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Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at

BackgroundRuxolitinib, a Janus kinase (JAK)-1/JAK-2 inhibitor that reduces T-cell activation and proliferation leading to a potent anti-inflammatory effect, was recently approved to treat steroid-refractory acute graft-versus-host disease (aGvHD) and is under study for chronic GvHD (cGvHD). However, there are few reports on its use in pediatric and young adult patients with GvHD.MethodsWe retrospectively report our single center experience with ruxolitinib in 15 patients aged 11-29 years: 5 patients with steroid refractory/steroid dependent aGvHD and 10 patients with cGvHD who had failed at least one systemic therapy.ResultsIn the aGvHD group, ruxolitinib led to an overall response rate (ORR) of 40% at day 28 and 80% with longer follow-up (complete response, CR 60%), with a median of 21 days (5-74) to achieve a response. In the cGvHD group (9 evaluable patients), we observed an ORR of 67% and a CR of 22%, with a median of 78 days (35-180) to achieve a response. Adverse events included cytopenias, gastrointestinal symptoms, infections, and an allergic reaction attributable to ruxolitinib.ConclusionsOverall, our results show that ruxolitinib is an effective salvage therapy for severe GvHD in pediatric and young adult patients. The toxicities noted warrant adequate antimicrobial prophylaxis and close monitoring of blood counts.

PURPOSE: The Primary Immune Deficiency Treatment Consortium (PIDTC) enrolled children with severe combined immunodeficiency (SCID) in a prospective natural history study of hematopoietic stem cell transplant (HSCT) outcomes over the last decade. Despite newborn screening (NBS) for SCID, infections occurred prior to HSCT. This study’s objectives were to define the types and timing of infection prior to HSCT in patients diagnosed via NBS or by family history (FH) and to understand the breadth of strategies employed at PIDTC centers for infection prevention. METHODS: We analyzed retrospective data on infections and pre-transplant management in patients with SCID diagnosed by NBS and/or FH and treated with HSCT between 2010 and 2014. PIDTC centers were surveyed in 2018 to understand their practices and protocols for pre-HSCT management. RESULTS: Infections were more common in patients diagnosed via NBS (55%) versus those diagnosed via FH (19%) (p = 0.012). Outpatient versus inpatient management did not impact infections (47% vs 35%, respectively; p = 0.423). There was no consensus among PIDTC survey respondents as to the best setting (inpatient vs outpatient) for pre-HSCT management. While isolation practices varied, immunoglobulin replacement and antimicrobial prophylaxis were more uniformly implemented. CONCLUSION: Infants with SCID diagnosed due to FH had lower rates of infection and proceeded to HSCT more quickly than did those diagnosed via NBS. Pre-HSCT management practices were highly variable between centers, although uses of prophylaxis and immunoglobulin support were more consistent. This study demonstrates a critical need for development of evidence-based guidelines for the pre-HSCT management of infants with SCID following an abnormal NBS. TRIAL REGISTRATION: NCT01186913

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Allogeneic hematopoietic stem cell transplant (HSCT) typically results in donor T-cell engraftment and function in patients with severe combined immunodeficiency (SCID), but humoral immunity, particularly when using donors other than matched siblings, is variable. B-cell function after HSCT for SCID depends on the genetic cause, the use of pre-HSCT conditioning, and whether donor B-cell chimerism is achieved. Patients with defects in IL2RG or JAK3 undergoing HSCT without conditioning often have poor B-cell function post-HSCT, perhaps as a result of impairment of IL-21 signaling in host-derived B cells. To investigate the effect of pre-HSCT conditioning on B-cell function, and the relationship of in vitro B-cell function to clinical humoral immune status, we analyzed 48 patients with IL2RG/JAK3 SCID who were older than 2 years after HSCT with donors other than matched siblings. T follicular helper cells (TFH) developed in these patients with kinetics similar to healthy young children; thus, poor B-cell function could not be attributed to a failure of TFH development. In vitro differentiation of B cells into plasmablasts and immunoglobulin secretion in response to IL-21 strongly correlated with the use of conditioning, donor B-cell engraftment, freedom from immunoglobulin replacement, and response to tetanus vaccine. Patients receiving immunoglobulin replacement who had normal serum immunoglobulin M showed poor response to IL-21 in vitro, similar to those with low serum IgM. In vitro response of B cells to IL-21 may predict clinically relevant humoral immune function in patients with IL2RG/JAK3 SCID after HSCT.

