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BackgroundWe explored the clinical significance of the residual hematoma cavity 1 year after minimally invasive intracerebral hemorrhage (ICH) evacuation.MethodsPatients presenting with spontaneous supratentorial ICH were evaluated for minimally invasive surgical evacuation. Inclusion criteria included age ≥18 years, preoperative hematoma volume (Hv) ≥15 mL, presenting National Institutes of Health Stroke Scale score ≥6, and premorbid modified Rankin Scale (mRS) score ≤3. Patients with longitudinal CT scans at least 3 months after evacuation were included in the study. Remnant cavity volumes (Cv) after evacuation were computed using semi-automatic volumetric segmentation software. Relative cavity volume (rCv) was defined as the ratio of the preoperative Hv to the remnant Cv.Results108 patients with a total of 484 head CT scans were included in the study. The median postoperative Cv was 2.4 (IQR 0.0–11) mL, or just 6% (0–33%) of the preoperative Hv. The median residual Cv on the final head CT scan a median of 13 months (range 11–27 months) after surgery had increased to 9.4 (IQR 3.1–18) mL, or 25% (10–60%) of the preoperative Hv. rCv on the final head CT scan was negatively associated with measures of operative success including evacuation percentage, postoperative Hv ≤15 mL, and decreased time from ictus to evacuation. rCv on the final head CT scan was also associated with a worse 6-month functional outcome (β per mRS point 17.6%, P<0.0001; area under the receiver operating characteristic curve 0.91).ConclusionAfter minimally invasive ICH evacuation the hematoma lesion decompresses significantly, with a residual Cv just 6% of the original lesion, but then gradually increases in size over time. Early and high percentage ICH evacuation may reduce the remnant Cv over time which, in turn, is associated with improved functional outcomes.

BackgroundDiffusion-weighted imaging (DWI) lesions have been linked to poor outcomes after intracerebral hemorrhage (ICH). We aimed to assess the impact of cerebral digital subtraction angiography (DSA) on the presence of DWI lesions in patients who underwent minimally invasive surgery (MIS) for ICH.MethodsRetrospective chart review was performed on ICH patients treated with MIS in a single health system from 2015 to 2021. One hundred and seventy consecutive patients who underwent postoperative MRIs were reviewed. Univariate analyses were conducted to determine associations. Variables with p<0.05 were included in multivariate analyses.ResultsDWI lesions were present in 88 (52%) patients who underwent MIS for ICH. Of the 83 patients who underwent preoperative DSA, 56 (67%) patients demonstrated DWI lesions. In this DSA cohort, older age, severe leukoaraiosis, larger preoperative hematoma volume, and increased presenting National Institutes of Health Stroke Score (NIHSS) were independently associated with DWI lesion identification (p<0.05). In contrast, of 87 patients who did not undergo DSA, 32 (37%) patients demonstrated DWI lesions on MRI. In the non-DSA cohort, presenting systolic blood pressure, intraventricular hemorrhage, and NIHSS were independently associated with DWI lesions (p<0.05). Higher DWI lesion burden was independently associated with poor modified Rankin Scale (mRS) at 6 months on a univariate (p=0.02) and multivariate level (p=0.02).ConclusionsIn this cohort of ICH patients who underwent minimally invasive evacuation, preprocedural angiography was associated with the presence of DWI lesions on post-ICH evacuation MRI. Furthermore, the burden of DWI lesions portends a worse prognosis after ICH.

BackgroundMinimally invasive evacuation may help ameliorate outcomes after intracerebral hemorrhage (ICH). However, hospital length of stay (LOS) post-evacuation is often long and costly.ObjectiveTo examine factors associated with LOS in a large cohort of patients who underwent minimally invasive endoscopic evacuation.MethodsPatients presenting to a large health system with spontaneous supratentorial ICH qualified for minimally invasive endoscopic evacuation if they met the following inclusion criteria: age ≥18, premorbid modified Rankin Scale (mRS) score ≤3, hematoma volume ≥15 mL, and presenting National Institutes of Health Stroke Scale (NIHSS) score ≥6. Demographic, clinical, radiographic, and operative characteristics were included in a multivariate logistic regression for hospital and ICU LOS dichotomized into short and prolonged stay at 14 and 7 days, respectively.ResultsAmong 226 patients who underwent minimally invasive endoscopic evacuation, the median intensive care unit and hospital LOS were 8 (4–15) days and 16 (9–27) days, respectively. A greater extent of functional impairment on presentation (OR per NIHSS point 1.10 (95% CI 1.04 to 1.17), P=0.007), concurrent intraventricular hemorrhage (OR=2.46 (1.25 to 4.86), P=0.02), and deep origin (OR=per point 2.42 (1.21 to 4.83), P=0.01) were associated with prolonged hospital LOS. A longer delay from ictus to evacuation (OR per hour 1.02 (1.01 to 1.04), P=0.007) and longer procedure time (OR per hour 1.91 (1.26 to 2.89), P=0.002) were associated with prolonged ICU LOS. Prolonged hospital and ICU LOS were in turn longitudinally associated with a lower rate of discharge to acute rehabilitation (40% vs 70%, P<0.0001) and worse 6-month mRS outcomes (5 (4–6) vs 3 (2–4), P<0.0001).ConclusionsWe present factors associated with prolonged LOS, which in turn was associated with poor long-term outcomes. Factors associated with LOS may help to inform patient and clinician expectations of recovery, guide protocols for clinical trials, and select suitable populations for minimally invasive endoscopic evacuation.

Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing to the risk of infections and malignant relapse. Restoration of T-cell immunity depends on tissue regeneration in the thymus, the primary site of T-cell development, although the capacity of the thymus to repair itself diminishes over its lifespan. However, although boosting thymic function and T-cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that zinc (Zn) is critically important for both normal T-cell development and repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

Supplemental Digital Content is available in the text.Thiopurines [e.g. 6-mercaptopurine (6MP)] are essential for the cure of acute lymphoblastic leukemia (ALL) but can cause dose-limiting hematopoietic toxicity. Germline variants in drug-metabolizing enzyme genes TPMTand NUDT15have been linked to the risk of thiopurine toxicity. However, the full spectrum of genetic polymorphism in these genes and their impact on the pharmacological effects of thiopurines remain unclear. Herein, we comprehensively sequenced the TPMTand NUDT15genes in 685 children with ALL from the Children’s Oncology Group AALL03N1 trial and evaluated their association with 6MP dose intensity. We identified 6 and 5 coding variants in TPMTand NUDT15respectively, confirming the association at known pharmacogenetic variants. Importantly, we discovered a novel gain-of-function noncoding variants in TPMTassociated with increased 6MP tolerance (rs12199316), with independent validation in 380 patients from the St. Jude Total Therapy XV protocol. Located adjacent to a regulatory DNA element, this intergenic variant was strongly associated TPMTtranscription, with the variant allele linked to higher expression (P= 2.6 × 10−9). For NUDT15, one noncoding common variant, rs73189762, was identified as potentially related to 6MP intolerance. Collectively, we described pharmacogenetic variants in TPMTand NUDT15associated with thiopurine sensitivity, providing further insights for implementing pharmacogenetics-based thiopurine individualization.

Inherited noncoding genetic variants confer significant disease susceptibility to childhood acute lymphoblastic leukemia (ALL) but the molecular processes linking germline polymorphisms with somatic lesions in this cancer are poorly understood. Through targeted sequencing in 5,008 patients, we identified a key regulatory germline variant in GATA3associated with Philadelphia chromosome-like ALL (Ph-like ALL). Using CRISPR–Cas9 editing and samples from patients with Ph-like ALL, we showed that this variant activated a strong enhancer that upregulated GATA3transcription. This, in turn, reshaped global chromatin accessibility and three-dimensional genome organization, including regions proximal to the ALL oncogene CRLF2. Finally, we showed that GATA3directly regulated CRLF2and potentiated the JAK–STAT oncogenic effects during leukemogenesis. Taken together, we provide evidence for a distinct mechanism by which a germline noncoding variant contributes to oncogene activation, epigenetic regulation and three-dimensional genome reprogramming.

Glucocorticoids (GCs) are a mainstay of contemporary, multidrug chemotherapy in the treatment of childhood acute lymphoblastic leukemia (ALL), and resistance to GCs remains a major clinical concern. Resistance to GCs is predictive of ALL relapse and poor clinical outcome, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in resistance. To address this, we mapped the gene regulatory response to GCs in two ALL cell lines using functional genomics and high-throughput reporter assays and identified thousands of GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and a depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. By integrating our GC response maps with genetic and epigenetic datasets in primary ALL cells from patients, we further uncovered cis-regulatory disruptions at GC-responsive genes that impact GC resistance in childhood ALL. Overall, these data indicate that GCs initiate pervasive effects on the leukemia epigenome, and that alterations to the GC gene regulatory network contribute to GC resistance.

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy comprised of molecular subtypes largely characterized by aneuploidy or recurring chromosomal rearrangements. Despite extensive information on the ALL transcriptome and methylome, there is limited understanding of the ALL chromatin landscape. We therefore mapped accessible chromatin in 24 primary ALL cell biospecimens comprising three common molecular subtypes (DUX4/ERG, ETV6-RUNX1 and hyperdiploid) from patients treated at St. Jude Children’s Research Hospital. Our findings highlight extensive chromatin reprogramming in ALL, including the identification ALL subtype-specific chromatin landscapes that are additionally modulated by genetic variation. Chromatin accessibility differences between ALL and normal B-cells implicate the activation of B-cell repressed chromatin domains and detail the disruption of normal B-cell development in ALL. Among ALL subtypes, we uncovered roles for basic helix-loop-helix, homeodomain and activator protein 1 transcription factors in promoting subtype-specific chromatin accessibility and distinct gene regulatory networks. In addition to chromatin subtype-specificity, we further identified over 3500 DNA sequence variants that alter the ALL chromatin landscape and contribute to inter-individual variability in chromatin accessibility. Collectively, our data suggest that subtype-specific chromatin landscapes and gene regulatory networks impact ALL biology and contribute to transcriptomic differences among ALL subtypes.

