Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare anemias characterized by ineffective erythropoiesis and hemolysis. The CDA registry in North America (CDAR; NCT02964494) was established to facilitate natural history studies. Participants who do not have a genetic diagnosis are offered family-trio whole genome sequencing (WGS) and elect to provide samples to CDAR biorepository to support collaborative mechanistic studies. CDA type II (CDA-II), the most common CDA type, is an autosomal recessive disease caused by biallelic SEC23Bvariants. CDA-II presents with hemolytic anemia with suboptimal reticulocytosis, iron overload, splenomegaly, and characteristic binucleation in 10-30% of the bone marrow (BM) erythroblasts. Although CDA-II is a recessive disorder, only one SEC23B variant is found in some patients. Here, we present the genetic and clinical data of CDA-II patients enrolled in CDAR so far. As of July 2023, 169 individuals (85 patients; 84 unaffected family members) enrolled in CDAR. Nine have a confirmed CDA-II diagnosis. Six patients have biallelic SEC23B pathogenic variants and 3 have only one variant identified. WGS for the 3 patients with a single variant revealed rare deep-intronic candidate variants. Since SEC23Bis ubiquitously expressed, we studied SEC23B protein levels in patient-derived lymphoblastoid cell lines generated for the CDAR biorepository. Patients with a single SEC23B variant exhibited an expression level comparable to those with biallelic mutations (Figure). The candidate deep intronic variants are currently being investigated for pathogenicity. Seven patients presented with hemolytic anemia during infancy, but the age of diagnosis was delayed by a mean of 12.5 yrs (0.1 to 55 y.o). Blood smears showed mild macrocytosis, poikilocytosis, and several spherocytes congruent with an osmotic gradient ektacytometry resembling mild hereditary spherocytosis. BM examination, when performed, exhibited erythroid hyperplasia, prominent dyserythropoiesis, and binucleation in ≥ 10% of erythroblasts. Anemia severity was variable: 6 patients were transfusion-dependent, 1 received intermittent transfusions, and 2 did not need blood transfusions. Two became transfusion-independent after splenectomy at ages 6 and 7 y.o, and two underwent successful matched-sibling BMT at age 10 and 26 y.o, after adequate chelation (liver iron concentration [LIC]<7 mg/g) Iron overload, disproportionate to transfusions, was present in 6 patients; 5 received iron chelation. All 5 patients with available MRI results had hepatic hemosiderosis (LIC range 3.4 to 21.8 mg/g) and evidence of myocardial hemosiderosis (T2* range 6.3 to 20 ms). Ferritin values were variable (range 58 to 1631.5 ng/ml) and discordant from LIC and T2* measurements. Notably, ferritin underestimated iron overload burden: 2 patients with normal ferritin (58 and 160 ng/mL) had hepatic hemosiderosis (LIC 6.1 and 3.4 mg/g respectively), and patients with severe hepatic and myocardial hemosiderosis (LIC 16.6-21.8 mg/g; T2* 6.3-7.8 ms) had modest ferritin values 1475-1631 ng/mL. Iron overload was present even in a non-transfusion-dependent patient and worsened in a patient post-splenectomy, suggesting a role for ineffective erythropoiesis. To further investigate this possibility, we measured erythroferrone (ERFE), GDF-15, and hepcidin levels in 2 patients so far. ERFE and GDF-15 levels were remarkably higher than controls, and hepcidin-to-ferritin ratios were significantly lower, suggesting that high ERFE, caused by ineffective erythropoiesis, inappropriately suppressed hepcidin for the body's iron levels. In summary, CDA-II diagnosis is often delayed despite early onset of anemia. Genetic testing may expedite the diagnosis. The severity of anemia is variable and there are several patients requiring transfusions. The risk of iron overload, including myocardial overload, is high and appears to be related to ineffective erythropoiesis. While frequency of transfusions may improve post-splenectomy, iron overload is not determined by blood transfusions alone and does not resolve post-splenectomy. Serum ferritin underestimated the burden of tissue iron in CDA-II, suggesting that MRI scans should be employed early. Future studies may identify a role for SEC23B deep intronic variants in CDA-II patients with heterozygous variants.

