Immune-mediated Cytopenias Research Articles

Daratumumab therapy for post-HSCT immune-mediated cytopenia: experiences from two pediatric cases and review of literature

BackgroundImmune-mediated cytopenias (AIC) are challenging complications following allogeneic hematopoietic stem cell transplantation (HSCT). While broad-acting immunosuppressive agents like corticosteroids are often standard of care, several novel therapies which target specific immunological pathways have recently been developed and provide hope for patients with steroid-refractory courses and may limit long-term toxicity. The successful off-label use of the plasma cell depleting anti-CD38 antibody daratumumab was published in several case reports, suggesting efficacy, i.e., in patients with antibody-mediated AIC refractory to previous B cell depletion. We want to share our experience with two children, whom we treated with daratumumab, including one fatal course with uncontrolled disease. Given the absence of substantial data from HSCT registries or prospective trials, we furthermore provide a critical review of the literature on daratumumab treatment of AIC.Case presentationsPatient 1 (P1), an 11-year-old girl with lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency who developed immune-mediated thrombocytopenia (AIT) from day +58 after HSCT, showed a complete response to daratumumab after the fourth of six total daratumumab doses. She remains transfusion independent for over a year of follow-up. Previously, her thrombocytopenia was refractory to corticosteroids, rituximab, intravenous immunoglobulins (IVIG), eltrombopag, cyclosporine A, and sirolimus. Patient 2 (P2), a 6-year-old boy with CD40 ligand (CD40L) deficiency, developed both AIT and hemolytic anemia (AIHA) after HSCT on days +58 and +83, respectively, and was also treated with daratumumab after being previously refractory to prednisolone, rituximab, and IVIG. Yet, he did neither respond to daratumumab nor the concomitantly administered methyprednisolone pulse, plasmapheresis, and eculizumab and succumbed due to refractory disease.ConclusionReviewing the literature on the use of daratumumab for refractory AIC post-HSCT, we consider daratumumab a promising agent for this life-threatening disorder: ten of the twelve patients reached transfusion independency in the literature. However, treatment failures are likely to be underreported. Thus, controlled trials are needed to explore the safety and efficacy of daratumumab in this rare post-HSCT complication.

Characterizing Immune-Mediated Cytopenias After Allogeneic Hematopoietic Cell Transplantation for Pediatric Nonmalignant Disorders

Immune-mediated cytopenias (IMC)-isolated or combined hemolytic anemia, thrombocytopenia, or neutropenia-are increasingly recognized as serious complications after allogeneic hematopoietic cell transplantation (HCT) for nonmalignant disorders (NMD). However, IMC incidence, duration, response to therapy, and risk factors are not well defined. This retrospective chart review identified cases of IMC with serologic confirmation among patients who underwent HCT for NMD at a single institution between 2010 and 2017. IMC after HCT for NMD in a large pediatric cohort (n=271) was common with a cumulative incidence of 18%, identified at a median of 136 days after HCT. Treatment included prolonged immune suppression (>3 months) in 58% of all IMC cases, 91% when multiple cell lines were affected. Multiple therapeutic agents were used for the majority affected, and median time to resolution of IMC was 118 days from diagnosis. Fine-Gray competing risk multivariate regression analysis identified a combined risk factor of younger age (<3 years) and inherited metabolic disorder, as well as hemoglobinopathy (at any age) associated with 1-year incidence of IMC (P < .01). We expand these findings with the observation of declining donor T-lymphoid chimerism from day 60 to 100 and lower absolute CD4+ counts at day 100 (P < .01), before median onset of IMC, for patients with IMC compared to those without. In this cohort, 4 deaths (8%) were associated with IMC, including 2 requiring second transplantation for secondary graft failure. Although the pathogenesis of IMC post-HCT for NMD remains elusive, further research may identify approaches to prevent and better treat this HCT complication.

Chronic Lymphocytic Leukaemia Presenting with Pancytopenia: ARare Haematological Coincidence

Chronic Lymphocytic Leukaemia (CLL) is commonly associated with autoimmune cytopenias but CLL presenting with pancytopenia is a rare occurrence. Here, we present a case of 56-year-old male patient, who presented with pallor, mild hepatosplenomegaly, right-sided pleural effusion in chest x-ray, pancytopenia and his bone marrow showed infiltration by mature appearing lymphoid cells making a diagnosis of CLL. Despite the frequency of these immune mediated cytopenia, only few cases of pancytopenia have been described so far to the best of our knowledge. Hence, to emphasise the rarity of such haematological coincidence we are presenting this case report. CLL generally presents with persistent absolute lymphocytosis on peripheral smear with an absolute monoclonal lymphocyte count of more than 5000/cumm. There is a common association with Autoimmune Haemolytic Anaemia (AIHA) and Immune Thrombocytopenia (ITP) and the association is called as Evan’s syndrome. Its pathogenesis is associated with autoimmune process. AIHA, ITP, and Pure Red Cell Aplasia (PRCA) are commonly associated complications seen in CLL. The pathogenesis of AIHA and ITP are antibody this inplace of the reactions and abnormal T cell activity is noted in the pathogenesis of PRCA. Despite of the frequency of these immune mediated cytopenias noted in CLL, bone marrow was seen to be hypercellular in most of the cases and marrow hypoplasia have been reported in only 2 cases reported in literature so far. In the present case, although pancytopenia was present on peripheral blood smear examination but bone marrow was hypercellular for age with diffuse infiltration by monoclonal mature appearing lymphoid cells which makes this case all the more interesting. Despite repeated blood transfusions,antiviral drug Tenofovir and aggressive supportive measures for 6 months, he developed right-sided pleural effusion and succumbed to his illness.

