The role of the conditioning regimen for autologous and ex vivo genetically modified hematopoietic stem cell-based therapies: recommendations from the ISCT stem cell engineering committee.

BACKGROUND: The advent of autologous gene modified cell therapies to treat monogenic disorders has been a major step forward for the field of hematopoietic stem cell transplantation (HCT) and cellular therapies. The need for disease-specific conditioning to enable these products to provide a potential cure has required extrapolation from experience in myeloablative and non-myeloablative HCT for these disorders. METHODS: In this manuscript, we review the current datasets and clinical experience using different conditioning regimens for autologous gene therapies in hemoglobinopathies, metabolic and lysosomal disorders, inborn errors of immunity (IEI) and bone marrow failure (BMF) syndromes. RESULTS: The disease specific and unique conditioning requirements of each disorder are considered in order to achieve maximal benefit while minimizing associated toxicities. CONCLUSIONS: Standardized recommendations based on these data are made for each set of disorders to harmonize treatment. Future directions and the possibility of non-genotoxic conditioning regimens for autologous gene therapies are also discussed. Ethical Statement: The authors followed all relevant ethical considerations in writing this manuscript.

Transplantation for immune dysregulatory disorders: current themes and future expectations.

PURPOSE OF REVIEW: Primary immune regulatory disorders (PIRDs) are an increasing indication for hematopoietic stem cell transplant (HCT) in pediatric patients. Here, we provide an updated overview of HCT for PIRDs, and discuss future avenues for improvement in outcomes. RECENT FINDINGS: There are now more than 50 described monogenic PIRDs, which impact all aspects of immune tolerance, regulation, and suppression. Disease characteristics are highly variable, and HCT remains the only option for cure. We review advances in targeted therapies for individual PIRDs, which have significantly improved outcomes and the ability to safely bridge to transplant. Additionally, advances in GVHD prevention, graft manipulation, personalized conditioning regimens, and supportive care have all increased survival after HCT. The high inflammatory state increases the risk of nonengraftment, rejection, and autologous reconstitution. Therapy to reduce the inflammatory state may further improve outcomes. In addition, although younger patients with fewer comorbidities have better outcomes, the clinical courses of these diseases may be extremely variable thereby complicating the decision to proceed to HCT. SUMMARY: HCT for PIRDs is a growing consideration in cell therapy. Yet, there remain significant gaps in our understanding of which patients this curative therapy could benefit the most. Here, we review the current data supporting HCT for PIRDs as well as areas for future improvement.

A semiautomated microfluidic ELISA for the detection of hemophagocytic lymphohistiocytosis biomarkers.

OBJECTIVES: Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by a massive overactivation of the immune system. Because the clinical findings are nonspecific, the development of assays to facilitate rapid diagnosis is critical for patient care. The objectives of this study were to evaluate the performance of a microfluidic enzyme-linked immunosorbent assay (ELISA) for HLH biomarkers and investigate the impact of insourcing this testing on workflow, cost, and turnaround time in a tertiary-care cancer hospital. METHODS: Trends in order volume were evaluated for C-X-C motif chemokine ligand 9 (CXCL9) and soluble interleukin 2 receptor ɑ (sIL2R), and a microfluidic ELISA was used to measure these analytes in serum samples. Analyte values, turnaround time, and costs were compared for this assay relative to reference laboratory testing. RESULTS: Test ordering has increased from 187 to 1030 requests annually over the past 5 years. Insourcing these analytes on a semiautomated ELISA can decrease time to result by approximately 2 days and generate a cost savings of roughly $140,000 annually within our laboratory. CONCLUSIONS: Using a semiautomated ELISA for sIL2R and CXCL9 may help physicians arrive at a diagnosis and monitor therapy for patients with HLH while decreasing turnaround time and costs within the clinical laboratory.

Reduced toxicity matched sibling bone marrow transplant results in excellent outcomes for severe congenital neutropenia.