Vitamin D receptor (VDR) has been implicated in fatty liver pathogenesis, but its role in the regulation of organismal energy usage remains unclear. Here, we illuminate the evolutionary function of VDR by demonstrating that zebrafish Vdr coordinates hepatic and organismal energy homeostasis through antagonistic regulation of nutrient storage and tissue growth. Hepatocyte-specific Vdr impairment increases hepatic lipid storage, partially through acsl4ainduction, while simultaneously diminishing fatty acid oxidation and liver growth. Importantly, Vdr impairment exacerbates the starvation-induced hepatic storage of systemic fatty acids, indicating that loss of Vdr signaling elicits hepatocellular energy deficiency. Strikingly, hepatocyte Vdr impairment diminishes diet-induced systemic growth while increasing hepatic and visceral fat in adult fish, revealing that hepatic Vdr signaling is required for complete adaptation to food availability. These data establish hepatocyte Vdr as a regulator of organismal energy expenditure and define an evolutionary function for VDR as a transcriptional effector of environmental nutrient supply.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the ERGgene (rs2836365; P= 3.76 × 10−8; odds ratio [OR] = 1.56), with independent validation (P= .01; OR = 1.43). Imputation analyses pointed to a single causal variant driving the association signal at this locus overlapping with putative regulatory DNA elements. The effect size of the ERGrisk variant rose with increasing Native American genetic ancestry. The ERGrisk genotype was underrepresented in ALL with the ETV6-RUNX1fusion (P< .0005) but enriched in the TCF3-PBX1subtype (P< .05). Interestingly, ALL cases with germline ERGrisk alleles were significantly less likely to have somatic ERGdeletion (P< .05). Our results provide novel insights into genetic predisposition to ALL and its contribution to racial disparity in this cancer.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the ERG gene (rs2836365; P = 3.76 × 10-8; odds ratio [OR] = 1.56), with independent validation (P = .01; OR = 1.43). Imputation analyses pointed to a single causal variant driving the association signal at this locus overlapping with putative regulatory DNA elements. The effect size of the ERG risk variant rose with increasing Native American genetic ancestry. The ERG risk genotype was underrepresented in ALL with the ETV6-RUNX1 fusion (P < .0005) but enriched in the TCF3-PBX1 subtype (P < .05). Interestingly, ALL cases with germline ERG risk alleles were significantly less likely to have somatic ERG deletion (P < .05). Our results provide novel insights into genetic predisposition to ALL and its contribution to racial disparity in this cancer.

The success of T cell immunotherapies in the solid tumor is impeded by challenges in part created by the secretion of metabolic byproducts from the tumor, which generate a hostile and immunosuppressive tumor microenvironment (TME) (Tan et al., 2021). Dysregulated tumor metabolism results in the accumulation of metabolites that are exported into the interstitial space in the tumor, where they act as signaling molecules promoting the immunosuppressive landscape, ultimately reducing anti-tumor responses to T cell therapies (Jiang et al., 2020; Joyce & Fearon, 2015). Tumor cells exhibit several advantages through their high metabolic plasticity, but metabolic aggression deprives surrounding immune cells of nutrients, leading to exhausted, non-functional, and suppressive phenotypes (Lim et al., 2020). Regulatory T cells (Tregs) are a central immunosuppressive component of the TMEthat can rewire their metabolism and obstruct anti-tumor immunity by suppressing proliferation and activation of CD8+ T cells and CD4+ helper T cells in the TME (Betts et al., 2012; McNally et al., 2011). High Treg accumulation is associated with reduced anti-tumor responses, reduced efficacy of TCR therapies and poor clinical outcomes (Onda et al., 2019; Preston et al., 2013; Tang et al., 2014). Therefore, there is an imminent clinical need for the development of more synergistic therapies that specifically target Tregs in the TME to successfully overcome the barriers of infiltration and function of antigen-specific T cells.

ABSTRACTThe OECD’s 2016 ‘Neurotechnology and Society: Strengthening Responsible Innovation in Brain Science’ International Workshop drew together scientists, policy-makers, industries, regulators, and academics for the purpose of discussing the present state of neurotechnology and exchanging ideas on how RRI can, and should, play a role in the rapidly evolving field. A myriad of technologies and devices were discussed during the workshop, but one area of neurotechnology received considerable attention: transcranial direct current stimulation (tDCS). In this discussion piece, I describe what tDCS technology is, give a brief overview of its history, introduce a number of scientific studies that suggest the efficacy of the technology and, most importantly, set out some of the concerns I have for its use. The piece concludes by making the argument that we, members of the neuroscience community, have an ethical duty to become more involved in this community so that we may help guide these people and potentially prevent any unintended brain damage.