Autoimmune cytopenias complicating hematopoietic cell transplantation

Immune cytopenias after allogeneic hematopoietic cell transplantation are rare, albeit increasingly recognized, complications. Autoimmune diseases are serious complications of HCT and include immune-mediated cytopenias i.e. autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP) and autoimmune neutropenia. Severe cytopenia is usually defined by decreases of hemoglobin concentration below 7 g/dL, of platelet count below 20 G/L, or of absolute granulocyte count below 0.5 G/L, and it is mediated by the presence of auto-antibodies. ITP occurring in combination with AIHA is known as Evans syndrome. Immune dysregulation is caused by impaired immune reconstitution and/or loss of self-tolerance. Primary risk factors of autoimmune cytopenias include: peripheral blood or cord blood as a stem cell source, unrelated HCT, non-malignant disease, use of alemtuzumab, acute/chronic graft-versus-host disease (GvHD), cytomegalovirus reactivation, infections, and, in pediatric settings, conditioning omitting total body irradiation. Diagnosis of autoimmune cytopenia is challenging due to a broad differential diagnosis: primary or secondary graft failure, infections, GvHD, disease relapse, drug-induced side effects, transplant-associated thrombotic microangiopathy, ABO-incompatibility, or disseminated intravascular coagulation. Treatment should be tailored to the individual patient, and ranges from watchful waiting to aggressive management in life-threatening situations. Apart from specific treatment adjusted for specific cytopenia, supportive care should include transfusions of leukocyte-reduced and irradiated red blood cell concentrates or pathogen-reduced platelet concentrates; treatment of infections and GvHD; modification of immunosuppression; and supplementation with microelements. Autoimmune cytopenias are usually highly resistant to standard therapy and are associated with increased risks of high morbidity and mortality, particularly when coexisting with other post-transplant complications.

Investigation of immune repertoires in thymoma.

e21117 Background: Paraneoplastic autoimmune manifestations are seen in 30 to 40% of thymoma cases. Thymus plays the main role in T- cell maturation with ‘negative selection’ of autoreactive T-cells. Due to reasons not well understood, this function is deranged in thymomas resulting in dysregulation of immune function, loss of ‘self-tolerance’ and autoreactivity. The low cost and high-throughput of next generation sequencing techniques now make it feasible to analyze extensive T-cell receptor(TCR) gene rearrangements in peripheral blood lymphocytes. Methods: 40 patients with thymoma were identified between December, 2010 and September, 2019 at Indiana University. Whole blood samples were collected after diagnosis of thymoma but prior to thymectomy. 20 patients developed paraneoplastic manifestations at some point during the disease course. Genomic DNA was isolated from whole blood samples and TCR analysis performed using a commercially available next generation sequencing assay. This was compared with the control population of 20 patients who did not demonstrate any paraneoplastic features during their disease course. Patients were matched based on age, clinical stage and WHO classification. Results: Paraneoplastic manifestations seen in the 20 patients included Myasthenia Gravis (n = 12), Good’s Syndrome (n = 2), immune mediated cytopenia (n = 3) including pure red cell aplasia, pure white cell aplasia and thrombocytopenia, neurologic manifestations such as encephalitis (n = 2), Autoimmune Autonomic Neuronopathy(n = 1) and Isaac’s Syndrome (n = 2). Some patients also demonstrated more than one paraneoplastic syndrome. The onset and course of these manifestations varied; onset prior to diagnosis of thymoma, onset after thymectomy and improving or worsening clinical features after thymectomy. Since the exact mechanism of autoimmunity is still unknown, we attempted to detect differences in TCR subsets between these two groups. Our study shows that it is technically feasible to interrogate the peripheral blood lymphocytes of thymoma patients for TCR gene clonality and diversity. Conclusions: Diverse autoimmune syndromes can be described in thymoma patients with varying clinical course and severity. Although our study is underpowered, TCR gene sequencing is efficient and may not only improve our understanding of these paraneoplastic syndromes but also identify biomarkers for their diagnosis. Our research could also potentially lead to development of targeted treatment options in the future based on TCR manipulation.

Immune-Mediated Cytopenias After Hematopoietic Cell Transplantation: Pathophysiology, Clinical Manifestations, Diagnosis, and Treatment Strategies.