Background Sickle cell disease (SCD) is a recessive monogenic disease caused by a single point mutation in which glutamic acid replaces valine in Codon 6 of the human beta-globin gene (HBB) leading to the production of abnormal globin chains (HbS) that polymerize and cause erythrocytes to sickle. This results in hemolytic anemia, vaso-occlusion and organ damage, which leads to lifelong complications and early mortality. Allogeneic hematopoietic stem cell transplant (allo-HSCT) is the only known cure for SCD, however, its use is limited by the lack of well-matched donors, need for immunosuppression, risk of graft versus host disease and graft rejection. GPH101 is an investigational, autologous, hematopoietic stem cell (HSC) drug product (DP) designed to correct the SCD mutation in the HBB gene ex vivo using a high fidelity Cas9 (CRISPR associated protein 9) paired with an AAV6 (adeno-associated virus type 6) delivery template, efficiently harnessing the natural homology directed repair (HDR) cellular pathway. This approach has the potential to restore normal adult hemoglobin (HbA) production while simultaneously reducing HbS levels. In preclinical studies, HBB gene correction in SCD donor HSCs resulted in ≥60% of gene-corrected alleles in vitro with minimal off-target effects. Gene corrected cells were successfully differentiated toward the erythroid lineage and produced ≥70% HbA in vitro. Long-term engraftment of gene-corrected HSCs was demonstrated in vivo, following transplant into immunodeficient mice, with multi-lineage allelic gene correction frequencies well above the predicted curative threshold of 20%, with potential of this approach to be equivalent or superior to allo-HSCT. In addition, HSC-based correction in an SCD mouse model led to stable adult hemoglobin production, increased erythrocyte lifespan and reduction in sickling morphology, demonstrating the therapeutic potential of this gene correction platform as a curative approach in SCD. Study Design and Methods CEDAR (NCT04819841) is a first-in-human, open-label, single-dose, multi-site Phase 1/2 clinical trial in participants with severe SCD designed to evaluate safety, efficacy and pharmacodynamics (PD) of GPH101. Approximately 15 adult (18-40 years) and adolescent (12-17 years) participants will be enrolled across 5 sites, with adolescent enrollment proceeding after a favorable assessment of adult safety data by a Safety Monitoring Committee. Participants must have a diagnosis of severe SCD (βS/βS), defined as ≥ 4 severe vaso-occlusive crises (VOCs) in the 2 years prior and/or ≥ 2 episodes of acute chest syndrome (ACS), in 2 years prior with at least 1 episode in the past year. Participants on chronic transfusion therapy may be eligible if required VOC and ACS criteria are met in the 2 years prior to the initiation of transfusions. Key exclusion criteria include availability of a 10/10 human leukocyte antigen-matched sibling donor, or prior receipt of HSCT or gene therapy. After eligibility confirmation including screening for pre-treatment cytogenetic abnormalities, participants will undergo plerixafor mobilization and apheresis, followed by CD34+ cell enrichment and cryopreservation, undertaken locally at each trial site before shipment to a centralized manufacturer for GPH101 production. After GPH101 release, participants will undergo eligibility reconfirmation prior to busulfan conditioning and DP infusion. Safety, efficacy and PD measurements will occur for 2 years post-infusion; a long-term follow up study will be offered to participants for an additional 13 years of monitoring. The primary endpoint for this study is safety, measured by the kinetics of HSC engraftment, transplant related mortality, overall survival and frequency and severity of adverse events. Secondary endpoints will explore efficacy and PD, including levels of globin expression as compared to baseline, gene correction rates, clinical manifestations of SCD (including VOC and ACS), laboratory parameters, complications and organ function. In addition, cerebral hemodynamics and oxygen delivery will be assessed by magnetic resonance techniques. Key exploratory endpoints include evaluation of patient-reported outcomes, erythrocyte function, on-target and off-target editing rates, and change from baseline in select SCD characteristics. Disclosures Kanter: Fulcrum Therapeutics, Inc.: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Forma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees; Beam: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Consultancy; GuidePoint Global: Honoraria; Fulcrum Tx: Consultancy. Thompson: Agios Pharmaceuticals: Consultancy; Graphite Bio: Research Funding; Vertex: Research Funding; Beam Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Biomarin: Research Funding; Baxalta: Research Funding; CRISPR Therapeutics: Research Funding; Global Blood Therapeutics: Current equity holder in publicly-traded company; bluebird bio: Consultancy, Research Funding; Novartis: Research Funding. Porteus: Versant Ventures: Consultancy; CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Intondi: Graphite Bio: Current Employment, Current equity holder in publicly-traded company; Global Blood Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Lahiri: Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Dever: Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Petrusich: bluebird bio: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Graphite Bio: Current Employment, Current equity holder in publicly-traded company. Lehrer-Graiwer: Global Blood Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Graphite Bio: Current Employment, Current equity holder in publicly-traded company.