Aim:Identify the functional status of the uridine-diphosphate glucuronyl transferase 1A1 (UGT1A1) -3279T>G (60) variant. Materials & methods:Retrospective review of clinically obtained serum bilirubin concentrations in pediatric patients to evaluate the association of the UGT1A1-3279T>G (60) variant with bilirubin concentrations and assessed linkage disequilibrium of the UGT1A1-3279T>G (60) and A(TA)7TAA (28) variants. Results:Total bilirubin concentration did not differ between patients who had a UGT1A11/1diplotype and patients homozygous for the UGT1A1-3279T>G (60/60) variant. Total bilirubin concentration was lower in patients homozygous for the UGT1A1-3279T>G (60/60) variant than in patients homozygous for the UGT1A1A(TA)7TAA (28/28) variant (p < 0.01). The -3279T>G (60) and A(TA)7TAA (28) variants were in strong incomplete linkage disequilibrium in both black and white patients. Conclusion:The presence of the UGT1A1-3279T>G (60) variant is not associated with increased bilirubin concentrations.

Introduction:Computed tomography perfusion (CTP) characterizes hemodynamic changes within brain tissue, particularly after stroke. This study aims to quantify cerebral blood volume (CBV) changes and identify predictors of CBV increase in the pericavity parenchyma after minimally invasive intracerebral hemorrhage evacuation (MIS for ICH).Methods:Thirty-two patients underwent MIS for ICH with pre/postoperative native CT imaging and intraoperative perfusion imaging (DynaCT PBV Neuro, Artis Q, Siemens). Scans were segmented using ITK-SNAP software to calculate hematoma volumes pre/post-evacuation and to delineate the pericavity tissue. Helical CT segmentations were registered to cone beam CT data using elastix software. Mean CBVs were computed inside subvolumes by dilating the segmentations with spheres with varying diameters.Results:In 27 patients with complete imaging, CTP analysis demonstrated significant increases in CBV from the 6 mm to 20 mm pericavity regions. CBV increased on average 32.7% from 2.27 mL/100mg (IQR 1.87-3.02) to 2.80 mL/100mg (2.41-3.61) in the 10 mm pericavity region (P=0.003). Factors associated with increased CBV in the univariate analysis (P≤0.10) included age (P=0.082) and percentage of hematoma evacuation (P=0.078). Upon multivariate linear regression, age (OR 4.49, [95% CI, 1.01-1.98], P=0.048) and percentage of hematoma evacuation (OR 4.09, [95% CI, 1.70-9.84], P=0.046) remained significantly predictive of increased CBV.Conclusions:CTP analysis demonstrated a significant increase in pericavity cerebral blood volume after MIS for ICH. Patient age and hematoma evacuation percentage may be predictive of CBV increase after MIS for ICH. Figure 1:Topographic map showing blood flow improvement at different distances from the lesion.

Osteonecrosis is a dose-limiting toxicity in the treatment of pediatric acute lymphoblastic leukemia (ALL). Prior studies on the genetics of osteonecrosis have focused on patients ≥10 years of age, leaving the genetic risk factors for the larger group of children <10 years incompletely understood. Here, we perform the first evaluation of genetic risk factors for osteonecrosis in children <10 years. The discovery cohort comprised 82 cases of osteonecrosis and 287 controls treated on Children’s Oncology Group (COG) standard-risk ALL protocol AALL0331 (NCT00103285, https://clinicaltrials.gov/ct2/show/NCT00103285), with results tested for replication in 817 children <10 years treated on COG protocol AALL0232 (NCT00075725, https://clinicaltrials.gov/ct2/show/NCT00075725). The top replicated single nucleotide polymorphisms (SNPs) were near bone morphogenic protein 7 [BMP7: rs75161997, P = 5.34 × 10−8 (odds ratio [OR] 15.0) and P = .0498 (OR 8.44) in the discovery and replication cohorts, respectively] and PROX1-antisense RNA1 (PROX1-AS1: rs1891059, P = 2.28 × 10−7 [OR 6.48] and P = .0077 [OR 3.78] for the discovery and replication cohorts, respectively). The top replicated nonsynonymous SNP, rs34144324, was in a glutamate receptor gene (GRID2, P = 8.65 × 10−6 [OR 3.46] and P = .0136 [OR 10.8] in the discovery and replication cohorts, respectively). In a meta-analysis, the BMP7 and PROX1-AS1 variants (rs75161997 and rs1891059, respectively) met the significance threshold of <5 × 10−8. Top replicated SNPs were enriched in enhancers active in mesenchymal stem cells, and analysis of annotated genes demonstrated enrichment in glutamate receptor and adipogenesis pathways. These data may provide new insights into the pathophysiology of osteonecrosis.