Abstract Background: Chimeric antigen receptor (CAR)-T cell therapy targeting the CD19 antigen has been effective in treating B-cell acute lymphoblastic leukemia. As CAR-T cells targeting new antigens are being explored for the treatment of other cancers in adults, parallel studies are warranted for pediatric cohorts. We have previously shown the safety and efficacy in adults of CAR-T cells targeting CD30, which is expressed in classical Hodgkin Lymphoma (HL) and in some Non-Hodgkin Lymphoma (NHL). We have therefore sought to study the feasibility and the safety of CD30.CAR-T cells in pediatric patients with relapsed/refractory CD30-expressing HL and Anaplastic Large Cell Lymphoma (ALCL). Design/Methods: Six pediatric patients (ages 9 to 17 years) with CD30+ HL (4) and NHL (2) were enrolled on two trials at the University of North Carolina. One NHL patient with ALK+ ALCL was enrolled on both trials. Two patients, one HL and one NHL, were enrolled on a phase I study and received 2x10 7 CD30.CAR-T cells/m 2 as consolidation for high-risk of relapse after autologous stem cell transplant (ASCT, NCT02663297). Five patients, 3 HL and 2 NHL, were enrolled on a phase Ib/II study and received 1x10 8 CD30.CAR-T cells/m 2, as treatment for relapsed disease, after lymphodepletion with bendamustine and fludarabine (NCT02690545). HL patients had failed multiple lines of therapies (5-6), including 2 with prior pembrolizumab, 2 with prior ASCT, and all 3 with prior brentuximab vedotin (BV) and radiation therapy. The two NHL patients both had ALCL, one was ALK positive and one was ALK negative. Both had been treated with prior BV. The ALK negative patient had been treated with 3 prior lines of therapy and the ALK positive patient had been treated with 6 lines of prior therapy including ASCT and two ALK inhibitors, crizotinib and brigatinib. The brigatinib was stopped 3 weeks prior to starting lymphodepletion. Results: CD30.CAR-T cells were successfully manufactured for all 7 patients and no differences were observed as compared to products manufactured for adults, based on cell number, transduction, potency or immunophenotype. For all patients, infusions were well-tolerated and no neurotoxicity experienced. On the post-ASCT study, 1 patient with HL and 1 with ALCL were treated. All adverse events (AE) were less than grade 4. The patient with HL remains in complete remission (CR) 41 months following therapy, while the patient with ALCL progressed. Five patients with relapsed/refractory disease (3 HL and 2 ALCL) were treated on the post-lymphodepletion study. Most grade 3 or higher AEs were anticipated hematologic toxicity secondary to lymphodepletion. The youngest HL patient on the study developed symptoms consistent with grade 2 cytokine release syndrome (CRS) and a concomitant pneumonia based on imaging, accompanied by a marked inflammatory response based on labs with maximum ferritin 9,920 ng/mL and CRP 150.7 mg/L. He responded to antibacterial agents and two doses of tocilizumab, as well as brief supplemental oxygen by nasal cannula. He did not require vasopressors. One patient with ALK+ ALCL had symptoms compatible with CRS and macrophage activation syndrome (maximum ferritin >100,000 ng/mL and CRP 39.7 mg/L), which were ultimately determined to be secondary to progressive disease, confirmed at autopsy 5 weeks post therapy. The other four patients (3 HL and 1 ALCL) achieved CR and remain in CR 4 to 27 months post CAR-T cell infusion. Of note, 2 of the HL patients chose to come off study while in CR to receive treatment with checkpoint inhibitor therapy. Conclusion: Our studies show that CD30.CAR-T cells are well tolerated in pediatric patients. CRs were observed in all heavily pre-treated and refractory HL patients, highlighting the potential of this strategy. All patients treated on both studies had previously received BV, which suggests CD30.CAR-T cells are effective even post BV progression. We continue to investigate how to better tailor CD30.CAR-T cells in NHL given the need for better therapies in ALCL, which is often aggressive at relapse. One patient with ALK negative ALCL remains in CR while the patient with ALK+ ALCL had rapid relapse. After study initiation, UNC entered into a research collaboration with Tessa Therapeutics. Disclosures Savoldo: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Dotti: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Grover: Tessa: Consultancy; Kite: Other: Advisory Board; Novartis: Consultancy; Genentech: Research Funding; ADC: Other: Advisory Board. Morrison: Vesselon: Consultancy. Riches: Jazz Pharmaceuticals: Other: Payment; ATARA Biotherapeutics: Other: Payment; BioIntelect: Membership on an entity's Board of Directors or advisory committees. Serody: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma.