Severe congenital neutropenia (SCN) is caused by germline mutations, most commonly in ELANE, impacting neutrophil maturation and leading to high risk of life-threatening infections. Most patients with ELANE-mutant SCN can achieve safe neutrophil counts with chronic Granulocyte-Colony Stimulating Factor (G-CSF). However, up to 10% of patients have neutropenia refractory to G-CSF and require allogeneic stem cell transplant. Traditional conditioning for these patients includes busulfan and cyclophosphamide which is associated with significant toxicities. We present five patients with SCN without myeloid malignancy transplanted using a reduced toxicity regimen of busulfan, fludarabine and thymoglobulin. 5 pediatric patients with SCN underwent matched sibling donor bone marrow transplant (MSD-BMT) between 2014-2022 on or per CHP14BT057 (NCT02928991), a prospective, single center trial testing elimination of cyclophosphamide from conditioning in pediatric patients with single lineage inherited BMF syndromes. All patients had MSDs and no evidence of MDS. Conditioning consisted of PK-adjusted busulfan, fludarabine, and thymoglobulin, with calcineurin inhibitor and mycophenolate mofetil GVHD prophylaxis. With median follow-up of 48.4 months, overall and event-free survival were 100%. There was no acute GVHD and one instance of chronic limited GVHD. Patients exhibited >95% donor myeloid chimerism at 5 years post-BMT. Two patients experienced CMV reactivation without end-organ disease, and no other viral reactivation or significant infections occurred. MSD-BMT with reduced toxicity myeloablation for SCN provides excellent outcomes while minimizing toxicity. These data suggest that busulfan, fludarabine, and ATG can be considered an efficacious, low-toxicity standard of care regimen for patients with SCN undergoing MSD-BMT.

Two novel assays demonstrate persistent daratumumab exposure in a pediatric patient with delayed engraftment following allogeneic hematopoietic stem cell transplantation.

BACKGROUND AIMS: Daratumumab, a human IgG monoclonal antibody targeting CD38, is a promising treatment for pediatric patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL). We describe a case of delayed engraftment following a mismatched, unrelated donor hematopoietic stem cell transplant (HSCT) in a 14-year-old female with relapsed T-ALL, treated with daratumumab and chemotherapy. By Day 28 post-HSCT, the patient had no neutrophil engraftment but full donor myeloid chimerism. METHODS: We developed two novel, semi-quantitative, antibody-based assays to measure the patient's bound and plasma daratumumab levels to determine if prolonged drug exposure may have contributed to her slow engraftment. RESULTS: Daratumumab levels were significantly elevated more than 30 days after the patient's final infusion, and levels inversely correlated with her white blood cell counts. To clear daratumumab, the patient underwent several rounds of plasmapheresis and subsequently engrafted. CONCLUSIONS: This is the first report of both delayed daratumumab clearance and delayed stem cell engraftment following daratumumab treatment in a pediatric patient. Further investigation is needed to elucidate the optimal dosing of daratumumab for treatment of acute leukemias in pediatric populations as well as daratumumab's potential effects on hematopoietic stem cells and stem cell engraftment following allogenic HSCT.

Osteopetrorickets: two contradictory patterns-one unifying diagnosis.

A 5-month-old infant with bone findings on x-ray presented an apparent contradiction including findings of both diffusely dense bones and rickets in the context of a history and laboratory investigation that suggested leukemia. Next generation gene panel sequencing revealed a TCIRG1 mutation which is consistent with autosomal recessive osteopetrosis. The paradoxical x-ray findings underscore a recently elucidated mechanism for the pathogenesis of a TCIRG mutation. This case highlights the importance of recognizing this radiographic, seeming contradictory, association in the context of a confusing clinical presentation. Failure to recognize this pattern promptly may lead to a delay in diagnosis, thus potentially permanent organ failure.

Uncovering the Genetic Etiology of Inherited Bone Marrow Failure Syndromes Using a Custom-Designed Next-Generation Sequencing Panel.

Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for ∼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.

Predictors of SARS-CoV-2 Omicron breakthrough infection after receipt of AZD7442 (tixagevimab-cilgavimab) for pre-exposure prophylaxis among hematologic malignancy patients.

