Ales Janda

Deficiency of Innate and Acquired Immunity Caused by an IKBKB Mutation

BACKGROUND Severe combined immunodeficiency (SCID) comprises a heterogeneous group of heritable deficiencies of humoral and cell-mediated immunity. Many patients with SCID have lymphocyte-activation defects that remain uncharacterized. METHODS We performed genetic studies in four patients, from four families of Northern Cree ancestry, who had clinical characteristics of SCID, including early onset of severe viral, bacterial, and fungal infections despite normal B-cell and T-cell counts. Genomewide homozygosity mapping was used to identify a candidate region, which was found on chromosome 8; all genes within this interval were sequenced. Immune-cell populations, signal transduction on activation, and effector functions were studied. RESULTS The patients had hypogammaglobulinemia or agammaglobulinemia, and their peripheral-blood B cells and T cells were almost exclusively of naive phenotype. Regulatory T cells and γδ T cells were absent. All patients carried a homozygous duplication--c.1292dupG in exon 13 of IKBKB, which encodes IκB kinase 2 (IKK2, also known as IKKβ)--leading to loss of expression of IKK2, a component of the IKK-nuclear factor κB (NF-κB) pathway. Immune cells from the patients had impaired responses to stimulation through T-cell receptors, B-cell receptors, toll-like receptors, inflammatory cytokine receptors, and mitogens. CONCLUSIONS A form of human SCID is characterized by normal lymphocyte development despite a loss of IKK2 function. IKK2 deficiency results in an impaired response to activation stimuli in a variety of immune cells, leading to clinically relevant impairment of adaptive and innate immunity. Although Ikk2 deficiency is lethal in mouse embryos, our observations suggest a more restricted, unique role of IKK2-NF-κB signaling in humans. (Funded by the German Federal Ministry of Education and Research and others.).

Clinical and immunological overlap between autoimmune lymphoproliferative syndrome and common variable immunodeficiency

Autoimmune lymphoproliferative syndrome (ALPS) is mainly caused by defects in the CD95 pathway. Raised CD3+TCRαβ+CD4-CD8- double negative T cells and impaired T cell apoptosis are hallmarks of the disease. In contrast, the B cell compartment has been less well studied. We found an altered distribution of B cell subsets with raised transitional B cells and reduced marginal zone B cells, switched memory B cells and plasma blasts in most of 22 analyzed ALPS patients. Moreover, 5 out of 66 ALPS patients presented with low IgG and susceptibility to infection revealing a significant overlap between ALPS and common variable immunodeficiency (CVID). In patients presenting with lymphoproliferation, cytopenia, hypogammaglobulinemia and impaired B cell differentiation, serum biomarkers were helpful in addition to apoptosis tests for the identification of ALPS patients. Our observations may indicate a role for apoptosis defects in some diseases currently classified as CVID.

Multicenter survey on the outcome of transplantation of hematopoietic cells in patients with the complete form of DiGeorge anomaly

Seventeen patients transplanted with hematopoietic cells to correct severe T lymphocyte immunodeficiency resulting from complete DiGeorge anomaly were identified worldwide, and retrospective data were obtained using a questionnaire-based survey. Patients were treated at a median age of 5 months (range, 2-53 months) between 1995 and 2006. Bone marrow was used in 11 procedures in 9 cases: 6 from matched unrelated donors, 4 from human leukocyte antigen (HLA)-identical siblings, and one haploidentical parent with T-cell depletion. Unmobilized peripheral blood was used in 8 cases: 5 from HLA-identical siblings, one from a matched unrelated donor, one from an HLA-identical parent, and one unrelated matched cord blood. Conditioning was used in 5 patients and graft-versus-host disease prophylaxis in 11 patients. Significant graft-versus-host disease occurred in 9 patients, becoming chronic in 3. Median length of follow-up was 13 months, with transplantation from HLA-matched sibling showing the best results. Median survival among deceased patients (10 patients) was 7 months after transplantation (range, 2-18 months). The overall survival rate was 41%, with a median follow-up of 5.8 years (range, 4-11.5 years). Among survivors, median CD3 and CD4 counts were 806 (range, 644-1224) and 348 (range, 225-782) cells/mm(3), respectively, CD4(+)/CD45RA(+) cells remained very low, whereas mitogen responses were normalized.

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation

Background: X‐linked hyper‐IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients. Objectives: We sought to collect data on the clinical presentation, treatment, and follow‐up of a large sample of patients with XHIGM to (1) compare long‐term overall survival and general well‐being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT. Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan‐Meier method compared with log‐rank tests and modeled by using proportional hazards regression. Results: Twenty‐eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow‐up and vital status information. Sixty‐seven (38%) patients received HCT. The average follow‐up time was 8.5 ± 7.2 years (range, 0.1‐36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987‐1995; the hazard ratio was significantly less than 1 for diagnosis years 1995‐1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2‐10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft‐versus‐host disease, and most deaths occurred within 1 year of transplantation. Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964‐2013). However, survivors treated with HCT experienced somewhat greater well‐being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates.

Unrelated partially matched lymphocyte infusions in a patient with complete DiGeorge/CHARGE syndrome.

