Results: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed., Conclusion: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency., (Copyright © 2021. Published by Elsevier Inc.)

Minimal residual disease (MRD) assessment plays a central role in risk stratification and treatment guidance in paediatric patients with acute lymphoblastic leukaemia (ALL). As such, MRD prior to haematopoietic stem cell transplantation (HSCT) is a major factor that is independently correlated with outcome. High burden of MRD is negatively correlated with post-transplant survival, as both the risk of leukaemia recurrence and non-relapse mortality increase with greater levels of MRD. Despite growing evidence supporting these findings, controversies still exist. In particular, it is still not clear whether multiparameter flow cytometry and real-time quantitative polymerase chain reaction, which is used to recognise immunoglobulin and T-cell receptor gene rearrangements, can be employed interchangeably. Moreover, the higher sensitivity in MRD quantification offered by next-generation sequencing techniques may further refine the ability to stratify transplant-associated risks. While MRD quantification from bone marrow prior to HSCT remains the state of the art, heavily pre-treated patients may benefit from additional staging, such as using 18 F-fluorodeoxyglucose positron emission tomography/computed tomography to detect focal residues of disease. Additionally, the timing of MRD detection (i.e., immediately before administration of the conditioning regimen or weeks before) is a matter of debate. Pre-transplant MRD negativity has previously been associated with superior outcomes; however, in the recent For Omitting Radiation Under Majority age (FORUM) study, pre-HSCT MRD positivity was associated with neither relapse risk nor survival. In this review, we discuss the level of MRD that may require pre-transplant therapy intensification, risking time delay and complications (as well as losing the window for HSCT if disease progression occurs), as opposed to an adapted post-transplant strategy to achieve long-term remission. Indeed, MRD monitoring may be a valuable tool to guide individualised treatment decisions, including tapering of immunosuppression, cellular therapies (such as donor lymphocyte infusions) or additional immunotherapy (such as bispecific T-cell engagers or chimeric antigen receptor T-cell therapy)., Competing Interests: JB has received personal fees, advisory board/steering committee honoraria, and nonfinancial support from Novartis; and advisory board honoraria from Pfizer, Kite, and Janssen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Merli, Ifversen, Truong, Marquart, Buechner, Wölfl and Bader.)

Infants with acute lymphoblastic leukaemia (ALL) have a high frequency of central nervous system (CNS) involvement. Flow cytometric analysis of cerebrospinal fluid (CSF) was recently demonstrated to be a sensitive method for detecting CNS involvement in childhood ALL. In the present study, CSF from 14 infants was collected at routine lumbar punctures and analysed by multicolour flow cytometry. At initial diagnosis, leukaemic blasts were detected in CSF by flow cytometry in 11 patients (78·6%) compared to seven patients (50%) by cytospin. Larger studies are needed to determine if CSF flow cytometry has prognostic value in infant ALL., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR 5y ) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR 5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10 -2 versus 5.2 × 10 -3 , p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR 5y  = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10 -4 associated with a CIR 5y  = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Truncation of asparaginase treatment due to asparaginase-related toxicities or silent inactivation (SI) is common and may increase relapse risk in acute lymphoblastic leukemia (ALL). We investigated relapse risk following suboptimal asparaginase exposure among 1401 children aged 1 to 17 years, diagnosed with ALL between July 2008 and February 2016, treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL2008 protocol (including extended asparaginase exposure [1000 IU/m2 intramuscularly weeks 5-33]). Patients were included with delayed entry at their last administered asparaginase treatment, or detection of SI, and followed until relapse, death, secondary malignancy, or end of follow-up (median, 5.71 years; interquartile range, 4.02-7.64). In a multiple Cox model comparing patients with (n = 358) and without (n = 1043) truncated asparaginase treatment due to clinical toxicity, the adjusted relapse-specific hazard ratio (HR; aHR) was 1.33 (95% confidence interval [CI], 0.86-2.06; P = .20). In a substudy including only patients with information on enzyme activity (n = 1115), the 7-year cumulative incidence of relapse for the 301 patients with truncation of asparaginase treatment or SI (157 hypersensitivity, 53 pancreatitis, 14 thrombosis, 31 other, 46 SI) was 11.1% (95% CI, 6.9-15.4) vs 6.7% (95% CI, 4.7-8.6) for the 814 remaining patients. The relapse-specific aHR was 1.69 (95% CI, 1.05-2.74, P=.03). The unadjusted bone marrow relapse-specific HR was 1.83 (95% CI, 1.07-3.14, P=.03) and 1.86 (95% CI, 0.90- 3.87, P=.095) for any central nervous system relapse. These results emphasize the importance of therapeutic drug monitoring and appropriate adjustment of asparaginase therapy when feasible. This trial was registered at www.clinicaltrials.gov as #NCT03987542., (© 2021 by The American Society of Hematology.)