Discuss the pathophysiology, clinical presentation, diagnosis, and treatment of immune-mediated cytopenias (IMC) after hematopoietic cell transplantation (HCT). Key risk factors for post-HCT IMC include younger age, non-malignant disease, and umbilical cord blood stem cell source. While anemia predominates, any or all three hematopoietic cell lines can be affected. In rare cases, IMC can cause graft failure or death. IMC is hypothesized to result from immune dysregulation upon reconstitution of donor hematopoietic cells (i.e., dysfunctional regulatory T cells). Aside from blood product transfusions, IMC treatment includes immune-suppressive or ablative agents. First-line therapies, including corticosteroids and intravenous immunoglobulin, are often inadequate, prompting use of additional agents aimed at antibody production/T cell dysfunction or direct antibody removal via plasmapheresis. IMC occurs in up to 20% of high-risk HCT populations. Morbidity and mortality from IMC post-HCT have been reduced by improved recognition and aggressive early interventions.

Immune cytopenia post–cord transplant in Hurler syndrome is a forme fruste of graft rejection

AbstractUmbilical cord blood (UCB) is the preferred donor cell source for children with Hurler syndrome undergoing transplant, and its use has been associated with improved “engrafted survival” rates. However, as in other pediatric recipients of UCB transplants for nonmalignant disease, immune-mediated cytopenia (IMC) is a significant complication. This article describes 8 episodes of IMC in 36 patients with Hurler syndrome undergoing UCB transplant. The incidence of IMC was increased in those with a higher preconditioning absolute lymphocyte count and in those conditioned with fludarabine-containing regimens rather than cyclophosphamide, and it included red cell alloantibodies directed at cord blood group antigens that are novel to the recipient. In several cases, IMC was a precursor to immune-mediated complete graft rejection. We describe IMC as part of a spectrum of graft rejection by a residual competent host immune system and a forme fruste of complete graft rejection.

Characterizing Immune Mediated Cytopenias Following Allogeneic Hematopoietic Cell Transplantation for Pediatric Non-Malignant Disorders

Background Immune mediated cytopenias (IMC) – isolated or combined hemolytic anemia, thrombocytopenia, and neutropenia - are increasingly recognized as potentially serious complications following allogeneic hematopoietic cell transplantation (HCT) for non-malignant disorders (NMD). However, IMC incidence, severity, response to therapy, and risk factors are not well defined. Methods Pediatric patients undergoing HCT for NMD between 2010 and 2017 at a single institution were identified, excluding those experiencing graft failure or death prior to day +100. Demographics and HCT characteristics were obtained from a prospectively-maintained database. A retrospective chart review identified cases of IMC with positive direct Coombs test, anti-platelet antibody, and/or anti-granulocyte antibody; and documented treatment and response. IMC severity was defined by months to resolution: mild (0-3), moderate (3-6), or severe (>6). Results Of 271 NMD HCT recipients, 52 (19%) exhibited IMC at a median of 130 days post-HCT (range, 30-279); 23 (44% of cases) had multiple cell lines affected. IMC disease was mild, moderate, or severe in 44%, 25%, and 31%, respectively, with no deaths attributed to IMC. For those with moderate or severe IMC, all received steroid therapy, with a response rate of 34%. An average of 2.0 unique agents were used to achieve IMC resolution, at a median of 117 days (range 17 – 639). Univariate analysis revealed statistically significant associations between IMC and younger age at HCT (median 3.7 vs. 7.0 years, p Conclusion Immune mediated cytopenias following HCT for NMD in a large pediatric cohort was common at 19%, with limited steroid responsiveness, use of multiple immunosuppressive agents and duration of therapy exceeding 3 months for the majority affected. Patient and transplant characteristics associated with IMC were consistent with previous reports, though we expand these findings with observation of declining donor T-lymphoid chimerism and CD4 counts by day +100, prior to median onset of IMC. While, the pathogenesis of IMC post-HCT for NMD remains elusive, further research may identify approaches to prevent this HCT complication.

Myeloid-Derived Suppressor Cells in Hematologic Diseases: Promising Biomarkers and Treatment Targets.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that exist at very low numbers in healthy subjects but can expand significantly in malignant, infectious, and chronic inflammatory diseases. These cells are characterized as early-MDSCs, monocytic-MDSCs, and polymorphonuclear-MDSCs and can be studied on the basis of their immunophenotypic characteristics and their functional properties to suppress T-cell activation and proliferation. MDSCs have emerged as important contributors to tumor expansion and chronic inflammation progression by inducing immunosuppressive mechanisms, angiogenesis and drug resistance. Most experimental and clinical studies concerning MDSCs have been mainly focused on solid tumors. In recent years, however, the implication of MDSCs in the immune dysregulation associated with hematologic malignancies, immune-mediated cytopenias and allogeneic hemopoietic stem cell transplantation has been documented and the potential role of these cells as biomarkers and therapeutic targets has started to attract a particular interest in hematology. The elucidation of the molecular and signaling pathways associated with the generation, expansion and function of MDSCs in malignant and immune-mediated hematologic diseases and the clarification of mechanisms related to the circulation and the crosstalk of MDSCs with malignant cells and other components of the immune system are anticipated to lead to novel therapeutic strategies. This review summarizes all available evidence on the implication of MDSCs in hematologic diseases highlighting the challenges and perspectives arising from this novel field of research.

