Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell rece... more 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 discriminat...
Introduction:The early treatment response, measured as minimal residual disease (MRD), is the mos... more Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity. Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients). Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts. Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations. Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (>20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments. Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells. The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. Disclosures No relevant conflicts of interest to declare.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with h... more 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 gd 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 gd 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,
Journal of Pediatric Hematology Oncology, Jun 1, 2009
Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered t... more Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10 À 3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10 À 3 , which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10 À 4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.
Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through rec... more Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through recognition and phagocytosis of pathogens, and mediation of acute inflammation through secretion of pro-inflammatory cytokines. Furthermore, monocytes constitute an important link between innate and adaptive immunity through antigen presentation to lymphocytes. Patients with chronic lymphocytic leukemia (CLL) have an increased risk of infection, despite often displaying normal neutrophil and monocyte counts. CLL-cells depend on interactions with the immune microenvironment (IME) for proliferation and survival, while inducing changes in surrounding immune cells. However, changes in myeloid cell function in patients with CLL remain sparsely documented. Since the btk-inhibitor ibrutinib impairs B-cell receptor signaling and disrupts CLL-IME interactions (Niemann et al, CCR, 2016), we investigated the impact of ibrutinib and venetoclax on monocyte- and neutrophil phenotype and function in CLL patients. Methods: Nine patients treated with ibrutinib 420 mg daily for 8 weeks followed by addition of venetoclax with an initial 5-week ramp up period were included. Blood samples were taken at baseline, after 8 weeks ibrutinib monotherapy, and after another 8 weeks of ibrutinib and venetoclax combination therapy. Immune phenotype was assessed in whole blood by an 8-tube, 10 color flow cytometry panel with custom designed lyophilized antibodies (Duraclone). Immune function was characterized using TruCulture, a whole blood-ligand stimulation assay applying the toll like receptor (TLR) ligands heat killed candida albicans (TLR 2,4,6), lipopolysaccharide (LPS; TLR4), resiquimod (single-stranded RNA-virus analog, TLR7,8), and Poly:IC (double-stranded RNA-virus analog, TLR3), after which the cytokine response was measured. Informed consent from patients and approval from the ethics committee was obtained. Results: Monocyte and neutrophil counts, as wells as distribution of mature and immature neutrophils, were within normal range at baseline (n=9) and remained unchanged throughout treatment. At baseline, expression of HLA-DR on monocytes was suppressed, but increased significantly upon combination treatment with ibrutinib and venetoclax (p=0.04). HLA-DR expression on neutrophils was high at baseline, remained unchanged upon ibrutinib treatment (n=8), but declined after addition of venetoclax (n=7) (p<0.01). LPS-stimulated TNF-α and IL-6 production was suppressed at baseline, IL-6 levels increased significantly upon ibrutinib monotherapy, and levels of both TNF-α and IL-6 almost normalized upon addition of venetoclax to ibrutinib (p=0.02 for TNF-α, p=0.009 for IL-6). Conclusion: Here we show for the first time that myeloid leukocytes in CLL patients exhibit an altered HLA-DR expression at baseline, which is paralleled by an impaired cytokine response to LPS stimulation. Following treatment with ibrutinib and venetoclax, HLA-DR expression increased on monocytes and decreased on neutrophils, both representing normalization. In parallel, LPS-induced TNF-α and IL-6 production improved, likely reflecting an improved monocytic function, as monocytes respond strongly to whole-blood LPS-stimulation. Increased monocyte HLA-DR expression has previously been linked to improved cytokine response (Belge et al, J Immunol, 2002). The contribution of neutrophils to the improved cytokine response is unclear, since the function of HLA-DR expressing neutrophils in CLL is largely unknown. We propose a model where ibrutinib and venetoclax eradicate CLL-cells, disrupt tumor-IME interactions, and maybe directly impact microenvironmental cell signaling, thereby restoring neutrophil and monocyte function and improving immune function. Further investigation of myeloid function in CLL at baseline and upon different treatment regimens is warranted to guide personalized treatment based on modulation of infection-risk. Disclosures Svanberg: Novo Nordisk Foundation: Research Funding; Abbvie: Other: Travel grant. Kater:Genentech: Research Funding; Roche: Other: Travel funding, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Niemann:Acerta: Consultancy, Research Funding; Roche: Other: Travel grant; Astra Zeneca: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding; CSL Behring: Consultancy; Janssen: Consultancy, Other: Travel grant, Research Funding; Gilead: Other: Travel grant; Sunesis: Consultancy; Abbvie: Consultancy, Other: Travel grant, Research Funding.