Bone marrow minimal residual disease (MRD) is the strongest predictor of relapse in children with acute lymphoblastic leukemia (ALL). 6-mercaptopurine (6MP) in ALL therapy has wide inter-individual variation in disposition and is strongly influenced by polymorphisms in the thiopurine methyltransferase ( TPMT ) gene. In 952 patients treated according to the NOPHO ALL2008 protocol, we explored the association between thiopurine disposition, TPMT genotypes and MRD levels after consolidation therapy with 6MP, high-dose methotrexate (HD-MTX), asparaginase, and vincristine. The levels of the cytotoxic DNA-incorporated thioguanine were significantly higher on day 70-79 in G460A/A719G TPMT heterozygous ( TPMT HZ ) compared to TPMT wild type ( TPMT WT ) patients (mean: 230.7 vs. 149.7 fmol/µg DNA, p = 0.002). In contrast, TPMT genotype did not associate with the end of consolidation MRD levels irrespective of randomization of the patients to fixed dose (25 mg/m 2 /day) or 6MP escalation (up to 50 or 75 mg/m 2 /day) during consolidation therapy.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and-for a subset of donors-also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαβ, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αβ cells. For TCR γδ as well as for TCR αβ cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Minculescu, Sengelov, Marquart, Ryder, Fischer-Nielsen and Haastrup.)

Background: Severe immunopathology may drive the deleterious manifestations that are observed in the advanced stages of coronavirus disease 2019 (COVID-19) but are poorly understood., Objective: Our aim was to phenotype leukocyte subpopulations and the cytokine milieu in the lungs and blood of critically ill patients with COVID-19 acute respiratory distress syndrome (ARDS)., Methods: We consecutively included patients less than 72 hours after intubation following informed consent from their next of kin. Bronchoalveolar lavage fluid was evaluated by microscopy; bronchoalveolar lavage fluid and blood were assessed by 10-color flow cytometry and a multiplex cytokine panel., Results: Four mechanically ventilated patients (aged 40-75 years) with moderate-to-severe COVID-19 ARDS were included. Immature neutrophils dominated in both blood and lungs, whereas CD4 and CD8 T-cell lymphopenia was observed in the 2 compartments. However, regulatory T cells and T H 17 cells were found in higher fractions in the lung. Lung CD4 and CD8 T cells and macrophages expressed an even higher upregulation of activation markers than in blood. A wide range of cytokines were expressed at high levels both in the blood and in the lungs, most notably, IL-1RA, IL-6, IL-8, IP-10, and monocyte chemoattactant protein-1, consistent with hyperinflammation., Conclusion: COVID-19 ARDS exhibits a distinct immunologic profile in the lungs, with a depleted and exhausted CD4 and CD8 T-cell population that resides within a heavily hyperinflammatory milieu., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chimerism analysis following hematopoietic stem cell transplantation (HSCT) for leukemia is routinely applied in parallel with quantification of minimal residual disease (MRD) to identify imminent relapse. In the past decades, new methods with a lower limit of detection compared to standard methods have been developed, so-called microchimerism analysis. Microchimerism analysis is fast, simple, applicable across pre-HSCT disease-type and can be applied on peripheral blood allowing frequent testing during follow-up. Monitoring of microchimerism in blood could replace repeated bone marrow analysis for MRD and allow earlier detection of imminent relapse or graft failure. Clinical studies in single center cohorts have shown conflicting but promising results. There is currently no consensus on the interpretation of microchimerism analysis and heterogeneity of studies remains a major obstacle for inter-study comparisons and meta-analysis in this field. We have conducted a systematic review of studies investigating associations between microchimerism and relapse of leukemia post-HSCT. We summarize current evidence and provide suggestions for future research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mature immunocompetent cells from the stem cell graft as well as early robust immune reconstitution are essential for the graft-vs. -tumor (GVT) effect to eliminate residual malignant cells after allogeneic hematopoietic stem cell transplantation (HSCT). In this prospective study we characterized graft composition of T- and NK cell subsets in 88 recipients of peripheral blood stem cell grafts with multicolor flowcytometry. Our primary aim was to analyze the impact of graft composition on immune reconstitution and clinical outcomes after transplantation. Patients transplanted with graft NK cell doses above the median value of 27 × 10 6 /kg had significantly increased relapse-free-survival compared to patients transplanted with lower doses, HR 2.12 (95% CI 1.01-4.45, p = 0.04) Peripheral blood concentrations of NK cells obtained from donors before G-CSF mobilization were significantly correlated to graft NK cell doses (Spearman's ρ 0.53, p = 0.03). The dose of transplanted NK cells/kg correlated significantly with NK cell concentrations in patients early after transplantation (Spearman's ρ 0.26, p = 0.02, and ρ = 0.35, p = 0.001 for days 28 and 56, respectively). Early immune reconstitution above median values of NK cells was significantly associated with improved relapse-free survival (HR 2.84 [95% CI 1.29-6.28], p = 0.01, and HR 4.19 [95% CI 1.68-10.4], p = 0.002, for day 28 and 56, respectively). Early concentrations above the median value of the mature effector CD56dim NK cell subset were significantly associated with decreased relapse incidences at 1 year, 7% (95% CI 1.8-17) vs. 28% (95% CI 15-42), p = 0.04, and 7% (95% CI 1.8-18) vs. 26% (95% CI 14-40) %, p = 0.03, for days 28 and 56, respectively. The results suggest a protective effect of high doses of NK cells in grafts and during early immune reconstitution and support the perception of NK cells as innate effector cells with anti-tumor effects in the setting of allogeneic stem cell transplantation., (Copyright © 2020 Minculescu, Fischer-Nielsen, Haastrup, Ryder, Andersen, Schjoedt, Friis, Kornblit, Petersen, Sengelov and Marquart.)