Background: Hematopoietic stem cell graft manipulation strategies, such as αβT-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT), address the lack of matched donors and reduce the incidence of severe acute graft-versus-host disease (aGvHD). However, grade II-IV aGvHD still occurs in 25-30% of αβhaplo-HSCT recipients . Studies aimed at understanding the pathogenesis underlying aGVHD in αβhaplo-HSCT are lacking. We hypothesized that αβT cells adoptively transferred with the HSCT ( Methods: Six patients with hematologic malignancies receiving fully myeloablative αβhaplo-HSCT at Lucile Packard Children's Hospital, Stanford, between 08/2018 and 05/2020 were enrolled upon signing IRB approved informed consent. Three patients developed grade II-IV aGvHD, while three did not. Aliquot of the graft and of peripheral blood collected at the time of aGvHD onset or at corresponding time points for the patients who did not develop aGvHD, were analyzed. Single sorted CD4 + and CD8 + T cells were profiled by single-cell barcode chip assay from IsoPlexis system (IsoPlexis, Branford, CT) after stimulation with PMA (50 ng/mL) and Ionomycin (1mcg/mL). Following the Human Adaptive Immune Panel, cytokines from CD4 + and CD8 + single T cells were captured by fluorescence ELISA, which measured the numbers of cytokine-producing cells (secretion frequency) and numbers of cytokines produced by individual cells across five functional groups: effector, stimulatory, chemoattractive, regulatory and inflammatory (Table 1). Polyfunctionality was defined as the secretion of 2+ cytokines from each CD4 + and CD8 + T cell. The T cell polyfunctional strength Index (PSI) was defined as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Additional statistical analysis was performed using the Student's t test. Results: We compared the combinatorial cytokine secretion signature of individual CD4 + and CD8 + T cells isolated from grafts infused into patients, who eventually did or didn't develop aGvHD. We are comparing the signature of post-HSCT CD4 + and CD8 + T cells isolated from patients who did or did not develop aGvHD. Collectively, we considered three variables: cytokine secretion frequency, numbers of cytokines produced by individual cells and characteristics of the cytokines secreted (functional group) upon stimulation. Single-cell functional heterogeneity evaluated by t-Distributed Stochastic Neighbor Embedding (t-SNE), showed higher CD4 + and CD8 + T-cell polyfunctionality (up to 4+ cytokines) with effector and stimulatory dominant functions in the grafts of patients who developed aGvHD, compared to those who did not develop aGvHD (Fig1). The average PSI (driven by Granzyme B, TNF-α, IFN-γ, MIP-1β, IL2, and IL-8) was found to be higher in both CD4 + and CD8 + T cells from the grafts of patients who developed aGvHD (Fig 2). Combinatorial cytokine secretion analysis showed that T cells from grafts of patients who did not develop aGvHD had unique signatures with CD4 + T cells having the predominant cytokine secretion signature of IL2 and TNF-α, and CD8 + T cells having three predominant cytokine secretion signatures: IL2, IL8, TNF-α; MIP-1β, IL8; and MIP-1β, IFN-γ (Fig3). Conclusions: Preliminary data from αβhaplo-HSCT pediatric recipients obtained using IsoPlexis single-cell functional proteomics for CD4 + and CD8 + T cells showed that an increased donor T-cell polyfunctionality with a Th1 dominant functional phenotype may be predictive of an increased risk of aGvHD, while CD4 + and CD8 + T cells infused into patients who didn't develop aGvHD, had combinations with limited cytokine secretion signatures. Ongoing analysis suggest that polyfunctional CD8 + T cells present in the graft of patients who developed aGvHD, are present at the time of aGvHD initiation, while the polyfunctional CD4 + T cell are not present at the onset of aGvHD. Correlation with ongoing studies on circulating cytokines and clonotypic analysis of αβT cells infused with the graft will be crucial to elucidate the cross talking between the donor's immune system and recipient's inflammatory milieu. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