A case of persistent B-cell lymphocytopenia in a 40-year-old woman with lymphoidepithelial thymom... more A case of persistent B-cell lymphocytopenia in a 40-year-old woman with lymphoidepithelial thymoma treated with chemotherapy, surgery and irradiation is described. The possible diagnosis of Good's syndrome (hypogammaglobulinaemia and thymoma) is discussed.
comparison had a lower P-value and had a higher degree of deregulation than the differentially ex... more comparison had a lower P-value and had a higher degree of deregulation than the differentially expressed genes from the comparison TET2 mutated versus wild type (Figure 1 b; Supplementary Figure 1 and Supplementary Tables 2 and 3). These results indicate that 5hmC levels are most likely a more relevant measurement to define biologically distinct secondary leukemia subtypes than the TET2 (or IDH1/2) mutational status. The fact that in some patient samples with low 5hmC levels neither TET2 nor IDH1/2 mutations could be identified suggests that additional genes might be directly or indirectly involved in the regulation of 5hmC levels. To further elucidate the regulation of 5hmC levels and their role in leukemogenesis, larger groups of sAML as well as de novo AML patients need to be studied.
Acute infectious spondylodiscitis (AIS) is a serious infection of the spine with rising incidence... more 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 mannosebinding 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.
Discordances between minimal residual disease estimates obtained by different methods are a probl... more Discordances between minimal residual disease estimates obtained by different methods are a problem in childhood acute lymphoblastic leukemia. We aimed to optimize methods allowing the biological exploration of such discrepancies, i.e. the combination of flow-sorting of small immunophenotypically defined cell populations with subsequent analyses of leukemiaassociated cytogenetic and molecular marker. The approaches described here optimize the use of the same tube of unfixed, antibody-stained BM cells for flow-sorting of small cell populations and subsequent exploratory FISH and PCR-based analyses.
Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute l... more Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.
response after induction is a prognostic factor for disease outcome in childhood acute myeloid le... more response after induction is a prognostic factor for disease outcome in childhood acute myeloid leukaemia (AML). Residual disease (RD) detection by multiparameter flow cytometry (MFC) was performed at day 15 and before consolidation therapy in 101 patients enrolled in the Nordic Society of Paediatric Haemato-Oncology AML 2004 study. A multicentre laboratory approach to RD analysis was used. Event-free survival (EFS) and overall survival (OS) was significantly different in patients with and without RD at both time points, using a 0Á1% RD cutoff level. RD-negative and-positive patients after first induction showed a 5-year EFS of 65 AE 7% and 22 AE 7%, respectively (P < 0Á001) and an OS of 77 AE 6% (P = 0Á025) and 51 AE 8%. RD-negative and-positive patients at start of consolidation therapy had a 5-year EFS of 57 AE 7% and 11 AE 7%, respectively (P < 0Á001) and an OS of 78 AE 6% and 28 AE 11%) (P < 0Á001). In multivariate analysis only RD was significantly correlated with survival. RD before consolidation therapy was the strongest independent prognostic factor for EFS [hazard ratio (HR):5Á0; 95% confidence interval (CI):1Á9-13Á3] and OS (HR:7Á0; 95%CI:2Á0-24Á5). In conclusion, RD before consolidation therapy identifies patients at high risk of relapse in need of intensified treatment. In addition, RD detection can be performed in a multicentre setting and can be implemented in future trials.
Mature immunocompetent cells from the stem cell graft as well as early robust immune reconstituti... more 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.
Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by t... more 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 phenotyp...
Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes se... more Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes severe ataxia, is a rare complication of severe immunodeficiency and caused by a variant of JCV with a VP1-deletion. JCV-GCN has not yet been reported in patients with autoimmune lymphoproliferative syndrome (ALPS-FAS). We report a 34-year old woman with autoimmune lymphoproliferative syndrome (ALPS-FAS) and common variable immunodeficiency (CVID) who developed JCV-GCN which progressed to fatal progressive multifocal leukoencephalopathy (PML).Methods: Extensive longitudinal virologic, genetic and immunologic characterisation by JCV CSF quantification and sequencing, peripheral blood flowcytometry and exome sequencing. Results: A diagnosis of JCV-GCN was established by findings of severe cerebellar vermis atrophy on MRI and a JCV variant with a VP1-deletion (JCV-VP1) in CSF. During a period of 18 months, at which treatment with high dose immunoglobulin, cidofovir, mirtazapine and high dose ...
Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell rece... more 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 discriminat...