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in childhood. Despite a significantly improved prognosis over the last decade with a 5-years survival rate of ~90%, treatment-related morbidity remains substantial and relapse occurs in 10-15% of patients (1). The most common site of relapse is the bone marrow, but early colonization and subsequent reoccurrence of the disease in the central nervous system (CNS) also occurs. Integrins are a family of cell surface molecules with a longstanding history in cancer cell adherence, migration and metastasis. In chronic lymphoblastic leukemia (CLL), the VLA-4 integrin has been acknowledged as a prognostic marker and mounting evidence indicates that this and other integrins may also play a role in acute leukemia, including ALL. Importantly, integrins engage in anti-apoptotic signaling when binding extracellular molecules that are enriched in the bone marrow and CNS microenvironments. Here, we review the current evidence for a role of integrins in the adherence of ALL cells within the bone marrow and their colonization of the CNS, with particular emphasis on mechanisms adding to cancer cell survival and chemoresistance., (Copyright © 2020 Scharff, Modvig, Marquart and Christensen.)

Risk stratification is essential for the delivery of optimal treatment in childhood acute lymphoblastic leukemia. However, current risk stratification algorithms dichotomize variables and apply risk factors independently, which may incorrectly assume identical associations across biologically heterogeneous subsets and reduce statistical power. Accordingly, we developed and validated a prognostic index (PIUKALL) that integrates multiple risk factors and uses continuous data. We created discovery (n = 2405) and validation (n = 2313) cohorts using data from 4 recent trials (UKALL2003, COALL-03, DCOG-ALL10, and NOPHO-ALL2008). Using the discovery cohort, multivariate Cox regression modeling defined a minimal model including white cell count at diagnosis, pretreatment cytogenetics, and end-of-induction minimal residual disease. Using this model, we defined PIUKALL as a continuous variable that assigns personalized risk scores. PIUKALL correlated with risk of relapse and was validated in an independent cohort. Using PIUKALL to risk stratify patients improved the concordance index for all end points compared with traditional algorithms. We used PIUKALL to define 4 clinically relevant risk groups that had differential relapse rates at 5 years and were similar between the 2 cohorts (discovery: low, 3% [95% confidence interval (CI), 2%-4%]; standard, 8% [95% CI, 6%-10%]; intermediate, 17% [95% CI, 14%-21%]; and high, 48% [95% CI, 36%-60%; validation: low, 4% [95% CI, 3%-6%]; standard, 9% [95% CI, 6%-12%]; intermediate, 17% [95% CI, 14%-21%]; and high, 35% [95% CI, 24%-48%]). Analysis of the area under the curve confirmed the PIUKALL groups were significantly better at predicting outcome than algorithms employed in each trial. PIUKALL provides an accurate method for predicting outcome and more flexible method for defining risk groups in future studies., (© 2020 by The American Society of Hematology.)