Immune Cytopenias after Ex Vivo CD34+-Selected Allogeneic Hematopoietic Cell Transplantation.

Immune-mediated cytopenias (ICs), such as immune thrombocytopenia and immune hemolytic anemia, are among the adverse events after allogeneic hematopoietic cell transplantation (allo-HCT). Previous reports suggest that in vivo T cell depletion may increase the incidence of IC after allo-HCT. We evaluated whether a strategy that reduces functional donor T cells via ex vivo CD34+-selection associates with the development of IC in a cohort of 408 patients who underwent allo-HCT for hematologic malignancy. The cumulative incidence of IC at 6, 12, and 36 months after the 30-day landmark post-HCT was 3.4%, 4.9%, and 5.8%, respectively. Among 23 patients who developed IC, 7 died of relapse-related mortality and 4 of nonrelapse mortality. A median 2 types of treatment (range, 1 to 5) was required to resolve IC, and there was considerable heterogeneity in the therapies used. In univariable analyses, a hematologic malignancy Disease Risk Index (DRI) score of 3 was significantly associated with an increased risk of IC compared with a DRI of 1 or 2 (hazard ratio [HR], 4.12; P = .003), and IC (HR, 2.4; P = .03) was associated with increased risk of relapse. In a multivariable analysis that included DRI, IC remained significantly associated with increased risk of relapse (HR, 2.4; P = .03). Our findings show that IC events occur with relatively similar frequency in patients after ex vivo CD34+-selected allo-HCT compared with unmodified allo-HCT, suggesting that reduced donor T cell immunity is not causative of IC. Moreover, we noted a possible link between its development and/or treatment and increased risk of relapse.

Immune Mediated Cytopenias Following Allogeneic Hematopoietic Cell Transplantation for Non-Malignant Disease: Risk Factors and Treatment Response in the Pediatric Population

Background: Immune mediated cytopenias (IMC) - isolated or combined hemolytic anemia, thrombocytopenia, and neutropenia - are increasingly recognized as potentially serious complications following allogeneic hematopoietic cell transplantation (HCT) for non-malignant disorders (NMD). However, IMC incidence, severity, response to therapy, and associated risk factors are not well defined.Methods: Pediatric patients undergoing HCT for NMD between 2010 and 2017 at a single institution were identified, excluding those experiencing graft failure or death prior to day +100. Demographics and HCT characteristics were obtained from a prospectively-maintained, institutional database. A retrospective chart review identified cases of IMC with positive direct antiglobulin test, anti-platelet antibody, and/or anti-granulocyte antibody; and documented treatment(s) and response. Composite IMC severity, incorporating peak acuity and response to therapy, was defined as mild (outpatient management with supportive care or a single course of therapy), moderate (outpatient management with 2 or more courses of therapy), or severe (inpatient management with 2 or more courses of therapy). Factors hypothesized to be associated with IMC included younger age, reduced intensity conditioning, mixed or declining donor chimerism, and inherited metabolic disorder as indication for HCT. For the first year after transplant, peripheral blood donor chimerism, B-cell reconstitution, and IgG and IgM levels were obtained.Results: A total of 271 NMD HCT recipients were included, with 53 (20%) exhibiting IMC, 20 (38% of cases) with multiple cell lines affected, diagnosed at a median of 131 days post-HCT (range, 30-264 days). IMC disease was mild, moderate, or severe in 56%, 32%, and 11%, respectively. For those with moderate or severe IMC, all received steroid therapy, with a response rate of 13%. Eighty-seven percent received additional therapy including IVIG, rituximab, bortezomib, and/or vincristine. Resolution of moderate IMC required an average of 2 individualized treatment courses, while severe IMC responded after an average of 6. Notably, no patients died of IMC. Univariate analysis revealed statistically significant association between IMC and younger age at HCT (median 3.1 vs. 7.1 years, p<0.01) and inherited metabolic disorder as the indication for HCT (p<0.01). Patient sex, recipient-donor HLA-match, stem cell graft source, intensity of conditioning, and presence of acute graft-versus-host disease were not significantly different between those with and those without IMC. Following HCT, a decline in peripheral blood donor CD3+ chimerism by at least 10% from day +60 to +100 was more common in those with IMC (p<0.01), although 50% of patients with IMC did not demonstrate a decrease in donor T-cell engraftment. The percent donor CD3+ chimerism at day +100 trended lower in the IMC group compared with the non-IMC group (71% vs 88.5%, p=0.10). The kinetics of patient B cell, IgG, or IgM recovery was not associated with IMC.Conclusion: Immune mediated cytopenias following HCT for NMD in a large pediatric cohort was common at 20%, with nearly half of those affected exhibiting clinically significant courses before disease resolution. Elucidation of risk factors may inform modifications to conditioning regimens or other approaches to prevent IMC. We found younger age at HCT, inherited metabolic disorder as HCT indication, and declining donor T-lymphoid chimerism as risk factors for IMC. However, the pathogenesis of IMC post-HCT for NMD remains elusive. Further insights may arise from investigation into the role of infections/molecular mimicry and complement activation. DisclosuresMiller:Sangamo Therapeutics: Employment.