Abstract:  We present an infant with cDGS overlapping with CHARGE syndrome, who suffered from T‐cell deficiency treated with screened healthy DLI from an unrelated donor (8/10 match). The first dose of DLI (1.1 × 106 CD3+/kg) was administered at the age of six months, the second one (0.9 × 106 CD3+/kg) 36 days later. No conditioning was employed, GvHD prophylaxis consisting of CsA was used only during the second infusion. Since day+10 after the first DLI, split chimerism showing T‐cell engraftment has been documented. Proliferative response to PHA was detected on day+145. The treatment was complicated by severe acute GvHD (grade II‐III) after the first DLI and prolonged chronic liver cholestatic GvHD developing after the second DLI. Vigorous EBV proliferation four wk after the second DLI was accompanied by peripheral expansion of CD8+ donor cells. The patient, 26‐months old, is clinically well and has slowly started to gain his developmental milestones. We believe that infusions of small doses of DLI from an unrelated donor represent a potentially helpful therapeutic option in patients with cDGS/CHARGE phenotype.

Characterization of Lymphocyte Subsets in Patients with Common Variable Immunodeficiency Reveals Subsets of Naive Human B Cells Marked by CD24 Expression

Increased proportions of naive B cell subset and B cells defined as CD27negCD21negCD38neg are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27negCD21negCD38neg B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21pos naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27negCD21negCD38neg B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.

Non‐anaplastic peripheral T‐cell lymphoma in children and adolescents – a retrospective analysis of the NHL‐BFM study group

Mature (peripheral) T‐cell lymphoma (PTCL) other than anaplastic large cell lymphoma is a heterogeneous group of diseases and exceedingly rare in children and adolescents. Survival rates range between 46% and 85%. This study reports the disease characteristics, treatment and outcome of all patients with the diagnosis of mature TCL registered in the Berlin‐Frankfurt‐Munster non‐Hodgkin lymphoma database between 1986 and 2012. All diagnoses were centrally reviewed and revised by clinico‐pathological correlation according to the criteria of the current World Health Organization classification. Of the 69 patients originally registered as having PTCL, the diagnosis was confirmed in 38 of them. Most patients were treated with an anaplastic large cell lymphoma (ALCL)‐like therapy regimen. Patients with PTCL‐not otherwise specified comprised the largest group and showed a 5‐year event‐free survival rate of 61 ± 11%. Patients suffering from Natural Killer/T‐cell‐ and hepatosplenic TCL had the poorest outcome. Our results suggest that the outcomes of children with mature TCL other than ALCL depend on the subtype and are worse than in all other paediatric lymphomas. The clinical experience presented in this largest study on paediatric mature TCL may serve as basis for future collaborative international prospective clinical trials.

Sequential decisions on FAS sequencing guided by biomarkers in patients with lymphoproliferation and autoimmune cytopenia

Clinical and genetic heterogeneity renders confirmation or exclusion of autoimmune lymphoproliferative syndrome difficult. To re-evaluate and improve the currently suggested diagnostic approach to patients with suspected FAS mutation, the most frequent cause of autoimmune lymphoproliferative syndrome, we prospectively determined 11 biomarkers in 163 patients with splenomegaly or lymphadenopathy and presumed or proven autoimmune cytopenia(s). Among 98 patients sequenced for FAS mutations in CD3+TCRα/β+CD4−CD8− “double negative” T cells, 32 had germline and six had somatic FAS mutations. The best a priori predictor of FAS mutations was the combination of vitamin B12 and soluble FAS ligand (cut-offs 1255 pg/mL and 559 pg/mL, respectively), which had a positive predictive value of 92% and a negative predictive value of 97%. We used these data to develop a web-based probability calculator for FAS mutations using the three most discriminatory biomarkers (vitamin B12, soluble FAS ligand, interleukin-10) of the 11 tested. Since more than 60% of patients with lymphoproliferation and autoimmune cytopenia(s) in our cohort did not harbor FAS mutations, 15% had somatic FAS mutations, and the predictive value of double-negative T-cell values was rather low (positive and negative predictive values of 61% and 77%, respectively), we argue that the previously suggested diagnostic algorithm based on determination of double-negative T cells and germline FAS sequencing, followed by biomarker analysis, is not efficient. We propose vitamin B12 and soluble FAS ligand assessment as the initial diagnostic step with subsequent decision on FAS sequencing supported by a probability-calculating tool.

Immunohistochemical Prognostic Markers in Intracranial Ependymomas: Systematic Review and Meta-Analysis

Distinction between grade II ependymomas and anaplastic ependymomas based on histopathological examination solely is problematic and, therefore, the management of intracranial ependymomas remains controversial. The aim of this study was to conduct a systematic review (SR) and meta-analysis (MA) of data published on immunohistochemical prognostic markers (IPM) in intracranial ependymomas (IE), and to establish an evidence-based perspective on their clinical value. Following the extensive search based on a strictly defined group of key words, 30 studies reporting results on IPM in IE were identified. Due to a pronounced inter-study heterogeneity, only 14 publications fulfilled the criteria for inclusion into SR. From the total of 67 immunohistochemical markers, 18 were found to correlate with prognosis. However, owing to inadequate data publishing, MA could be performed only with data on proliferation marker MIB-1 (Ki-67) from 5 publications, including 337 patients: The pooled hazard ratio for overall survival was 3.16 (95% confidence interval = 1.96–5.09; p < 0.001) implicating that patients suffering from tumors with higher immunohistochemical expression of MIB-1 had a significantly worse outcome. Marked inter-study heterogeneity and incomplete data publishing in primary studies significantly limited extent of the SR, and the possibility of performing MA. Although the prognostic impact of MIB-1 immunoexpression in IE could be confirmed, there remains lack of further reliable IPM that could be used in routine diagnosis. We encourage to search for new, useful markers, as well as to standardize lab-techniques and data interpretation algorithms across laboratories in order to increase data compatibility.

Loss of B cells and their precursors is the most constant feature of GATA-2 deficiency in childhood myelodysplastic syndrome

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).