The importance of hematopoietic stem and progenitor cell (HSPC) dose in the outcome of hematopoietic stem cell transplant (HSCT) has been demonstrated by analyses of the threshold dose of CD34+ cells required to achieve donor engraftment, usually defined as endpoints of donor-derived neutrophil (PMN) or platelet (PLT) absolute numbers, and RBC transfusion independence. We recently described the heterogeneous HSPC composition of prospectively obtained umbilical cord blood samples and lack of correlation between CD34+ cell dose and the frequency of hematopoietic stem cells (HSC)(Mantri S et al, Blood Adv 2020). We describe here a pilot analysis of the relationship between HSPC graft composition and engraftment after HSCT. The study population is comprised of 17 children (3.4-22 years, median 13 years) treated with αβT/CD19B - cell depleted mobilized peripheral blood stem cell (PBSC) HSCT (αβhaplo-HSCT). Eight patients had acute lymphoblastic leukemia, seven had acute myeloid leukemia, and two myelodysplastic syndrome. All patients received serotherapy with Thymoglobulin and Rituximab, and a myeloablative conditioning consisting of either TBI 1200 cGy in combination with Fludarabine/Thiotepa (15 patients), Melphalan/Thiotepa (1 patient), or TBI (400 cGy) with Fludarabine/Thiotepa (1 patient receiving a second HSCT). Only 2/17 patients received 1-2 doses of G-CSF before Day+30. The CD34+ Lin- cells in the PBSC products were analyzed for HSPC composition, including HSC (CD38- CD90+ CD45RA-), Multipotent Progenitors (MPP, CD38- CD90- CD45RA-), Common Myeloid Progenitors (CMP, CD38+ CD123+ CD45RA-), Granulocyte-Monocyte Progenitors (GMP, CD38+ CD123+ CD45RA+), Megakaryocyte-Erythroid Progenitors (MEP, CD38+ CD123- CD45RA-) and Common Lymphoid Progenitors (CLP, CD38+ CD127+). Similar HSPC analyses were performed on marrow obtained at Days +30, +60, and +90 post-HSCT. The CD34+ cell dose in the grafts ranged from 8.5-40 x 10e6/kg recipient body weight (mean 15.6). The median time to Absolute Neutrophil Count (ANC) > 500 or 1000/mm 3 were Days +12 and +14, respectively, and for PLT > 50K or 100K/mm 3 Days +14 and +15, respectively. The time to either PMN or PLT engraftment did not correlate with the HSC dose. In contrast, the GMP dose was predictive of PMN engraftment: 7/8 patients receiving the highest GMP dose achieved ANC > 500 at 0-3 days before the median day of engraftment, while PMN engraftment was delayed by 1-5 days beyond the median in 6 of the 9 receiving the lowest GMP dose (χ 2 = 8.14, p=0.004)(Fig A). Of the patients with the highest MEP dose, 7/8 achieved PLT >50K/mm 3 0-4 days before the median, while 5/9 receiving the lowest dose engrafted 1-6 days beyond the median (χ 2 4.9, p=0.026) (Fig B). In the first 100 days post-HSCT, naïve CD4+ T-cells were all CD31+ CD45RA+ Recent Thymic Emigrants (RTE), indicating that they were newly produced T cells and not adoptively transferred from the αβhaplo-HSCT. The HSC dose did not correlate with the number of naïve CD4+ T cells until Day+180 (Fig C). Like PMN and PLT recovery, early T lymphoid recovery after HSCT was mainly derived from infused CLP, and likely switched to HSC-derived lymphopoiesis by Day +180. Consistent with this concept, HSC dose in the PBSC products was unrelated to the numbers of committed progenitors, e.g., CMP, MEP, CLP, in early (Day +30) post-transplant marrow samples. These data are consistent with clonal analyses of patients receiving lentiviral gene therapy and murine experiments demonstrating prolonged steady-state contribution of committed progenitors to peripheral blood cell maintenance (Biasco L et al, Cell Stem Cell 2016; Sun J et al, Nature 2014). While post-HSCT PMN or PLT numbers are frequently equated with "HSC engraftment" and naïve T-cell numbers with HSC-derived immune reconstitution, these early events reflect blood cell production by committed progenitors, which are variably present in the grafts. Although CD34+ cell dose is currently used to predict post-transplant engraftment and to qualify stem cell products for release, more accurate clinical predictions may be determined by the HSPC grafts' composition. Further, engineering of the progenitor composition of clinical HSCT products, e.g., co-transplantation of additional committed progenitors like GMP or CLP, could be strategically used to control post-transplant lymphohematopoietic recovery. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

Purpose/Objective(s) Late toxicity of treatment remains a major consideration among survivors of childhood leukemia. Pediatric patients with leukemia who receive fractionated total body irradiation (fTBI) as conditioning prior to stem cell transplantation (SCT) are frequently treated with a 4 Gy testicular boost in addition to fTBI to 12-13.2 Gy to reduce the risk of testicular relapse in this “sanctuary site.” However, total body irradiation can be associated with risk of impaired hormonal function; it is unclear if the 4 Gy testicular boost is needed to reduce risk of testicular relapse, and whether it contributes to the toxicity beyond that imparted by fTBI. This study investigated patients both with and without a boost for risk of recurrence, hormonal function, and fertility. Materials/Methods We retrospectively reviewed records of boys treated for leukemia with fTBI as part of conditioning for SCT at our institution from 1989-present. We included patients with both AML and ALL in our analysis. We excluded patients who died in the peri-transplant period. Cumulative incidence was tested using Gray's Test for Equality of Cumulative Incidence Functions. Endocrine outcomes were tested using chi square test. Results We identified 91 boys (age range 9 months – 22 years), 62 with ALL and 29 with AML. Median follow up was 43.4 months. In addition to fTBI to 12-13.2 Gy, 52 patients received a midplane testicular dose of 4 Gy (49 with ALL, 3 with AML), while 39 patients did not receive the testicular boost (13 with ALL, 26 with AML). There was no difference in time to first relapse or cumulative incidence of relapse between the boost and no-boost groups. There were 2 testicular relapses, one ALL patient who received boost and one AML patient who did not receive boost. The risk of abnormal luteinizing hormone (LH) was 65% in the boost group vs 26% in the non-boost group, P = 0.0063. Serum follicle-stimulating hormone, total testosterone, free testosterone, abnormal sperm studies did not differ between groups, nor did the percentage of patients who required endocrine or fertility treatments. We further investigated the subset of patients with ALL and similarly found no difference in risk of relapse between patients who had received a testicular boost and those who had not boost. Conclusion Omission of boost was associated with comparable risk of recurrence and improved endocrine outcomes, specifically LH levels. Our data suggest that omission of testicular boost in the fTBI program is oncologically safe and results in improved hormonal function.