Osteonecrosis is a dose-limiting toxicity in the treatment of pediatric acute lymphoblastic leukemia (ALL). Prior studies on the genetics of osteonecrosis have focused on patients ≥10 years of age, leaving the genetic risk factors for the larger group of children <10 years incompletely understood. Here, we perform the first evaluation of genetic risk factors for osteonecrosis in children <10 years. The discovery cohort comprised 82 cases of osteonecrosis and 287 controls treated on Children's Oncology Group (COG) standard-risk ALL protocol AALL0331 (NCT00103285, https://clinicaltrials.gov/ct2/show/NCT00103285), with results tested for replication in 817 children <10 years treated on COG protocol AALL0232 (NCT00075725, https://clinicaltrials.gov/ct2/show/NCT00075725). The top replicated single nucleotide polymorphisms (SNPs) were near bone morphogenic protein 7 [BMP7: rs75161997, P= 5.34 × 10−8(odds ratio [OR] 15.0) and P= .0498 (OR 8.44) in the discovery and replication cohorts, respectively] and PROX1-antisense RNA1 (PROX1-AS1: rs1891059, P= 2.28 × 10−7[OR 6.48] and P= .0077 [OR 3.78] for the discovery and replication cohorts, respectively). The top replicated nonsynonymous SNP, rs34144324, was in a glutamate receptor gene (GRID2, P= 8.65 × 10−6[OR 3.46] and P= .0136 [OR 10.8] in the discovery and replication cohorts, respectively). In a meta-analysis, the BMP7and PROX1-AS1variants (rs75161997 and rs1891059, respectively) met the significance threshold of <5 × 10−8. Top replicated SNPs were enriched in enhancers active in mesenchymal stem cells, and analysis of annotated genes demonstrated enrichment in glutamate receptor and adipogenesis pathways. These data may provide new insights into the pathophysiology of osteonecrosis.

Glucocorticoids are important therapy for acute lymphoblastic leukemia (ALL) and their major adverse effect is osteonecrosis. Our goal was to identify genetic and nongenetic risk factors for osteonecrosis. We performed a genome-wide association study of single nucleotide polymorphisms (SNPs) in a discovery cohort comprising 2285 children with ALL, treated on the Children's Oncology Group AALL0232 protocol (NCT00075725), adjusting for covariates. The minor allele at SNP rs10989692 (near the glutamate receptor GRIN3Alocus) was associated with osteonecrosis (hazard ratio = 2.03; P= 3.59 × 10−7). The association was supported by 2 replication cohorts, including 361 children with ALL on St. Jude's Total XV protocol (NCT00137111) and 309 non-ALL patients from Vanderbilt University's BioVU repository treated with glucocorticoids (odds ratio [OR] = 1.87 and 2.26; P= .063 and .0074, respectively). In a meta-analysis, rs10989692 was also highest ranked (P= 2.68 × 10−8), and the glutamate pathway was the top ranked pathway (P= 9.8 × 10−4). Osteonecrosis-associated glutamate receptor variants were also associated with other vascular phenotypes including cerebral ischemia (OR = 1.64; P= 2.5 × 10−3), and arterial embolism and thrombosis (OR = 1.88; P= 4.2 × 10−3). In conclusion, osteonecrosis was associated with inherited variations near glutamate receptor genes. Further understanding this association may allow interventions to decrease osteonecrosis. These trials are registered at www.clinicaltrials.govas #NCT00075725 and #NCT00137111.

Glucocorticoids are important therapy for acute lymphoblastic leukemia (ALL) and their major adverse effect is osteonecrosis. Our goal was to identify genetic and nongenetic risk factors for osteonecrosis. We performed a genome-wide association study of single nucleotide polymorphisms (SNPs) in a discovery cohort comprising 2285 children with ALL, treated on the Children’s Oncology Group AALL0232 protocol (NCT00075725), adjusting for covariates. The minor allele at SNP rs10989692 (near the glutamate receptor GRIN3A locus) was associated with osteonecrosis (hazard ratio = 2.03; P = 3.59 × 10−7). The association was supported by 2 replication cohorts, including 361 children with ALL on St. Jude’s Total XV protocol (NCT00137111) and 309 non-ALL patients from Vanderbilt University’s BioVU repository treated with glucocorticoids (odds ratio [OR] = 1.87 and 2.26; P = .063 and .0074, respectively). In a meta-analysis, rs10989692 was also highest ranked (P = 2.68 × 10−8), and the glutamate pathway was the top ranked pathway (P = 9.8 × 10−4). Osteonecrosis-associated glutamate receptor variants were also associated with other vascular phenotypes including cerebral ischemia (OR = 1.64; P = 2.5 × 10−3), and arterial embolism and thrombosis (OR = 1.88; P = 4.2 × 10−3). In conclusion, osteonecrosis was associated with inherited variations near glutamate receptor genes. Further understanding this association may allow interventions to decrease osteonecrosis. These trials are registered at www.clinicaltrials.gov as #NCT00075725 and #NCT00137111.

Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children’s Research Hospital (SJCRH) protocols Total XIIIA (n = 154), Total XV (n = 498), and Total XVI (n = 271), or Children’s Oncology Group protocols POG 9906 (n = 222) and AALL0232 (n = 2163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome BeadChip array. In multivariate logistic regression, the intronic rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) had the strongest association with hypersensitivity (P = 4.1 × 10−8; odds ratio [OR] = 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruba HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2 expression compared with noncarriers (P = 1.1 × 10−3 and 0.03, respectively). The top ranked nonsynonymous polymorphism was rs17885382 in HLA-DRB1 (P = 3.2 × 10−6; OR = 1.63), which is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.

Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols Total XIIIA (n = 154), Total XV (n = 498), and Total XVI (n = 271), or Children's Oncology Group protocols POG 9906 (n = 222) and AALL0232 (n = 2163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome BeadChip array. In multivariate logistic regression, the intronic rs6021191 variant in nuclear factor of activated T cells 2 (NFATC2) had the strongest association with hypersensitivity (P= 4.1 × 10−8; odds ratio [OR] = 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruba HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2expression compared with noncarriers (P= 1.1 × 10−3and 0.03, respectively). The top ranked nonsynonymous polymorphism was rs17885382 in HLA-DRB1(P= 3.2 × 10−6; OR = 1.63), which is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.

A murine model was developed that recapitulates key features of clinical hypersensitivity to Escherichia coliasparaginase. Sensitized mice developed high levels of anti-asparaginase IgG antibodies and had immediate hypersensitivity reactions to asparaginase upon challenge. Sensitized mice had complete inhibition of plasma asparaginase activity (P= 4.2 × 10−13) and elevated levels of mouse mast cell protease 1 (P= 6.1 × 10−3) compared with nonsensitized mice. We investigated the influence of pretreatment with triprolidine, cimetidine, the platelet activating factor (PAF) receptor antagonist CV-6209 [2-(2-acetyl-6-methoxy-3,9-dioxo-4,8-dioxa-2,10-diazaoctacos-1-yl)-1-ethyl-pyridinium chloride], or dexamethasone on the severity of asparaginase-induced allergies. Combining triprolidine and CV-6209 was best for mitigating asparaginase-induced hypersensitivity compared with nonpretreated, sensitized mice (P= 1.2 × 10−5). However, pretreatment with oral dexamethasone was the only agent capable of mitigating the severity of the hypersensitivity (P= 0.03) and partially restoring asparaginase activity (P= 8.3 × 10−4). To rescue asparaginase activity in sensitized mice without requiring dexamethasone, a 5-fold greater dose of asparaginase was needed to restore enzyme activity to a similar concentration as in nonsensitized mice. Our results suggest a role of histamine and PAF in asparaginase-induced allergies and indicate that mast cell–derived proteases released during asparaginase allergy may be a useful marker of clinical hypersensitivity.

Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P ≤ 5 × 10−8 in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P = 2.8 × 10−10) and rs3781093, OR, 1.73 (P = 3.2 × 10−9). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non–Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P = 6.29 × 10−11). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.

Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P≤ 5 × 10−8in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P= 2.8 × 10−10) and rs3781093, OR, 1.73 (P= 3.2 × 10−9). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non–Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P= 6.29 × 10−11). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children’s Research Hospital (n = 541) and the Children’s Oncology Group (n = 1329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P = 7.5 × 10−5, odds ratio [OR] = 1.64; P = 1.4 × 10−5, OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1 alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P = 3.3 × 10−4, OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1 alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. These trials were registered at www.clinicaltrials.gov as NCT00137111, NCT00549848, NCT00005603, and NCT00075725.

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children's Research Hospital (n = 541) and the Children's Oncology Group (n = 1329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P= 7.5 × 10−5, odds ratio [OR] = 1.64; P= 1.4 × 10−5, OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P= 3.3 × 10−4, OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. These trials were registered at www.clinicaltrials.govas NCT00137111, NCT00549848, NCT00005603, and NCT00075725.

Methotrexate clearance can influence the cure of and toxicity in children with acute lymphoblastic leukemia (ALL). We estimated methotrexate plasma clearance for 1279 patients with ALL treated with methotrexate (24-hour infusion of a 1 g/m2 dose or 4-hour infusion of a 2 g/m2 dose) on the Children's Oncology Group P9904 and P9905 protocols. Methotrexate clearance was lower in older children (P = 7 × 10−7), girls (P = 2.7 × 10−4), and those who received a delayed-intensification phase (P = .0022). A genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in the organic anion transporter gene SLCO1B1 (P = 2.1 × 10−11). This replicates findings using different schedules of high-dose methotrexate in St Jude ALL treatment protocols; a combined meta-analysis yields a P value of 5.7 × 10−19 for the association of methotrexate clearance with SLCO1B1 SNP rs4149056. Validation of this variant with 5 different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance. This study is registered at http://www.clinicaltrials.gov as NCT00005585 and NCT00005596.