Immune Mediated Cytopenia's Following Cord Blood Transplantation for Hurler's Syndrome: A Failure of Pre-Transplant Immune Suppression

Background: Hurler's syndrome is the most severe phenotype of Mucopolysaccharidosis 1 (MPS1H) with severe deficiency of the lysosomal enzyme alpha-L-iduronidase causing heparan and dermatan sulfate accumulation, CNS and somatic manifestations and premature death. Haematopoeitic Stem Cell Transplant (HSCT) alters the disease phenotype and prevents early death, and is standard of care in this condition. The use of an established ‘donor hierarchy’ recommending Unrelated Cord Blood Transplantation (UCBT) in the absence of a HLA-matched, non-carrier sibling, has resulted in improved engrafted and surviving rates and better post-enzyme levels with improved somatic and CNS clinical outcomes. However, UCBT for malignant and non-malignant conditions has led to high rates of post-transplant autoimmune disease, most frequently Autoimmune Cytopenia (AIC). We report a large cohort of MPS1H patients receiving busulfan-based UCBT and show that AIC is a frequently encountered complication of treatment with significant associated morbidity and we identify important risk factors for its occurrence.Methods: This was a retrospective observational study of patients undergoing first UCBT for Hurler's Syndrome at the Royal Manchester Children's Hospital between 30th September 2004 and 15th March 2018. An episode of AIC was defined as an episode of single or multi-lineage cytopenia occurring post-engraftment, confirmed by the presence of antibodies or diagnosed on the basis of clinical, biochemical and pathologic features. All patients received pK targeted busulfan to achieve myeloablative levels between 80-90 mg/L x hr. Conditioning between 2004 and May 2010 was with Busulfan and Cyclophosphamide (BuCy), and Fludarabine and Busulfan (FluBu) thereafter based on the reported equivocal efficacy and favourable toxicity profile of FluBu conditioning. All patients received serotherapy with Anti-Thymocyte Globulin (ATG) 10mg/kg for In Vivo T-Cell depletion. Timing of ATG was either distal (day -4 to -1) or proximal (day - 9 to -6) in accordance with international consensus at the time. Variables assessed in statistical analysis were conditioning drugs, ATG timing, Absolute Lymphocyte Count pre-conditioning, age-at-transplant, Graft-versus-Host Disease (GVHD) and Total Nucleated Cell dose.Results and Discussion: Thirty-six patients underwent first UCBT for Hurler's Syndrome. There were 8 episodes of AIC (22%), with median onset post-UCBT of 66 days (range 22-236 days). The median number of therapies required was 4 (range 0-8), with duration of illness ranging from 10 to 215 days. There were no deaths, though there were 2 episodes of life-threatening bleeding. Absolute Lymphocyte Count pre-conditioning (p=0.004), FluBu conditioning (p=0.03) and use of proximal-ATG (p=0.016) were significant risk factors for AIC in univariate analysis(see figure 1). Absolute Lymphocyte Count pre-conditioning was the most significant predictor in multivariable analysis (AOR 5.796, CI 0.921-36.474). This confirms that AIC is a frequent, serious but survivable complication of UCBT for Hurler's Syndrome. This data indicates that the balance between recipient immunity and graft immunity impacts AIC risk and severity, mandating a larger international review of registry data to define the frequency of AIC in this population, and an analysis of the risk factors that predispose to it. By better defining these risk factors, we can identify patients in whom conditioning might be altered to reduce risk.Figure 1. Univariate analysis of variables predicting an episode of Autoimmune Cytopenia. ALC (p=0.004), ATG timing (0.016) and Conditioning drugs (p=0.032) were significant risk factors. GVHD= Graft-versus-Host Disease ATG= Anti-Thymocyte Globulin [Display omitted] DisclosuresWynn:Orchard SAB: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Equity Ownership; Chimerix: Research Funding; Genzyme: Honoraria; Bluebird Bio: Consultancy; Orchard Therapeutics: Consultancy; Chimerix: Consultancy.

Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation

Gain-of-function (GOF) mutations in the human signal transducer and activator of transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity with impaired TH17cell differentiation and exaggerated responsiveness to type I and II interferons. Allogeneic bone marrow transplantation has been attempted in severely affected patients, but outcomes have been poor. We sought to define the effect of increased STAT1 activity on T helper cell polarization and to investigate the therapeutic potential of ruxolitinib in treating autoimmunity secondary to STAT1 GOF mutations. We used invitro polarization assays, as well as phenotypic and functional analysis of STAT1-mutated patient cells. We report a child with a novel mutation in the linker domain of STAT1 who had life-threatening autoimmune cytopenias and chronic mucocutaneous candidiasis. Naive lymphocytes from the affected patient displayed increased TH1 and follicular T helper cell and suppressed TH17cell responses. The mutation augmented cytokine-induced STAT1 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reduced hyperresponsiveness to type I and II interferons, normalized TH1 and follicular T helper cell responses, improved TH17 differentiation, cured mucocutaneous candidiasis, and maintained remission of immune-mediated cytopenias. Autoimmunity and infection caused by STAT1 GOF mutations are the result of dysregulated T helper cell responses. Janus kinase inhibitor therapy could represent an effective targeted treatment for long-term disease control in severely affected patients for whom hematopoietic stem cell transplantation is not available.