AZD7442 (tixagevimab-cilgavimab) is a combination of two human monoclonal antibodies for pre-exposure prophylaxis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among high-risk patients who do not mount a reliable vaccine response. Foremost among these are hematologic malignancy patients with limited clinical trial or realworld experience to assess the effectiveness of this combination treatment since the emergence of Omicron and its subvariants. We performed a retrospective study of 892 high-risk hematologic malignancy patients who received AZD7442 at Memorial Sloan Kettering Cancer Center in New York City from January 1, 2022 to July 31, 2022. We evaluated demographic, clinical, and laboratory characteristics and performed regression analyses to evaluate risk factors for breakthrough infection. We also evaluated the impact of updated AZD7442 dosing regimens on the risk of breakthrough infection. Among 892 patients, 98 (10.9%) had a breakthrough infection during the study period. A majority received early outpatient treatment (82%) and eventually eight (8.2%) required hospitalization for management of Coronavirus Disease 2019 (COVID-19), with a single instance of severe COVID-19 and death. Patients who received a repeat dose or a higher firsttime dose of AZD7442 had a lower incidence of breakthrough infection. Univariate analyses did not reveal any significant predictors of breakthrough infection. While AZD7442 is effective at reducing SARS-CoV-2 breakthrough infection in patients with hematologic malignancies, no risk factors reliably predicted risk of infection. Patients who received updated dosing regimens as per Food and Drug Administration guidelines had better protection against breakthrough infection.

Virus-specific T-cells from third party or transplant donors for treatment of EBV lymphoproliferative diseases arising post hematopoietic cell or solid organ transplantation.

EBV+ lymphomas constitute a significant cause of morbidity and mortality in recipients of allogeneic hematopoietic cell (HCT) and solid organ transplants (SOT). Phase I and II trials have shown that in HCT recipients, adoptive transfer of EBV-specific T-cells from the HCT donor can safely induce durable remissions of EBV+ lymphomas including 70->90% of patients who have failed to respond to treatment with Rituximab. More recently, EBV-specific T-cells generated from allogeneic 3rd party donors have also been shown to induce durable remission of EBV+ lymphomas in Rituximab refractory HCT and SOT recipients. In this review, we compare results of phase I and II trials of 3rd party and donor derived EBV-specific T-cells. We focus on the attributes and limitations of each product in terms of access, safety, responses achieved and durability. The limited data available regarding donor and host factors contributing to T cell persistence is also described. We examine factors contributing to treatment failures and approaches to prevent or salvage relapse. Lastly, we summarize strategies to further improve results for virus-specific immunotherapies for post-transplant EBV lymphomas.

Pathogenicity and impact of HLA class I alleles in aplastic anemia patients of different ethnicities.

Acquired aplastic anemia (AA) is caused by autoreactive T cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some patients with AA. However, pathogenicity, structural characteristics, and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 patients with AA from 2 multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a potentially novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or transplant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult patients with AA. Our study provides insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved understanding of clonal evolution in AA.

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

Population based frequency of naturally occurring loss-of-function variants in genes associated with platelet disorders.

Essentials The frequency of predicted loss-of-function (pLoF) variants in platelet-associated genes is unknown in the general population. Datasets like Genome Aggregation Database allow us to analyze pLoF variants with increased resolution. Expected prevalence of significant pLoF variants in platelet-associated genes in 0.329% in the general population. Platelet-associated genes that cause phenotypes due to haploinsufficiency are significantly depleted for deleterious variation. ABSTRACT: Background Inherited platelet disorders are being recognized more frequently as advanced sequencing technologies become more commonplace in clinical scenarios. The prevalence of each inherited platelet disorder and the disorders in aggregate are not known. This deficit in the field makes it difficult for clinicians to discuss results of sequencing assays and provide appropriate anticipatory guidance. Objectives In this study, we aim to calculate the prevalence of predicted loss-of-function variants in platelet-associated genes in the general population. Methods Here, we leverage the aggregation of exomes from the general population in the form of Genome Aggregation Database to assess 58 platelet-associated genes with phenotypic correlates. We use the loss-of-function transcript effect estimator (LOFTEE) to identify predicted loss-of-function mutations in these platelet-associated genes. These variants are curated and we then quantify the frequency of predicted loss-of-function variants in each gene. Results Our data show that 0.329% of the general population have a clinically meaningful predicted loss-of-function variant in a platelet-associated gene. Thus, these individuals are at risk for bleeding disorders that can range from mild to severe. Conclusions These data provide a novel lens through which clinicians can analyze sequencing results in their patients as well as an additional method to curate newly discovered platelet-associated genes in the future.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

Human mutational constraint as a tool to understand biology of rare and emerging bone marrow failure syndromes.