Central nervous system (CNS) involvement by cytospin is associated with increased risk of relapse in childhood acute lymphoblastic leukemia. We investigated if flow cytometric analysis of cerebrospinal fluid (CSF) at diagnosis improves the prediction of relapse. This prospective cohort study included patients (1.0-17.9 years) treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol. CSF flow cytometry samples were obtained at 17 centers, preserved with Transfix®, and analyzed at a central laboratory. One-hundred and seventy-one (25.4%) of 673 patients were positive by flow cytometry (CNS flow+ ). The 4-year cumulative incidence of relapse was higher for patients with cytospin positivity (CNS cyto+ ) (17.1% vs. 7.5%), CNS flow+ (16.5% vs. 5.6%), and cytospin and/or flow positivity (CNS comb+ ) (16.7% vs. 5.1%). In Cox regression analysis stratified by immunophenotype and minimal residual disease day 29 and adjusted by sex, predictors of relapse were age (hazard ratio [HR] 1.1, 95% CI 1.1-1.2, P < 0.001), white blood cell count at diagnosis (HR 1.4, 95% CI 1.1-1.6, P < 0.001), and CNS comb+ (HR 2.2, 95% CI 1.0-4.7, P = 0.042). Flow cytometric analysis of CSF improves detection of CNS leukemia, distinguishes patients with high and low risk of relapse, and may improve future risk stratification and CNS-directed therapy.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1-45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69-0.81), being 0.82 (0.74-0.88) for patients 1.0-9.9 years, 0.76 (0.66-0.86) for those 10.0-17.9 years, and 0.65 (0.55-0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1-9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1-45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

Analysis of chimerism in blood post-HCT using STR-PCR is routinely applied in parallel with quantification of MRD to predict relapse of leukemia. RQ-PCR chimerism is 10- to 100-fold more sensitive, but clinical studies in children are sparse. We analyzed IMC in blood samples following transplantation for acute lymphoblastic or myeloid leukemia in 56 children. IMC was defined as a minimum increase of (a) 0.1% or (b) 0.05% recipient DNA between two samples. The risk of relapse was higher in children with IMC of both 0.1% and 0.05% compared to children without IMC (HR 12.8 [95% CI: 3.9-41.4; P < .0001] and 7.6 [95% CI: 2.2-26.9; P < .01], respectively). The first IMC was detected at a median of 208 days prior to relapse. The 5-year cumulative incidence of relapse for children with a single IMC was 45.5% (CI 12.3-74.4) and 41.0% (14.2-66.6) for IMC above 0.1% and 0.05%, respectively. However, in 47 and 38 children never attaining IMC > 0.1% and >0.05%, 10 and 8 children relapsed, respectively. In a landmark analysis, no association was found between IMC prior to 90 days post-HCT and subsequent relapse by either classification of IMC and AUC for RQ-PCR chimerism was 54.2% (95 CI 27.7- 84.8). Although limited by a retrospective design, these results indicate that monitoring of RQ-PCR chimerism in peripheral blood may have a role in early detection of relapse in acute childhood leukemia., (© 2019 Wiley Periodicals, Inc.)