The ability to accurately predict leukemic relapse post-HSCT would improve outcomes by allowing pre-emptive therapeutic strategies. Recent studies have identified post-transplant T- and CD34 cell chimerism as predictors of relapse in patients, who had undergone HSCT for hematologic malignancies (Preuner et al, 2016; Lee et al, 2015). However, these studies assess relapse risk looking at only a single threshold of chimerism using standard regression analysis, which permits only limited consideration of other patient variables. As the result, the findings of these analysis are frequently not applicable to patients generally. Machine learning methods offer the possibility to capture nonlinear relationships and simultaneous interactions between multiple variables, thus better recapitulate the dynamics and nuances of the relapse process in different patients. We use machine learning methods, specifically random forest classification (RF), to build a predictive model of post-transplant relapse and to analyze the data from a cohort of 46 pediatric patients, who received HSCT for acute lymphoblastic leukemia (ALL) and had serial lineage-specific chimerism testing post-transplant. Our model achieved 58 % sensitivity and 98% specificity at predicting relapses in cross validation compared to a baseline model (24% sensitivity, 76% specificity). Consistent with previous reports, our model implicates both peripheral blood (PB) donor CD34 and CD3 chimerism as important variables for relapse. More importantly, the RF showed how different variables interacted with each other, providing additional insights into how to best interpret post-transplant chimerism results. To our knowledge, this is the first study featuring RF machine learning methods in the clinical setting of relapse after HSCT. We use a dataset of patients with ALL undergoing HSCT at Lucile Packard Children's Hospital from 2012 to 2018. Variables collected are summarized in Table 1. The analytical sensitivity of STR-based chimerism testing is 1%. Chimerism results on the same day of relapse were excluded from the analysis. The RF model is based on a set of 500 individual decision trees, each based on a bootstrapped sample of the patient data. A 5-fold cross-validation was used to test predictive skill, with 20% of patients excluded from each fold. We compared results with a Monte Carlo baseline model in which relapse status was repeatedly assigned randomly to each patient with a probability based on the prevalence of relapse in our cohort. Patients, transplantation, and relapse characteristics are summarized in Table 2. Chimerism data are summarized in Table 3. The cross-validation results show a robust predictive skill of relapse within 2 years post-transplant. Our RF achieved 58% sensitivity and 98% specificity, greatly improving the predictive values from the base model (Table 4). Variable importance, the ability of a variable to decrease the error of the prediction model, was calculated for all variables used in our RF (Figure 1). Our analysis shows that the age at the time of transplant has the highest importance, followed by PB donor CD34 chimerism. Bone marrow chimerism generally has lower importance suggesting PB monitoring only is adequate in the clinical setting. We showcase the relationships of 1) age at transplant, 2) donor PB CD34, and 3) donor PB CD3 chimerism to the odds of relapse using a partial dependence plot. Younger patients relapse less often. Donor PB CD34 chimerism exhibits a threshold effect, in which the odds of relapse dramatically decreases when it is above 95% while donor PB CD3 chimerism has a more gradual linear profile (Figure 2). 2D dependence plot of donor PB CD34 and PB CD3 chimerism shows the interaction of the two variables (Figure 3) as continuous variables; relapse risk remaining low with even if donor PB CD3 chimerism is as low as 50% as long as donor PB CD34 chimerism is > 95%. Our study shows that machine learning methods such as RF can be very useful at making accurate predictive model of post-HSCT complications that incorporates multiple variables, allowing for more granular differentiation between different patients. Such analyses can enable more effective deployment of risk-adapted, personalized treatment. By building hundreds of independent decision trees, the RF is also able provide useful insights to the interaction between different variables in a clinically relevant manner. Disclosures No relevant conflicts of interest to declare.

Background With >36 years of data collected, PIDTC prospective (6901) and retrospective (6902) natural history studies provide an unprecedented opportunity to study hematopoietic cell transplantation (HCT) outcomes for SCID over time. Methods Patients in this 6901/6902 analysis met PIDTC diagnostic criteria for SCID and underwent HCT. Categorial variables were analyzed between decades (a) 1982-89 (b) 1990-99 (c) 2000-09 (d) 2010-18 using the chi-square test. Continuous outcomes were compared using the Kruskal-Wallis test. Kaplan-Meier method was used for estimates of overall survival (OS). Results 896 children with typical (n=742) and atypical (n=154) SCID requiring HCT between 1982-2018 were enrolled. Diagnosis of SCID for reasons other than family history or newborn screening was common (60%) in early cohorts (a-c) dropping to 30% in cohort (d). Distribution of SCID genotypes changed over time (Fig 1), and novel/unknown genotypes also decreased from (a) 53% to (d) 13%, p Conclusion This longitudinal data set, spanning the implementation of NBS and advancements in diagnostics and supportive care, highlights improved OS after HCT for SCID, including alternative donors. Exploration of decreased toxicity approaches that maintain high OS and engraftment are warranted.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients 95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.)