Introduction:The early treatment response, measured as minimal residual disease (MRD), is the mos... more Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity. Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients). Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts. Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations. Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments. Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells. The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. Disclosures No relevant conflicts of interest to declare.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with h... more 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 gd 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 gd 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,
Journal of Pediatric Hematology Oncology, Jun 1, 2009
Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered t... more Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10 À 3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10 À 3 , which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10 À 4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.
Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through rec... more Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through recognition and phagocytosis of pathogens, and mediation of acute inflammation through secretion of pro-inflammatory cytokines. Furthermore, monocytes constitute an important link between innate and adaptive immunity through antigen presentation to lymphocytes. Patients with chronic lymphocytic leukemia (CLL) have an increased risk of infection, despite often displaying normal neutrophil and monocyte counts. CLL-cells depend on interactions with the immune microenvironment (IME) for proliferation and survival, while inducing changes in surrounding immune cells. However, changes in myeloid cell function in patients with CLL remain sparsely documented. Since the btk-inhibitor ibrutinib impairs B-cell receptor signaling and disrupts CLL-IME interactions (Niemann et al, CCR, 2016), we investigated the impact of ibrutinib and venetoclax on monocyte- and neutrophil phenotype and function in CLL patients. Methods: Nine patients treated with ibrutinib 420 mg daily for 8 weeks followed by addition of venetoclax with an initial 5-week ramp up period were included. Blood samples were taken at baseline, after 8 weeks ibrutinib monotherapy, and after another 8 weeks of ibrutinib and venetoclax combination therapy. Immune phenotype was assessed in whole blood by an 8-tube, 10 color flow cytometry panel with custom designed lyophilized antibodies (Duraclone). Immune function was characterized using TruCulture, a whole blood-ligand stimulation assay applying the toll like receptor (TLR) ligands heat killed candida albicans (TLR 2,4,6), lipopolysaccharide (LPS; TLR4), resiquimod (single-stranded RNA-virus analog, TLR7,8), and Poly:IC (double-stranded RNA-virus analog, TLR3), after which the cytokine response was measured. Informed consent from patients and approval from the ethics committee was obtained. Results: Monocyte and neutrophil counts, as wells as distribution of mature and immature neutrophils, were within normal range at baseline (n=9) and remained unchanged throughout treatment. At baseline, expression of HLA-DR on monocytes was suppressed, but increased significantly upon combination treatment with ibrutinib and venetoclax (p=0.04). HLA-DR expression on neutrophils was high at baseline, remained unchanged upon ibrutinib treatment (n=8), but declined after addition of venetoclax (n=7) (p&lt;0.01). LPS-stimulated TNF-α and IL-6 production was suppressed at baseline, IL-6 levels increased significantly upon ibrutinib monotherapy, and levels of both TNF-α and IL-6 almost normalized upon addition of venetoclax to ibrutinib (p=0.02 for TNF-α, p=0.009 for IL-6). Conclusion: Here we show for the first time that myeloid leukocytes in CLL patients exhibit an altered HLA-DR expression at baseline, which is paralleled by an impaired cytokine response to LPS stimulation. Following treatment with ibrutinib and venetoclax, HLA-DR expression increased on monocytes and decreased on neutrophils, both representing normalization. In parallel, LPS-induced TNF-α and IL-6 production improved, likely reflecting an improved monocytic function, as monocytes respond strongly to whole-blood LPS-stimulation. Increased monocyte HLA-DR expression has previously been linked to improved cytokine response (Belge et al, J Immunol, 2002). The contribution of neutrophils to the improved cytokine response is unclear, since the function of HLA-DR expressing neutrophils in CLL is largely unknown. We propose a model where ibrutinib and venetoclax eradicate CLL-cells, disrupt tumor-IME interactions, and maybe directly impact microenvironmental cell signaling, thereby restoring neutrophil and monocyte function and improving immune function. Further investigation of myeloid function in CLL at baseline and upon different treatment regimens is warranted to guide personalized treatment based on modulation of infection-risk. Disclosures Svanberg: Novo Nordisk Foundation: Research Funding; Abbvie: Other: Travel grant. Kater:Genentech: Research Funding; Roche: Other: Travel funding, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Niemann:Acerta: Consultancy, Research Funding; Roche: Other: Travel grant; Astra Zeneca: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding; CSL Behring: Consultancy; Janssen: Consultancy, Other: Travel grant, Research Funding; Gilead: Other: Travel grant; Sunesis: Consultancy; Abbvie: Consultancy, Other: Travel grant, Research Funding.