T-cell receptor (TCR) γδ cells are perceived as innate-like effector cells with the possibility of mediating graft-vs. -tumor (GVT) without causing graft-vs.-host disease (GVHD) in the setting of hematopoietic allogeneic stem cell transplantation (HSCT). We conducted a prospective study to assess the clinical impact of TCR γδ cell immune reconstitution on overall survival, relapse-free-survival, relapse and GVHD. The impact of CD3, CD4, and CD8 T cells together with NK cells including subtypes were analyzed in parallel. A total of 108 patients with hematological malignancies transplanted with HLA-matched, T cell replete stem cell grafts were included for analyses of absolute concentrations of CD3, CD4, and CD8 positive T cells and NK cells together with a multi-color flow cytometry panel with staining for TCRαβ, TCRγδ, Vδ1, Vδ2, CD3, CD4, CD8, HLA-DR, CD196, CD45RO, CD45RA, CD16, CD56, CD337, and CD314 at 28, 56, 91, 180, and 365 days after transplantation. Immune reconstitution data including subsets and differentiation markers of T and NK cells during the first year after transplantation was provided. Patients with TCR γδ cell concentrations above the median value of 21 (0-416) × 10 6 cells/L 56 days after transplantation had significantly improved overall survival ( p = 0.001) and relapse-free survival ( p = 0.007) compared to patients with concentrations below this value. When day 56 cell subset concentrations were included as continuous variables, TCR γδ cells were the only T cell subsets with a significant impact on OS and RFS; the impact of TCR γδ cells remained statistically significant in multivariate analyses adjusted for pre-transplant risk factors. The risk of death from relapse was significantly decreased in patients with high concentrations of TCR γδ cells 56 days after transplantation ( p = 0.003). Also, the risk of acute GVHD was significantly lower in patients with day 28 TCR γδ cell concentrations above the median of 18 × 10 6 cells/L compared to patients with low concentrations ( p = 0.01). These results suggest a protective role of TCR γδ cells in relapse and GVHD and encourage further research in developing adaptive TCR γδ cell therapy for improving outcomes after HSCT.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10 -3 ) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10 -3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10 -3 compared with MRD < 10 -3 . Patients stratified to intermediate-risk therapy on day 29 with MRD 10 -4 -<10 -3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10 -4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10 -4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.

The population-based Nordic/Baltic acute lymphoblastic leukaemia (ALL) Nordic Society for Paediatric Haematology and Oncology (NOPHO) ALL2008 protocol combined minimal residual disease (MRD)-driven treatment stratification with very intense first line chemotherapy for patients with high risk ALL. Patients with MRD ≥5% at end of induction or ≥10 -3 at end of consolidation or following two high risk blocks were eligible for haematopoietic cell transplantation (HCT) in first remission. After at least three high risk blocks a total of 71 children received HCT, of which 46 had MRD ≥5% at end of induction. Ten patients stratified to HCT were not transplanted; 12 received HCT without protocol indication. Among 69 patients with evaluable pre-HCT MRD results, 22 were MRD-positive, one with MRD ≥10 -3 . After a median follow-up of 5·5 years, the cumulative incidence of relapse was 23·5% (95% confidence interval [CI]: 10·5-47·7) for MRD-positive versus 5·1% (95% CI: 1·3-19·2), P = 0·02) for MRD-negative patients. MRD was the only variable significantly associated with relapse (hazard ratio 9·1, 95% CI: 1·6-51·0, P = 0·012). Non-relapse mortality did not differ between the two groups, resulting in disease-free survival of 85·6% (95% CI: 75·4-97·2) and 67·4% (95% CI: 50·2-90·5), respectively. In conclusion, NOPHO block treatment efficiently reduced residual leukaemia which, combined with modern transplant procedures, provided high survival rates, also among pre-HCT MRD-positive patients., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

With the discovery that the highly effective anti-CD20 antibody therapies developed to deplete CD20+ B cells deplete CD20+ T cells equally well, a great interest in the biological properties of CD20+ T cells has emerged. In this study we show that CD20+ T cells have a proinflammatory Th1/Tc1 phenotype with a high proliferative capacity to CNS antigens. We also found that the percentage of CD20+ T cells is increased in the blood of patients with multiple sclerosis and are enriched in the CSF of the patients. Furthermore, we found a positive correlation between CD20+ T cells in the CSF and multiple sclerosis disease severity and see that regulation of CD20+ T cells likely contributes to the positive treatment effect of the multiple sclerosis treatment alemtuzumab. These data represent an important contribution to the understanding of the nature of CD20+ T cells and strongly suggests a role of CD20+ T cells in the pathogenesis of multiple sclerosis.