Methotrexate clearance can influence the cure of and toxicity in children with acute lymphoblastic leukemia (ALL). We estimated methotrexate plasma clearance for 1279 patients with ALL treated with methotrexate (24-hour infusion of a 1 g/m2dose or 4-hour infusion of a 2 g/m2dose) on the Children's Oncology Group P9904 and P9905 protocols. Methotrexate clearance was lower in older children (P= 7 × 10−7), girls (P= 2.7 × 10−4), and those who received a delayed-intensification phase (P= .0022). A genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in the organic anion transporter gene SLCO1B1(P= 2.1 × 10−11). This replicates findings using different schedules of high-dose methotrexate in St Jude ALL treatment protocols; a combined meta-analysis yields a Pvalue of 5.7 × 10−19for the association of methotrexate clearance with SLCO1B1SNP rs4149056. Validation of this variant with 5 different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance. This study is registered at http://www.clinicaltrials.govas NCT00005585 and NCT00005596.

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)–stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV+ lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV+ LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility. The current trial was registered at www.clinicaltrials.gov as #NCT00058812.

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)–stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV+lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV+LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility. The current trial was registered at www.clinicaltrials.govas #NCT00058812.

There is a growing interest in using antigen-specific T cells for the treatment of human malignancy. For example, adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been effective prophylaxis and treatment of EBV-associated lymphoproliferative disease in immunocompromised patients. For all immunotherapies, however, there has been a hypothetical concern that mutations in tumor-specific antigens may lead to tumor escape. We now demonstrate that such events may indeed occur, with lethal outcome. A patient who developed lymphoma after marrow transplantation received donor-derived, EBV-specific CTLs but died with progressive disease. The tumor cells proved substantially less sensitive to cytolysis than the EBV-transformed B-cell line used for CTL generation. The major cytolytic activity of the donor CTL was directed against 2 HLA-A11–restricted epitopes in the viral EBNA-3B antigen. Sequence analysis of this gene in the tumor virus revealed a 245–base pair deletion, which removed these 2 CTL epitopes. Hence, the viral antigen in the tumor had mutated in a way that allowed escape from CTLs. Analysis of EBV polymorphisms demonstrated that before CTL infusion, more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant strain in vivo. Such an occurrence, even when polyclonal CTL lines are used against genetically stable virus antigens, suggests that escape mutants may be a serious problem when CTL therapy is directed against more unstable tumor cell–derived targets.

A young woman with DiGeorge anomaly showed normal immune tests as a child and did not experience the symptoms of profound T cell immunodeficiency. However, she had chronic pulmonary infections which led to bronchiectasis. At age 14, she developed an Epstein–Barr virus-induced lymphoma and her T cell function tests were markedly abnormal. After intensive chemotherapy, she received an organ-cultured thymus transplant but because of an abnormally high EBV DNA titer was also given autologous EBV-specific cytotoxic T cells, prepared prior to transplant. Titers fell from 80,000 genome copies/mg DNA to 2000 within 6 weeks. She was clinically well and her T cell tests improved. Sixteen months after the transplant, however, her tumor returned; EBV DNA levels had risen to 40,000 copies/mg DNA. She again received autologous EBV-specific cytotoxic T lymphocytes and valcyclovir and Cytogam as well. Her tumor resolved on this therapy and she has remained well to this date, 29 months after the recurrence. T cell tests, which had deteriorated with the recurrence of the tumor, now show normal responses. This experience records a number of unique features of thymus transplantation. This is the first recorded successful thymus transplant in a patient with partial T cell immunity and thus expands the potential of this treatment modality to patients other than infants with complete DiGeorge anomaly. The patient demonstrates cytotoxic activity against mouse cells, demonstrating the ability to respond to a new antigen which requires host antigen presenting cells. Measurement of α 1 TRECs (T cell receptor excision circles) shows evidence of increasing and sustained thymopoiesis since the transplant at a level consistent with the age of the transplant donor rather than that of the recipient.