Development of red blood cell autoantibodies following treatment with checkpoint inhibitors: a new class of anti-neoplastic, immunotherapeutic agents associated with immune dysregulation

Ipilimumab, nivolumab, and pembrolizumab represent a new class of immunotherapeutic drugs for treating patients with advanced cancer. Known as checkpoint inhibitors, these drugs act to upregulate the cellular and humoral immune response to tumor antigens by inhibiting T-cell autoregulation. As a consequence, they can be associated with immune-related adverse events (irAEs) due to loss of self-tolerance, including rare cases of immune-related cytopenias. We performed a retrospective clinical chart review, including serologic, hematology, and chemistry laboratory results, of two patients who developed red blood cell (RBC) autoantibodies during treatment with a checkpoint inhibitor. Serologic testing of blood samples from these patients during induction therapy with ipilimumab and nivolumab, respectively, showed their RBCs to be positive by the direct antiglobulin test (IgG+, C3+) and their plasma to contain panreactive RBC autoantibodies. Neither patient had evidence of hemolysis. Both patients developed an additional irAE during treatment. A literature review for patients who had developed immune-mediated cytopenia following treatment with a checkpoint inhibitor was performed. Nine other patients were reported with a hematologic irAE, including six with anemia attributable to autoimmune anemia, aplastic anemia, or pure RBC aplasia. Hematologic irAEs tend to occur early during induction therapy, often coincident with irAEs of other organs. In conclusion, checkpoint inhibitors can be associated with the development of autoantibodies, immune-mediated cytopenias, pure RBC aplasia, and aplastic anemia. Immunohematology reference laboratories should be aware of these agents when evaluating patients with advanced cancer and new-onset autoantibodies, anemia, and other cytopenias.

High-Exposure, Targeted Daily Busulfan and Fludarabine-Based Conditioning for Children Undergoing Hematopoietic Stem Cell Transplantation for Inherited Metabolic Disorders: Outcomes at a Single Center

Allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as standard therapy for children suffering from various life-threatening inherited metabolic disorders (IMD). Historically, successful outcomes have been limited by high rates of both graft failure and regimen-related toxicity. Real-time busulfan pharmacokinetic monitoring, combined with once daily dosing, has allowed for more precise delivery of targeted exposures during transplant conditioning. Recently, the Utrecht group reported highly favorable outcomes following a high-dose busulfan and fludarabine-based preparative regimen in patients with a variety of diagnoses, particularly in comparison to historical regimens that employ two or more alkylating agents. We report our experience using the same regimen specifically in patients with IMD. Since July 2014, 24 children undergoing first HSCT for IMD were conditioned with once daily IV busulfan (4 days; total regimen exposure of 90 mg x h/L) and once daily IV fludarabine (40 mg/m2/day x 4 days). Busulfan pharmacokinetic monitoring was performed on each of the first 3 doses (days); subsequent doses were altered to achieve the desired total regimen exposure. Serotherapy use was donor-dependent: HLA-matched related donor (MRD, n = 5), none; unrelated UCB (n = 16), thymoglobulin (2.5 mg/kg/day, days - 8 to -5); HLA-matched unrelated donor (MUD, n = 1), thymoglobulin (2.5 mg/kg/day, days - 5 -to -2); and, related HLA haploidentical marrow (n = 2), alemtuzumab (0.5 mg/kg divided days -11 and -10), +/- rituximab (375 mg/m2, day -12). All patients received CSA for GvHD prophylaxis. Additional GvH prophylaxis depended upon the allograft source: UCB, prednisolone; MRD/MUD, MMF; related haploidentical, MMF and post-transplant cyclophosphamide. Growth factor support was administered to recipients of UCB or haploidentical grafts. IMD diagnoses were as follows: Hurler syndrome (n = 10); cerebral adrenoleukodystrophy (n = 9); metachromatic leukodystrophy (n = 1); osteopetrosis (n = 1); fucosidosis (n = 1); alpha-mannosidosis (n = 1); and, beta-mannosidosis (n = 1). At a median follow-up of 229 days (range, 15 - 743), 22 of 24 patients survive, with a Kaplan Meier estimate of survival at one year of 86%. Causes of death were pulmonary bronchiolitis obliterans (BOS, n = 1) and graft failure (n = 1). Twenty-two patients had initial donor neutrophil recovery at a median day +14 (range, +11 to +21); for these patients, the median duration of severe neutropenia (ANC < 500/μL) was 7.5 days (range, 4 to 16 days). Of 20 patients who at the time of analysis are ≥ 30 days from HSCT, 18 had platelet engraftment (≥20,000/μL, transfusion unsupported) at a median day +22.5 (range, +10 to +94). One patient developed Grade II aGvHD (haploidentical marrow graft) which was successfully treated; no patients developed Grade III-IV aGvHD. One patient developed chronic GvHD with fatal BOS (UCB). Regimen-related complications were as follows: veno-occlusive disease, n = 0; hemorrhagic cystitis, n = 1; diffuse alveolar hemorrhage n = 1; idiopathic pneumonia syndrome, n = 1; immune-mediated cytopenias, n = 2; CMV viremia, n = 3; and, adenovirus infection, n = 1. Twenty patients (83%) have demonstrated durable, complete donor myeloid engraftment following the regimen. Of these, all have demonstrated mixed donor-recipient T-cell chimerism. Four patients, (all UCB grafts), have experienced graft failure: aplastic graft failure, n = 3, and autologous hematopoietic recovery, n = 1. Three of these patients underwent second HSCT and 2 are alive and engrafted while the other died of complications following second transplant. One patient awaits second transplant. In summary, this regimen of high-exposure, targeted daily busulfan and fludarabine in children with IMD provides favorable engrafted survival with minimal toxicities. DisclosuresNo relevant conflicts of interest to declare.