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for pediatric patients with high risk or refractory leukemia. In the absence of related or unrelated HLA-matched donors, alternative approaches such as HLA-haploidentical HSCT have been implemented. Our group has developed one such approach, αβ T-cell/CD19 B-cell depletion (αβhaplo-HSCT), and demonstrated its clinical efficacy (Bertaina A, Blood 2018). In αβhaplo-HSCT, anti-thymocyte globulin (ATG) is used for preventing graft rejection and graft-versus-host disease (GvHD). However, the optimal dose still needs to be elucidated. Here, we present the first analysis comparing 2 different Thymoglobulin® ATG doses: one fixed at 3.75 mg/Kg, as established in previous European studies with the closely related ATG Grafalon®, and one based on a newly developed algorithm that also integrates absolute lymphocyte count (ALC). METHODS: Between March 2017 and April 2020, 27 pediatric patients (median age 12 years) with hematological malignancies were transplanted at Stanford University's Lucile Packard Children's Hospital. Importantly, 60% of these patients were in CR2 or greater and 2/27 had active disease at the time of HSCT. All children received a fully myeloablative conditioning regimen. On days -9/-8/-7 before αβhaplo-HSCT, patients were given a regimen of Thymoglobulin® ATG. No patient received post-HSCT pharmacological GvHD prophylaxis. The fixed dosage was given to 14 patients in cohort 1 (ATG: 3.75 mg/Kg) and the novel ALC-based dosage to 13 in cohort 2 (ATG ranging between 3-6 mg/Kg). All patients enrolled in this study (BMT Protocol 179 and 351 approved from our IRB) had a minimum follow-up of 100 days, evaluated at Day 30 and 90. Following Admiraal R (Lancet Hematology 2015), we defined CD4 immune reconstitution (IR) as more than 50 CD4 T-cells/ul twice within the first 100 days after HSCT. PBMC were enriched by Ficoll-Hypaque (Sigma Aldrich) density gradient centrifugation. Flow analyses were performed on fresh cells resuspended in PBS 2% FBS on Cytek DxP 10 flow-cytometer. At least 5x104 events of total cells were acquired and analyzed using FloJo software. RESULTS: With a median follow-up of 555 and 124 days for cohort 1 and 2 respectively, 12 patients (85.7%) in cohort 1 and 7 patients (54%) in cohort 2 achieved CD4 immune reconstitution. In cohort 2, CD3 αβ T cells were significantly lower at both Day 30 and Day 90 (P=0.0003, Figure 1A). At Day 90, both the CD4 and CD8 subpopulations were significantly depressed (P=0.01 and P=0.056, respectively, Figure 1C). In both subpopulations, the memory compartment was the most reduced (Figure 1D). The absolute numbers of CD3 γδ T cells did not differ between the cohorts at either Day 30 or 90 (Figure 1B). Viral reactivations were higher in cohort 1 (10/14, 71%) than in cohort 2 (6/13, 46%, P=NS). Half of the reactivations were CMV reactivations, but no patient developed organ disease. There was no statistically significant difference in overall survival and the incidence of relapse in the two cohorts. Three patients developed grade III-IV aGvHD: 2 in cohort 1 (14%) and 1 in cohort 2 (8%, P=NS). Remarkably, the only patient, who developed grade IV aGvHD in cohort 2, did not experience symptoms until Adenovirus reactivation 138 days after HSCT. CONCLUSION: Our analysis confirms that the optimal dose of ATG Thymoglobulin® before αβhaplo-HSCT remains elusive. There were no significant clinical differences between the 2 ATG regimens. However, the ALC-based regimen resulted in the more pronounced reduction of donor-derived memory T cells. Our analysis suggests two intriguing explanations for the observed pattern of results. First, the selective depletion of the memory compartment in both CD4 and CD8 T cells may well be due to a priming effect of ATG Thymoglobulin® on the few αβ T cells left over in the graft. Second, the equivalent reconstitution of naive T cells in the 2 cohorts is likely because the ATG has no impact on the thymus-dependent IR. Remarkably, in our overall cohort, 70% of the patients achieved CD4 IR by 90 days after αβhaplo-HSCT. This result is superior to the best results from other ex vivo T-cell depleted approaches 54%, recently reported by Van Roessel (Cytotherapy 2020). In vivo studies of the pharmacokinetics of ATG in αβhaplo-HSCT recipients and a comparison with the use of Grafalon® are required to shed more light on this crucial topic. Disclosures No relevant conflicts of interest to declare.

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

In a 250 patient cohort from the US and Canada in the current era (2010–2018), we show that over 90% of patients with severe combined immunodeficiency (SCID) can be genetically-characterized.