Intro/Objectives Warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome is a rare primary immunodeficiency defined by its symptoms and CXC chemokine receptor 4 (CXCR4) gene mutation. This leads to myelokathexis in which mature neutrophils cannot exit the bone marrow, causing severe neutropenia. Use of hematopoietic cell transplant (HCT) to correct a primary immunodeficiency is well accepted; yet, due to the rarity of WHIM, data on HCT in this setting are limited. We present a successful matched sibling donor (MSD) bone marrow (BM) transplant in a 6 year old male with WHIM who developed unexpected respiratory complications post HCT. Methods/Results The patient was initially followed for congenital neutropenia and therapy consisted of weekly filgrastim. At age 5 years, he was diagnosed with WHIM by CXCR4 sequencing. Treatment was changed to plerixafor, but he required frequent dose escalation. HLA typing revealed his brother was an 8/8 allele match. Patient's history was significant for asthma, but no recent flares. He was unable to perform pulmonary function tests due to age. At 6 years old he received an MSD BM graft after myeloablative conditioning with busulfan, cyclophosphamide, and ATG. GVHD prophylaxis consisted of cyclosporine and short course methotrexate. Myeloid engraftment ocurred on day +20 with >95% donor chimerism after day +55. On days +76, +87, and +104, he was admitted for respiratory distress. Infectious evaluations only revealed EBV PCR in a BAL specimen obtained during the second admission. For this he received four weeks of rituximab. On each admission, he improved quickly with albuterol and systemic steroids, but symptoms recurred soon after returning home. During his third admission it was found that he was exposed to an African Grey Parrot in his home. Due to the close association of his symptoms with returning home, and his exquisite response to steroids, Pulmonology consultation lead to a diagnosis of hypersensitivity pneumonitis. The bird was relocated, the home extensively cleaned, and while staying in temporary housing from day +110 to +275 the patient did not relapse. After returning home the patient had no further respiratory events. The bird was allowed to return on day +367. Patient has remained well on inhaled corticosteroids as of day +473. Conclusion Hypersensitivity pneumonitis is a type IV hypersensitivity reaction that can be linked to bird exposure. It is T cell mediated and IgE independent. Acquisition of IgE mediated allergies has been reported after HCT, but there are no reports of new type IV hypersensitivity development. Neither patient nor donor had previous hypersensitivity to the bird. Due to the transient nature of the pneumonitis in our patient, this likely represents an unusual case of abnormal T cell maturation post HCT. This case also emphasizes the importance of environmental exposures in post HCT respiratory symptoms.

Background: Oligoclonality of the T-lymphocyte repertoire is seen early after hematopoietic stem cell transplantation (HSCT). Increasing T-cell receptor (TCR) diversity is part of immune reconstitution after HSCT and can be measured by TCR spectratype. Recovery of gamma-delta (GD) T-cells is associated with good outcomes post-HSCT. However, TCR GD diversity after HSCT is not extensively reported, and its relationship with acute graft-versus-host disease (aGVHD) is unclear. Methods: TCR GD spectratype was analyzed in pediatric allogeneic HSCT recipients with nonmalignant disorders enrolled on clinical trial NCT01962415. Patients received reduced-intensity conditioning with alemtuzumab, hydroxyurea, fludarabine thiotepa, and melphalan. Prophylaxis for aGVHD consisted of tacrolimus and mycophenolate mofetil. Spectratype was analyzed at Day 100 and 180 using reverse transcriptase polymerase chain reaction with primers for TCR loci gamma 2-5, gamma 8-9, and delta 1-3. A spectratype complexity score (SCS) was then calculated for each TCR GD subfamily. Results: Samples were available for 17 patients (10 female), median age 2.4 years (range .4-33.9). Donor source was marrow (N = 3) or umbilical cord blood (N = 14), 94% unrelated. Incidence of aGVHD Grade 2-4 was 24% and Grade 3-4 was 18%. The mean SCS of the gamma and delta subfamilies was significantly lower at Day 100 (Table 1) in those with aGVHD Grade 3-4 and significantly lower at Day 180 (Table 2) in those with aGVHD Grade 2-4 and 3-4, compared to those with maximum aGVHD Grade 0-1. Increasing aGVHD severity was associated with a progressive decrease in mean SCS (Figure 1).Table 1Mean SCS at Day 100LocusMaximum aGVHD grade0-12-43-4G24.631.5G33.93.31G43.72.71.5G52.21.31G83.42.3.5G953.32.5Total gamma3.72.71.3 (P < .01)D14.720D254.33D32.81.7.5Total delta4.12.71.2 (P = .02) Open table in a new tab Table 2Mean SCS at Day 180LocusMaximum aGVHD grade0-12-43-4G25.42.3G34.52.81G4410G52.3.50G82.41.8.7G94.11.81Total gamma3.81.6 (P = .001).5 (P < .001)D15.310D24.82.81D33.8.80Total delta4.31.5 (P = .02).3 (P = .002) Open table in a new tab Discussion: Decreased TCR GD diversity at Day 100 and Day 180 was associated with the severity of aGVHD. Possible causes include immunosuppressive GVHD therapy and multifactorial interference with donor T-cell maturation. Theoretically TCR GD rearrangement occurs earlier in T-cell maturation than that of TCR alpha-beta, making the GD population sensitive to early post-HSCT changes in immune homeostasis. Factors affecting the maturation and immunoregulatory properties of GD T-cells require further investigation, as does the prognostic role of TCR GD diversity in aGVHD.