Editorial: Current Challenges in Immune and Other Acquired Cytopenias of Childhood.

Editorial: Current Challenges in Immune and Other Acquired Cytopenias of Childhood.

Alemtuzumab in T-cell large granular lymphocytic leukaemia: interim results from a single-arm, open-label, phase 2 study

BackgroundT-cell large granular lymphocytic leukemia (T-LGL) is a lymphoproliferative disease presenting with immune-mediated cytopenias and characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes. Methotrexate, cyclosporine, or cyclophosphamide improve cytopenias in 50% of patients as first therapy, but the activity of an anti-CD52 monoclonal antibody, alemtuzumab, is not defined in T-LGL.MethodsTwenty-five consecutive subjects with T-LGL were enrolled from October 2006 to March 2015 at the National Institutes of Health (www.clinicaltrials.gov-NCT00345345). Alemtuzumab was administered at 10 mg/day intravenously for 10 days. The primary endpoint was haematologic response at 3 months. Analysis was intention to treat. Here we report the protocol specified interim benchmark of a phase II clinical trial using alemtuzumab in T-LGL.FindingsIn this heterogeneous, previously treated cohort, 14/25 (56%; 95% CI, 37–73%) subjects had a haematological response at 3 months. In T-LGL cases not associated with myelodysplasia or marrow transplantation, the response rate was 14/19 (74%; 95% CI, 51–86%). First dose infusion reactions were common which improved with symptomatic therapy. EBV and CMV reactivations were common and subclinical. In only 2 patients pre-emptive anti-CMV therapy was instituted. There were no cases of EBV or CMV disease. Alemtuzumab induced sustained reduction of absolute clonal population of T-cytotoxic lymphocytes, as identified by TCRBV-receptor phenotype, but the abnormal clone serendipitously persisted in responders. STAT3 mutations in the SH2 domain, identified in ten subjects, did not correlate with response. When compared with healthy volunteers, T-LGL subjects showed a distinct plasma cytokine and JAK-STAT signature prior to treatment, but neither correlated to response.InterpretationThis is the largest and only prospective cohort of T-LGL subjects treated with alemtuzumab yet reported. The high activity with a single course of a lymphocytotoxic agent in a mainly relapsed and refractory suggests that haematologic response outcomes can be accomplished without the need for continued use of oral immunosuppression.FundingThis research was supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute.

Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis.

BackgroundTargeting CTLA-4 is a recent strategic approach in cancer control: blocking CTLA-4 enhances an antitumor immunity by promoting T-cell activation and cytotoxic T-lymphocyte proliferation. This induction of a tolerance break against the tumor may be responsible for immune-related adverse events (irAEs). Our objective was to assess the incidence and nature of irAEs in oncologic patients receiving anti-CTLA-4 antibodies (ipilimumab and tremelimumab).MethodsA systematic search of literature up to February 2014 was performed in MEDLINE, EMBASE, and Cochrane databases to identify relevant articles. Paired reviewers independently selected articles for inclusion and extracted data. Pooled incidence was calculated using R©, package meta.ResultsOverall, 81 articles were included in the study, with a total of 1265 patients from 22 clinical trials included in the meta-analysis. Described irAEs consisted of skin lesions (rash, pruritus, and vitiligo), colitis, and less frequently hepatitis, hypophysitis, thyroiditis, and some rare events such as sarcoidosis, uveitis, Guillain-Barré syndrome, immune-mediated cytopenia and polymyalgia rheumatic/Horton. The overall incidence of all-grade irAEs was 72 % (95 % CI, 65–79 %). The overall incidence of high-grade irAEs was 24 % (95 % CI, 18–30 %). The risk of developing irAEs was dependent of dosage, with incidence of all-grade irAEs being evaluated to 61 % (95 % CI, 56–66 %) for ipilimumab 3 mg/kg and 79 % (95 % CI, 69–89 %) for ipilimumab 10 mg/kg. Death due to irAEs occurred in 0.86 % of patients.The median time of onset of irAEs was about 10 weeks (IQR, 6–12) after the onset of treatment, corresponding with the first three cycles but varied according to the organ system involved. Such immune activation could also be indicative for tumor-specific T-cell activation and irAE occurrence was associated with clinical response to CTLA-4 blocking in 60 % of patients.ConclusionThe price of potential long-term survival to metastatic tumors is an atypical immune toxicity, reflecting the mechanism of action of anti-CTLA-4 antibodies. A better knowledge of these irAEs and its management in a multidisciplinary approach will help to reduce morbidity and therapy interruptions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0455-8) contains supplementary material, which is available to authorized users.