Ruxolitinib was recently approved as a treatment for steroid refractory acute GVHD (aGVHD) and is used in adults for the treatment of chronic GVHD (cGVHD). As a JAK-1/JAK-2 inhibitor, it reduces T-cell proliferation and trafficking and leads to a potent anti-inflammatory effect. There is limited data of its use in pediatric and adolescent/young adult (AYA) patients. We report our single center experience with ruxolitinib in this population. Data on patients treated with ruxolitinib at Lucile Packard Children's Hospital between 8/1/18 and 8/31/19 were retrospectively analyzed after institutional IRB approval. aGVHD was graded according to the Modified Glucksberg scale, while cGVHD was scored as limited or extensive based on Seattle criteria. Only patients on ruxolitinib for ≥14 days were evaluated for response, which was defined as a reduction by at least one grade in severity of aGVHD. Ability to reduce other GVHD medications, especially steroids, and/or symptomatic improvement was considered a response in cGVHD. 15 patients (median age 18, range: 11-29 years) received ruxolitinib: 5 for aGVHD and 10 for cGVHD. Standard starting dose was 5 mg twice a day. Patients in the aGVHD group were steroid refractory with ≥ Grade III GVHD and had received a median of 3 (range: 2-5) different agents prior to ruxolitinib. Patients received treatment for a median of 46 days (range: 23-332). 4 of 5 patients had a partial response (PR) with one non-responder, for an overall response rate (ORR) of 80%. All patients experienced at least one episode of infection- CMV reactivation (n=2), adenovirus (n=1), viral gastroenteritis (n=1), bacterial infections (n=3) and probable fungal infection (n=1). Dose-limiting cytopenia was the only toxicity observed in 3 patients (60%), requiring dose reduction or discontinuation. 2 patients had TRM, both related to GVHD and infections. 10 patients treated for cGVHD (6 limited, 4 extensive) had received a median of 3 (range: 2-6) different agents prior to ruxolitinib. One patient required early cessation of therapy due to a severe allergic reaction and in another patient ruxolitinib was stopped due to leukemia relapse. Of the eight patients evaluable for response, treatment duration was for a median of 195 days (range: 59-372) with an ORR of 88%. 5 (63%) patients developed infections: CMV reactivation (n=2), respiratory infections (n=4), bacterial infection (n=1). Only one patient required dose reduction due to GI side effects and cytopenias. Ruxolitinib is an effective salvage therapy for severe GVHD. It was well tolerated in our patient population, with myelosuppression as the dose limiting toxicity, especially in aGHVD patients. A high rate of infections was noted during ruxolitinib treatment and concomitant antimicrobial prophylaxis is strongly recommended.

The leukodystrophies are a heterogeneous group of inherited diseases characterized by progressive demyelination of the central nervous system leading to devastating neurologic symptoms and premature death. Hematopoietic stem cell transplantation (HSCT) has been successfully used to treat certain leukodystrophies, including adrenoleukodystrophy, globoid leukodystrophy (Krabbe disease), and metachromatic leukodystrophy, over the past 30 years. To date, these complex patients have primarily been transplanted at a limited number of pediatric centers. As the number of cases identified through pregnancy and newborn screening is increasing, additional centers will be required to treat these children. Hunter's Hope created the Leukodystrophy Care Network in part to create and standardize high-quality clinical practice guidelines to guide the care of affected patients. In this report the clinical guidelines for the care of pediatric patients with leukodystrophies undergoing treatment with HSCT are presented. The initial transplant evaluation, determination of patient eligibility, donor selection, conditioning, supportive care, and post-transplant follow-up are discussed. Throughout these guidelines the need for early detection and treatment and the role of the partnership between families and multidisciplinary providers are emphasized.

Inherited leukodystrophies are a group of diseases affecting central nervous system myelin that lead to death or significant health problems. Although for most leukodystrophies there are no curative treatments, for a handful of diseases hematopoietic stem cell transplantation (HSCT; bone marrow transplant) can stop disease progression, and if initiated in a timely fashion, prevent many or all neurologic and other systems involvement. However, HSCT is a complex procedure with significant morbidity and mortality risks. The study goal was to determine whether HSCT was being more widely used outside of those leukodystrophies for which HSCT is typically employed. The authors conducted a 2-year retrospective review of HSCT performed across the United States in 51 children’s hospitals that are part of the Pediatric Health Information System. The authors screened for 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) codes for leukodystrophies in which HSCT is “nonstandard,” including sphingolipidoses, Fabry disease, Gaucher disease, and Niemann-Pick disease, and excluded patients who had ICD-10 codes for leukodystrophies that are HSCT candidates, specifically X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease, and Hurler disease. The authors identified 91 patients (from a total cohort of 937) with one of the nonstandard leukodystrophies who had HSCT. HSCT was performed at 20 of the hospitals, with the majority performed at only 6 hospitals. Average costs ($786 846) per patient were more than 6 times higher than patients who did not have HSCT. The data show that an unexpectedly large number of leukodystrophy patients are receiving transplants for conditions in which HSCT is not typically used, and which are associated with high medical costs.

The use of machine learning (ML) and deep learning (DL) methods in hematology includes diagnostic, prognostic, and therapeutic applications. This increase is due to the improved access to ML and DL tools and the expansion of medical data. The utilization of ML remains limited in clinical practice, with some disciplines further along in their adoption, such as radiology and histopathology. In this review, we discuss the current uses of ML in diagnosis in the field of hematology, including image-recognition, laboratory, and genomics-based diagnosis. Additionally, we provide an introduction to the fields of ML and DL, highlighting current trends, limitations, and possible areas of improvement.