A case of persistent B-cell lymphocytopenia in a 40-year-old woman with lymphoidepithelial thymom... more A case of persistent B-cell lymphocytopenia in a 40-year-old woman with lymphoidepithelial thymoma treated with chemotherapy, surgery and irradiation is described. The possible diagnosis of Good's syndrome (hypogammaglobulinaemia and thymoma) is discussed.
comparison had a lower P-value and had a higher degree of deregulation than the differentially ex... more comparison had a lower P-value and had a higher degree of deregulation than the differentially expressed genes from the comparison TET2 mutated versus wild type (Figure 1 b; Supplementary Figure 1 and Supplementary Tables 2 and 3). These results indicate that 5hmC levels are most likely a more relevant measurement to define biologically distinct secondary leukemia subtypes than the TET2 (or IDH1/2) mutational status. The fact that in some patient samples with low 5hmC levels neither TET2 nor IDH1/2 mutations could be identified suggests that additional genes might be directly or indirectly involved in the regulation of 5hmC levels. To further elucidate the regulation of 5hmC levels and their role in leukemogenesis, larger groups of sAML as well as de novo AML patients need to be studied.
Acute infectious spondylodiscitis (AIS) is a serious infection of the spine with rising incidence... more 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 mannosebinding 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.
Discordances between minimal residual disease estimates obtained by different methods are a probl... more Discordances between minimal residual disease estimates obtained by different methods are a problem in childhood acute lymphoblastic leukemia. We aimed to optimize methods allowing the biological exploration of such discrepancies, i.e. the combination of flow-sorting of small immunophenotypically defined cell populations with subsequent analyses of leukemiaassociated cytogenetic and molecular marker. The approaches described here optimize the use of the same tube of unfixed, antibody-stained BM cells for flow-sorting of small cell populations and subsequent exploratory FISH and PCR-based analyses.
Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute l... more Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.
response after induction is a prognostic factor for disease outcome in childhood acute myeloid le... more response after induction is a prognostic factor for disease outcome in childhood acute myeloid leukaemia (AML). Residual disease (RD) detection by multiparameter flow cytometry (MFC) was performed at day 15 and before consolidation therapy in 101 patients enrolled in the Nordic Society of Paediatric Haemato-Oncology AML 2004 study. A multicentre laboratory approach to RD analysis was used. Event-free survival (EFS) and overall survival (OS) was significantly different in patients with and without RD at both time points, using a 0Á1% RD cutoff level. RD-negative and-positive patients after first induction showed a 5-year EFS of 65 AE 7% and 22 AE 7%, respectively (P < 0Á001) and an OS of 77 AE 6% (P = 0Á025) and 51 AE 8%. RD-negative and-positive patients at start of consolidation therapy had a 5-year EFS of 57 AE 7% and 11 AE 7%, respectively (P < 0Á001) and an OS of 78 AE 6% and 28 AE 11%) (P < 0Á001). In multivariate analysis only RD was significantly correlated with survival. RD before consolidation therapy was the strongest independent prognostic factor for EFS [hazard ratio (HR):5Á0; 95% confidence interval (CI):1Á9-13Á3] and OS (HR:7Á0; 95%CI:2Á0-24Á5). In conclusion, RD before consolidation therapy identifies patients at high risk of relapse in need of intensified treatment. In addition, RD detection can be performed in a multicentre setting and can be implemented in future trials.
Mature immunocompetent cells from the stem cell graft as well as early robust immune reconstituti... more 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.
Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by t... more 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 phenotyp...
Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes se... more Background: JC-virus (JCV) associated granular cerebellar neuronopathy (JCV-GCN), which causes severe ataxia, is a rare complication of severe immunodeficiency and caused by a variant of JCV with a VP1-deletion. JCV-GCN has not yet been reported in patients with autoimmune lymphoproliferative syndrome (ALPS-FAS). We report a 34-year old woman with autoimmune lymphoproliferative syndrome (ALPS-FAS) and common variable immunodeficiency (CVID) who developed JCV-GCN which progressed to fatal progressive multifocal leukoencephalopathy (PML).Methods: Extensive longitudinal virologic, genetic and immunologic characterisation by JCV CSF quantification and sequencing, peripheral blood flowcytometry and exome sequencing. Results: A diagnosis of JCV-GCN was established by findings of severe cerebellar vermis atrophy on MRI and a JCV variant with a VP1-deletion (JCV-VP1) in CSF. During a period of 18 months, at which treatment with high dose immunoglobulin, cidofovir, mirtazapine and high dose ...
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Papers by Hanne Marquart