Assessment of Mutation Status in a Large Series of Patients with Cytopenias with Normal Karyotypes and without Increased Blasts

Background: Myelodysplastic syndromes (MDS) are clonal stem cell disorders that can variably affect the erythroid, myeloid, and megakaryocytic hematopoietic lineages producing persistent cytopenias. Distinguishing MDS without increased blasts from immune-mediated cytopenias is difficult as criteria for dysplasia lack specificity and karyotypic changes are frequently absent. Given the need for better diagnostic assays to identify the clonal hematopoietic populations characteristic of MDS, we examined mutation status of 8 genes that are commonly mutated in the early stages of MDS. We profiled a group of 505 samples from patients with cytopenia(s) that were clinically and/or morphologically suspicious for MDS but had no increase in blasts and normal karyotypes.Methods: All 505 samples analyzed met these criteria: 1) clinical suspicion of MDS; 2) concurrent complete blood count with cytopenia(s); 3) bone marrow biopsy without increase in blasts; 4) normal karyotype (ie normal diploid, constitutional changes or –Y). In 170 cases, a FISH panel for 6 MDS changes was also performed and was negative. DNA was extracted from bone marrow aspirate (n=461) or blood (n =44). Sequencing of EZH2, TET2, exon 12 of ASXL1, JAK2 codon 617 and mutation hotspots in IDH1, IDH2, KRAS and NRAS was performed on the Ion Torrent PGM platform and analyzed using SeqNext software. To exclude artifacts and constitutional and low-level background alterations, a positive result required confirmation by Sanger sequencing (or pyrosequencing) and was defined as frameshift change, nonsense or previously characterized missense change, or novel missense change with high mutation probability based on level and structural features. All single nucleotide polymorphisms (SNPs) seen in normal controls or public databases were excluded. Ninety-two cases were subsequently analyzed for mutations in 21 additional MDS-associated genes using a TruSeq assay on the MiSeq platform.Results: Among 505 cases, M:F ratio was 1.02; 90.9% of patients were ≥50 years old. The frequency of MDS-associated mutations in cytopenic samples that had normal karyotype was 19.6% (99/505); mutations were identified in TET2 (63 cases), ASXL1 (36), IDH1 or IDH2 (8), NRAS (5), EZH2 (4), JAK2 V617F (4) and KRAS (1). The lowest mutation rate was in samples with isolated neutropenia (8.8%) with any cases showing neutropenia having an unusually high frequency of IDH1 or IDH2 mutation (7/39, 17.9%); other CBC parameters did not correlate significantly with incidence or type of mutation (Table). Multiple mutations were seen in 40 (7.9%) samples, with dual TET2 and ASXL1 mutation in 13. Among cases with several mutations, unequivocal subclonal mutation changes were seen in 17. Among 69 cases with mutations detected by the 8-gene panel, a larger sequencing panel detected additional mutations in 49 (71%), most commonly SRSF2 (9) and RUNX1 (3). 6/23 cases (26.1%) that were negative in the 8-gene panel had MDS-associated mutation(s) identified in the larger panel.Table.Frequency of mutations (mut) by cytopenia.CBC findingNumber of casesAny mutation% of all casesTET2mut% of mut casesASXL1mut% of mut casesAll cases5059919.66363.63636.4Pancytopenia911718.71376.5423.52 cytopenias1663722.32362.21848.6Anemia only1753620.61952.81336.1Macrocytic anemia55814.5450.0337.5Thrombocytopenia only39615.46100.0116.7Neutropenia only3438.8266.7133.3Conclusions: Using a targeted 8-gene panel, we identified MDS-associated mutations in approximately 20% of samples suspicious for MDS based on cytopenia(s) but with normal karyotype and no increase in blasts. In this community-based, unselected series, mutation detection rate was largely independent of hematologic pattern. Mutation density and subclonal complexity were relatively low even using a larger panel of genes. The high detection rate illustrates the enhanced clinical utility of targeted sequencing panels for identifying clonal alterations in early/low-grade MDS. Further work is needed to delineate the prognostic significance of mutations identified in minimally atypical cytopenic bone marrow samples. DisclosuresLee:Quest Diagnostics: Employment. Wang:Quest Diagnostics: Employment. Maiese:AmeriPath: Employment. Arvai:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Mehta:Quest Diagnostics: Employment. Caruthers:Quest Diagnostics: Employment. Gersen:AmeriPath: Employment. Billouin-Frazier:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

Impaired receptor editing and heterozygous RAG2 mutation in a patient with systemic lupus erythematosus and erosive arthritis

Impaired receptor editing and heterozygous RAG2 mutation in a patient with systemic lupus erythematosus and erosive arthritis