Introduction:Portable stroke detection devices may serve as important tools in stroke triage. With the capacity to differentiate stroke type and severity, these devices may allow for reductions in time-to-treatment, leading to improved patient outcomes in time sensitive neurologic diseases. One such device is the Cerebrotech Visor, an FDA-approved technology that aims to identify severe stroke by detecting bioimpedance asymmetry in brain tissue across a wide range of electromagnetic wavelengths. This study aims to evaluate the ability of this device to detect stroke types in a pilot set of patients presenting as acute stroke codes.Methods:Trained operators performed scans using the Visor during stroke codes from November 2020 to July 2021 on eligible patients. Clinical and radiologic characteristics as well as the Visor output, an algorithmically derived asymmetry score, were prospectively entered into a quality assurance database. CT findings and subsequent physician notes were used to classify patient stroke type and severity. T-tests were performed to assess the ability of the Visor score to detect various types of CT-confirmed stroke.Results:Seventy-eight patients were included and scanned. Using CT results, patients were classified into categories: ischemic stroke (IS) (16), intracerebral hemorrhage (ICH) (12), subdural hematoma (2), brain tumor (0), prior neurosurgical procedure (0), intraventricular hemorrhage (2), hydrocephalus (1), and no findings (49), with 3 patients experiencing a combination of these neurological findings. Visor scores for IS and ICH were individually compared to the cohort without IS and ICH, respectively. A t-test comparing patients with IS to the rest of the cohort showed significantly higher Visor scores (9.66 vs 7.13, p = 0.014). Although patients with ICH also had higher Visor scores, the difference was not statistically significant (8.16 vs 7.55, p = 0.30).Conclusion:The Cerebrotech Visor was able to differentiate between IS and non-IS with the current bioimpedance asymmetry-based device algorithm. Future studies aim to increase patient cohort size and use this patient-centered data to develop a machine learning algorithm to better differentiate other stroke types with this bioimpedance technology.

Introduction:Stroke is a leading cause of morbidity and mortality, with worse outcomes associated with increased time-to-treatment. As stroke subtypes are currently not confirmed until in-hospital imaging, portable stroke detection technology can reduce time to stroke diagnosis and treatment. The Cerebrotech Visor is an FDA-approved portable bioimpedance device that has been shown to differentiate severe stroke from minor stroke. This study aims to evaluate the accuracy of the Visor device in severe stroke detection in patients presenting as acute stroke codes.Methods:All patients presenting to the emergency department and receiving a stroke code at two hospitals from November 2020 to July 2021 were eligible. Trained operators completed device scans on eligible patients. The device functioned by detecting asymmetry in bioimpedance over a range of wavelengths to provide an algorithmically derived score. Visor scores, time to scan, and in-hospital imaging findings were recorded prospectively in a quality assurance database. T-tests were performed.Results:Eighty-seven patient scans were attempted. Patients without successful scans, due to reasons like patient motion and low patient tolerance of the device, were excluded. Patients with underlying diseases noted in scan comments that would alter the baseline brain landscape, such as advanced dementia, were also excluded. In total, 78 patients were included, with a mean age of 69.2 years (σ = 14.3). Scans were most frequently performed in the emergency department and took an average of 105.6 seconds. Using CT data, patients were divided into two groups based on normal or abnormal CT findings. Patients with abnormal CT results have significantly higher Visor scores (8.56 vs 7.11, p = 0.0495).Conclusion:The Cerebrotech Visor is a portable stroke detection device with the ability to detect alterations in brain bioimpedance in under 2 minutes. Though a large-scale validation study must be performed to assess the accuracy of the device to diagnose specific neurologic conditions, this pilot series of patients undergoing assessment as acute stroke codes demonstrates that this device may provide a rapid method for the in-hospital triage of severe stroke, reducing time-to-treatment for these patients.

Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing toward the risk of infections and malignant relapse. Restoration of T cell immunity is dependent on tissue regeneration in the thymus, the primary site of T cell development; although the capacity of the thymus to repair itself diminishes over lifespan. However, although boosting thymic function and T cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that Zinc (Zn), is critically important for both normal T cell development as well as repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell-production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation; although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration, but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients.

The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised.

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4+ and CD8+) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell–depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing theneo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2,000 genome copies/106 mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease. © 1998 by The American Society of Hematology.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (EBV-LPD) is a frequently fatal complication of organ transplantation and human immunodeficiency virus (HIV) infection. We have studied the safety and efficacy of adoptively transferred, gene-marked virus-specific cytotoxic T lymphocytes (CTLs) as prophylaxis and treatment of EBV-LPD in recipients of T-cell-depleted allogeneic bone marrow. In 42 patients treated prophylactically, no toxicity was experienced. None of these patients developed EBV-LPD, in contrast with eight of 53 (15%) patients who did not receive prophylactic CTL. Three patients who had not received CTL developed aggressive disease and received CTL as treatment. Gene-marked CTL homed to tumor sites and selective accumulation of marker gene was detected in tumor tissues. Tumors regressed completely in two patients, but the third died of respiratory failure. Infused CTLs persisted for up to 3 years in vivo, they rapidly reconstituted EBV-specific immune responses to levels seen in normal individuals, and they reduced high viral titers by two to three logs. We are now using autologous EBV-specific CTL to treat patients with relapsed EBV-positive Hodgkins disease and we are developing methods for the generation of antigen-specific lines. This approach could be applied to patients with HIV who develop EBV-LPD, using CTL derived early in the course of HIV infection.