Mucormycosis is uncommon in patients with chronic granulomatous disease (CGD). We report a 7-year-old boy with X-linked CGD and absent oxidative burst who developed fatal Lichtheimia ramosa infection with fungal thrombosis of the kidneys, spleen and other organs after hematopoietic stem cell transplantation. Lichtheimia infection is rarely reported in patients with CGD and could be related to iatrogenic immunosuppression.

Moyamoya syndrome occurs in sickle cell disease (SCD) as a secondary complication of large-artery stenosis. Moyamoya increases the risk of stroke, but its optimal management in SCD is not established.Encephaloduroarteriosynangiosis (EDAS) is a neurosurgical revascularization procedure for moyamoya whose use has been reported in SCD patients. We report the outcomes of 11 patients with SCD systematically evaluated for EDAS by a multidisciplinary team and compare the rate of stroke in patients who received EDAS to those who did not. Moyamoya syndrome was diagnosed by flow abnormalities on magnetic resonance angiography at median age of 8.2years. Four patients deferred surgery. Seven patients underwent EDAS at median age of 19years. There were no intraoperative complications, perioperative strokes, or deaths. Transient postoperative complications occurred in six cases (86%). On follow-up, three patients (43%) had no evidence of flow in their EDAS grafts, and one later developed a hemorrhagic stroke. Five EDAS patients (71%) had radiographically stable vasculopathy. Compared to the four patients who deferred surgery, the incidence of stroke in EDAS group was no different. The optimal use of EDAS in patients with SCD-associated moyamoya syndrome requires further investigation by a prospective, controlled clinical trial.

The tempo of immunologic reconstitution after hematopoietic stem cell transplantation (HSCT) for thalassemia and sickle cell disease (SCD) has not been reported. Immunologic reconstitution and recovery of adaptive immunity are important for long-term health-related quality of life in HSCT recipients. Based on observations in allogeneic HSCT for other disorders, we predict full immunological recovery after HSCT for hemoglobinopathy. We reviewed all patients with SCD or thalassemia who underwent HSCT at Children's Hospital and Research Center Oakland. In patients who stopped immunosuppression, we analyzed lymphocyte subsets and response to mitogens and microbial antigens as part of standard clinical management. One patient enrolled in an autologous gene-therapy trial was excluded. Thirty-six patients (50% female, 50% with SCD) received transplants between 2000 and 2014. Median age at transplant was 5.8 years, and median follow-up was 51 months. Event-free survival was 81%, disease-free survival was 89%, and overall survival was 94%. Lymphocyte phenotype analyses were available in 16 patients not receiving immunosuppressive therapy, and lymphocyte proliferation studies were available in 15. Table 1 summarizes the tempo of reconstitution in these patients. Lymphocyte subsets and proliferation normalized a median of 4 to 18 weeks after stopping immunosuppression; B-cell function, based on antibody to H. influenzae polyribosylribitol phosphate (PRP), recovered more slowly. Time to reconstitution did not differ significantly with donor type, cell source, or underlying diagnosis, although recovery of lymphocyte proliferation to mitogens showed a trend toward variability (see Figure 1). Of 12 SCD patients with evidence of asplenia before HSCT, 5 (42%) had normal pitted erythrocyte counts post-transplant, suggesting recovery of splenic function. HSCT for hemoglobin disorders was associated with excellent survival. T-cell function recovered within weeks of stopping immunosuppressive therapy, which suggests that extending the course of post-HSCT immunosuppression to prevent late graft rejection does not cause a delay in immune reconstitution. Transplantation also improved splenic function in some SCD patients.Table 1Time to immunologic recovery after stopping immunosuppression.Analysis (N)Median time, weeks (IQR)Lymphocyte population Total T-cells (13)13 (3 – 26) Total B-cells (16)5 (2 – 26) NK cells (13)4 (2 – 25)Lymphocyte proliferation Mitogens (15)8 (4 – 29) C. albicans (14)5 (4 – 29) Cytomegalovirus (12)8 (6 – 24) Herpes simplex virus (7)18 (7 – 20) Varicella-zoster virus (9)7 (5 – 18) PRP (10)17 (5 – 94)IQR: Interquartile range. Open table in a new tab