Background The prognosis of children who relapse after allogeneic hematopoietic cell transplant (alloHCT) for myeloid malignancies remains poor. Procedure To describe the safety and feasibility of post-transplant azacitidine for relapse prevention, we retrospectively reviewed the charts of 18 children undergoing alloHCT for myeloid malignancies. Results There were 15 evaluable patients since three patients did not receive planned azacitidine due to early relapse or TRM. Azacitidine (32 mg/m2 /dose for 5 days, in 28-day cycles as tolerated up to 1 year post-transplant) was started at a median of 66 days post-transplant (range 42-118). Two-thirds (10/15) of patients received eight or more cycles. Five patients stopped therapy early, only one attributable to toxicity. Mild myelosuppression was the most common reason for cycle delays. Dose modifications were made in three patients. There were three relapses, two of which occurred in patients in CR2 and one in CR1, with a median follow-up of 20 months (range 12.5-28), and no TRM in patients who received azacitidine. Conclusions Post-transplant azacitidine in children is safe and feasible, with most patients successfully receiving all planned cycles. Despite the limitations of a small cohort, low relapse incidence suggests a potential benefit in disease control that warrants further investigation.

Relapse remains the most common cause of treatment failure after hematopoietic cell transplantation for acute myeloid leukemia. Inability to achieve hematologic complete remission has been a barrier to transplant for patients with refractory disease. We describe six children with refractory myeloid disease undergoing transplant in chemotherapy-induced aplasia, as a strategy to facilitate curative therapy in refractory patients. Clofarabine- or high-dose cytarabine-based chemotherapy regimens were used to achieve marrow aplasia, followed by reduced-intensity conditioning and allogeneic transplant before hematologic recovery. Long-term disease control was achieved in five, with one transplant-related mortality, suggesting the feasibility of this approach.

Previous studies of 5,10,15,20-tetraarylporphyrins have shown that the barrier for meso aryl-porphyrin rotation (DeltaG++(ROT)) varies as a function of the core substituent M and is lower for a small metal (M = Ni) compared to a large metal (M = Zn) and for a dication (M = 4H(2+)) versus a free base porphyrin (M = 2H). This has been attributed to changes in the nonplanar distortion of the porphyrin ring and the deformability of the macrocycle caused by the core substituent. In the present work, X-ray crystallography, molecular mechanics (MM) calculations, and variable temperature (VT) (1)H NMR spectroscopy are used to examine the relationship between the aryl-porphyrin rotational barrier and the core substituent M in some novel 2,3,5,7,8,10,12,13,15,17,18,20-dodecaarylporphyrins (DArPs), and specifically in some 5,10,15,20-tetraaryl-2,3,7,8,12,13,17,18-octaphenylporphyrins (TArOPPs), where steric crowding of the peripheral groups always results in a very nonplanar macrocycle. X-ray structures of DArPs indicate differences in the nonplanar conformation of the macrocycle as a function of M, with saddle conformations being observed for M = Zn, 2H or M = 4H(2+) and saddle and/or ruffle conformations for M = Ni. VT NMR studies show that the effect of protonation in the TArOPPs is to increase DeltaG++(ROT), which is the opposite of the effect seen for the TArPs, and MM calculations also predict a strikingly high barrier for the TArOPPs when M = 4H(2+). These and other findings suggest that the aryl-porphyrin rotational barriers in the DArPs are closely linked to the deformability of the macrocycle along a nonplanar distortion mode which moves the substituent being rotated out of the porphyrin plane.

With the aim of better understanding the electronic and structural factors which govern electron-transfer processes in porphyrins, the electrochemistry of 29 nickel(II) porphyrins has been examined in dichloromethane containing either 0.1 M tetra-n-butylammonium perchlorate (TBAP) or tetra-n-butylammonium hexafluorophosphate (TBAPF(6)) as supporting electrolyte. Half-wave potentials for the first oxidation and first reduction are only weakly dependent on the supporting electrolyte, but E(1/2) for the second oxidation varies considerably with the type of supporting electrolyte. E(1/2) values for the first reduction to give a porphyrin pi-anion radical are effected in large part by the electronic properties of the porphyrin macrocycle substituents, while half-wave potentials for the first oxidation to give a pi-cation radical are affected by the substituents as well as by nonplanar deformations of the porphyrin macrocycle. The potential difference between the first and second oxidations (Delta/Ox(2) - Ox(1)/) is highly variable among the 29 investigated compounds and ranges from 0 mV (two overlapped oxidations) to 460 mV depending on the macrocycle substituents and the anion of the supporting electrolyte. The magnitude of Delta/Ox(2) - Ox(1)/ is generally smaller for compounds with very electron-withdrawing substituents and when TBAP is used as the supporting electrolyte. This behavior is best explained in terms of differences in the binding strengths of anions from the supporting electrolyte (ClO(4)(-) or PF(6)(-)) to the doubly oxidized species. A closer analysis suggests two factors which are important in modulating Delta/Ox(2) - Ox(1)/ and thus the binding affinity of the anion to the porphyrin dication. One is the type of pi-cation radical (a proxy for the charge distribution in the dication), and the other is the conformation of the porphyrin macrocycle (either planar or nonplanar). These findings imply that the redox behavior of porphyrins can be selectively tuned to display separate or overlapped oxidation processes.