Inherited bone marrow failure (IBMF) syndromes are rare blood disorders characterized by hematopoietic cell dysfunction and predisposition to hematologic malignancies. Despite advances in the understanding of molecular pathogenesis of these heterogeneous diseases, genetic variant interpretation, genotype-phenotype correlation, and outcome prognostication remain difficult. As new IBMF and other myelodysplastic syndrome (MDS) predisposition genes continue to be discovered (frequently in small kindred studies), there is an increasing need for a systematic framework to evaluate penetrance and prevalence of mutations in genes associated with IBMF phenotypes. To address this need, we analyzed population-based genomic data from >125 000 individuals in the Genome Aggregation Database for loss-of-function (LoF) variants in 100 genes associated with IBMF. LoF variants in genes associated with IBMF/MDS were present in 0.426% of individuals. Heterozygous LoF variants in genes in which haploinsufficiency is associated with IBMF/MDS were identified in 0.422% of the population; homozygous LoF variants associated with autosomal recessive IBMF/MDS diseases were identified in only .004% of the cohort. Using age distribution of LoF variants and 2 measures of mutational constraint, LOEUF ("loss-of-function observed/expected upper bound fraction") and pLI ("probability of being loss-of-function intolerance"), we evaluated the pathogenicity, tolerance, and age-related penetrance of LoF mutations in specific genes associated with IBMF syndromes. This analysis led to insights into rare IBMF diseases, including syndromes associated with DHX34, MDM4, RAD51, SRP54, and WIPF1. Our results provide an important population-based framework for the interpretation of LoF variant pathogenicity in rare and emerging IBMF syndromes.

STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Mitochondria Biogenesis Modulates Iron-Sulfur Cluster Synthesis to Increase Cellular Iron Uptake.

Iron-sulfur (Fe-S) clusters are required for mitochondrial function. Fe-S cluster synthesis occurs in the mitochondria and iron uptake is required for mitochondrial biogenesis. However, Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfR1), whereas lack of the Fe-S cluster stimulates TfR1 expression. Yet, it is unclear whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, whether this negatively modulates TfR1 expression. We manipulated peroxisome proliferator-activated receptor-gamma coactivator-1α expression to control mitochondrial biogenesis in a variety of cell types, including erythroid cells. We demonstrated that Fe-S cluster synthesis increases with mitochondria biogenesis but does not interfere with increasing TfR1 expression. In fact, TfR1 expression is stimulated through alternative means to meet iron requirement for mitochondria biogenesis. Furthermore, under enhanced mitochondria biogenesis, increased Fe-S cluster synthesis inhibits the function of iron-regulating protein (IRP)1 and hence stimulates the expression of 5'-aminolevulinate synthase 2 (ALAS2), a target of IRP1 and rate-limiting enzyme in erythroid heme biogenesis. Increased ALAS2 expression leads to enhanced heme production, hemoglobinization, and erythropoiesis. Therefore, our study also provides a mechanism to link mitochondrial biogenesis with erythropoiesis and has a potential therapeutic value in the treatment of blood disorders.

Exceptional Response to Nivolumab in a 13-Year-Old Female with Metastatic HPV-Negative Cervical Carcinoma.

Cervical carcinoma is associated with high-risk human papillomavirus (HPV) DNA integration and usually occurs after age 21 (peak 45 years), as reflected in screening guidelines. Between 1999 and 2008, cervical carcinoma rate in adolescents aged 15-19 years was 0.15 per 100,000. HPV-negative cervical carcinoma is rare in adolescents. The youngest previously reported case was 15 years old. Treatment options for cervical carcinoma are limited after first-line therapy. Immune checkpoint inhibitors blocking programmed death receptor (PD-1) and its ligand, PD-L1, have shown objective clinical responses and are tolerable in adults with gynecologic cancers. This class of agents is well tolerated in pediatric patients. PD-1/PD-L1 is commonly expressed in gynecologic cancers but its expression may not predict clinical response. We describe an exceptional response to single agent nivolumab postradiation therapy in a 13-year-old adolescent with poorly differentiated cervical carcinoma and widespread metastatic disease.