The contribution of B cells to the pathogenesis of relapsing-remitting multiple sclerosis (RRMS) is currently of great interest due to the positive outcomes of treatment with B cell-depleting monoclonal antibodies. In this exploratory study we examined the phenotype and cytokine response of B cells from untreated patients with RRMS and healthy controls. The CNS migration potential of the individual blood B cell subpopulations was evaluated according to the expression of CD49d, ALCAM, CXCR3, and CCR7, and cerebrospinal fluid (CSF) samples were analyzed to establish the phenotype of migrated B cells. The frequency of the individual blood B cell subsets expressing CD5, CD43, CD69, CD80, CD83, DC-SIGN and CD138 was similar in patients with RRMS and healthy controls. However, a higher percentage of CD27 - IgD - IgM + memory B cells were found in the blood of patients with RRMS, a population also identified in the CSF samples. We also observed an increased percentage of B cells producing LTα and a higher level of TGFβ1 in patients with RRMS. Altogether, we found that patients with RRMS have an increased frequency of blood CD27 - IgD - IgM + memory B cells that are recruited to the CSF together with other memory B cell populations. Furthermore, we report an increased B cell production of LTα and TGFβ1 in patients with RRMS., (Copyright © 2018. Published by Elsevier B.V.)

Acute infectious spondylodiscitis (AIS) is a serious infection of the spine with rising incidence and a mortality of 3-6%. The role of the immune system in AIS is largely unknown. We performed extensive B and T-lymphocyte phenotyping in patients with AIS at diagnosis and after treatment cessation. In this prospective multicentre study, flow cytometric analysis of T and B-lymphocyte subsets was performed in 35 patients at diagnosis and 3 months after treatment cessation. We additionally analysed levels of immunoglobulins and IgG subclasses, serum level and genetic variants of mannose-binding lectin, and somatic hypermutation. A total of 22 (61%) patients had B-lymphocytes below reference limit at baseline, persisting in 7 (30%) patients at follow-up. We found a lower proportion of CD19 + CD27 + IgD+ marginal zone B-lymphocytes and a higher proportion of γδ+ T-lymphocyte receptors compared with controls at both time points. Immunoglobulin levels were elevated at baseline compared to follow-up, and not associated with absolute B-lymphocyte count. In conclusion, a large proportion of AIS patients presented with profound B-lymphocyte deficiency, only partly reversible at follow-up. Identification of immune dysfunction related to AIS may allow for future targeted therapeutic interventions to restore host immunity.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19 + leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19 + ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19 - and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial., (© 2018 by The American Society of Hematology.)

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1-45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1-9 years (A), 266 were 10-17 years (B) and 221 were 18-45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1-3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS 5y ) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.

Hypogammaglobulinemia (HGG) is well-characterized as a common phenomenon after kidney transplantation. However, no reports of pre-existing HGG from kidney transplantation seem to be available. We have reviewed three patients who developed HGG prior to kidney transplantation, and all three were treated successfully with immunoglobulin replacement therapy before and after kidney transplantation. The kidney grafts were functioning at follow-up 1.5-8 years (mean: 3.6 years) after transplantation, and there were no diagnosed episodes of clinical rejections and no severe infection complications post-transplantation., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

Early immune reconstitution plays a critical role in clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Natural killer (NK) cells are the first lymphocytes to recover after transplantation and are considered powerful effector cells in HSCT. We aimed to evaluate the clinical impact of early NK cell recovery in T cell-replete transplant recipients. Immune reconstitution was studied in 298 adult patients undergoing HSCT for acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome from 2005 to 2013. In multivariate analysis NK cell numbers on day 30 (NK30) > 150 cells/µL were independently associated with superior overall survival (hazard ratio, .79; 95% confidence interval, .66 to .95; P = .01). Cumulative incidence analyses showed that patients with NK30 > 150 cells/µL had significantly less transplant-related mortality (TRM), P = .01. Patients with NK30 > 150 cells/µL experienced significantly lower numbers of life-threatening bacterial infections as well as viral infections, including cytomegalovirus. No association was observed in relation to relapse. These results suggest an independent protective effect of high early NK cell reconstitution on TRM that translates into improved overall survival after T cell-replete HSCT., (Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

A small group of children with acute lymphoblastic leukemia (ALL) have a preleukemic phase of pancytopenia followed by a period of spontaneous remission before the diagnosis (pre-ALL). A 6-year-old girl presented with pancytopenia, fever, and myelodysplasia. Following transient remission pre-B ALL was diagnosed 14 months later. Clonal B-lineage blasts at the period of pancytopenia were identified retrospectively. The interval between pre-ALL and ALL-diagnosis was longer than previously reported. The infection was clinically severe and might have induced a significant endogenous corticosteroids production resulting in the long-lasting remission. The case supports the adrenal and the Coley's toxin hypothesis in leukemogenesis.