Identification of a Multipotent Progenitor Population in the Spleen That Is Regulated by NR4A1.

The developmental fate of hematopoietic stem and progenitor cells is influenced by their physiological context. Although most hematopoietic stem and progenitor cells are found in the bone marrow of the adult, some are found in other tissues, including the spleen. The extent to which the fate of stem cells is determined by the tissue in which they reside is not clear. In this study, we identify a new progenitor population, which is enriched in the mouse spleen, defined by cKit+CD71lowCD24high expression. This previously uncharacterized population generates exclusively myeloid lineage cells, including erythrocytes, platelets, monocytes, and neutrophils. These multipotent progenitors of the spleen (MPPS) develop from MPP2, a myeloid-biased subset of hematopoietic progenitors. We find that NR4A1, a transcription factor expressed by myeloid-biased long term-hematopoietic stem cells, guides the lineage specification of MPPS. In vitro, NR4A1 expression regulates the potential of MPPS to differentiate into erythroid cells. MPPS that express NR4A1 differentiate into a variety of myeloid lineages, whereas those that do not express NR4A1 primarily develop into erythroid cells. Similarly, in vivo, after adoptive transfer, Nr4a1-deficient MPPS contribute more to erythrocyte and platelet populations than do wild-type MPPS. Finally, unmanipulated Nr4a1-/- mice harbor significantly higher numbers of erythroid progenitors in the spleen compared with wild-type mice. Together, our data show that NR4A1 expression by MPPS limits erythropoiesis and megakaryopoeisis, permitting development to other myeloid lineages. This effect is specific to the spleen, revealing a unique molecular pathway that regulates myeloid bias in an extramedullary niche.

Clinical and immunologic impact of CCR5 blockade in graft-versus-host disease prophylaxis.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Lymphocyte trafficking via chemokine receptors such as CCR5 plays a critical role in alloreactive responses, and previous data suggest that CCR5 blockade with maraviroc results in a low incidence of visceral GVHD. However, the full scope of clinical and immunologic effects of CCR5 blockade in HSCT has not been described. We compared a cohort of patients enrolled on a trial of reduced-intensity allo-HSCT with standard GVHD prophylaxis plus maraviroc to a contemporary control cohort receiving standard GVHD prophylaxis alone. Maraviroc treatment was associated with a lower incidence of acute GVHD without increased risk of disease relapse, as well as reduced levels of gut-specific markers. At day 30, maraviroc treatment increased CCR5 expression on T cells and dampened T-cell activation in peripheral blood without impairing early immune reconstitution or increasing risk for infections. Patients who developed acute GVHD despite maraviroc prophylaxis showed increased T-cell activation, naive T-cell skewing, and elevated serum CXCL9 and CXCL10 levels. Collectively, these data suggest that maraviroc effectively protects against GVHD by modulating alloreactive donor T-cell responses, and that CXCR3 signaling may be an important resistance mechanism to CCR5 blockade in GVHD.

The orphan nuclear receptor NR4A1 specifies a distinct subpopulation of quiescent myeloid-biased long-term HSCs.

Hematopoiesis is maintained throughout life by self-renewing hematopoietic stem cells (HSCs) that differentiate to produce both myeloid and lymphoid cells. The NR4A family of orphan nuclear receptors, which regulates cell fate in many tissues, appears to play a key role in HSC proliferation and differentiation. Using a NR4A1(GFP) BAC transgenic reporter mouse we have investigated NR4A1 expression and its regulation in early hematopoiesis. We show that NR4A1 is most highly expressed in a subset of Lin(-) Sca-1(+) c-Kit(+) CD48(-) CD150(+) long-term (LT) HSCs, and its expression is tightly associated with HSC quiescence. We also show that NR4A1 expression in HSCs is induced by PGE2, a known enhancer of stem cell engraftment potential. Finally, we find that both NR4A1(GFP+) and NR4A1(GFP-) HSCs successfully engraft primary and secondary irradiated hosts; however, NR4A1(GFP+) HSCs are distinctly myeloid-biased. These results show that NR4A1 expression identifies a highly quiescent and distinct population of myeloid-biased LT-HSCs.

Infusion of CD3/CD28 costimulated umbilical cord blood T cells at the time of single umbilical cord blood transplantation may enhance engraftment.

Limited cell numbers in umbilical cord blood (UCB) grafts present a major impediment to favorable outcomes in adult transplantation, largely related to delayed or failed engraftment. The advent of UCB transplantation (UCBT) using two grafts successfully circumvents this obstacle, despite the engraftment of only one unit. Preclinical models suggested that the addition of UCB T cells at the time of transplant can enhance engraftment. We tested whether ex vivo activation by CD3/CD28 costimulation and expansion of T cells from a single UCB graft would be safe and feasible in adults with advanced hematologic malignancies, with an overall objective of optimizing engraftment in single unit UCBT. In this phase 1 study, recipients of single UCB units were eligible if the unit was stored in two adequate fractions. Dose limiting toxicity was defined as grade 3 or grade 4 GVHD within 90 days of UCBT. Four patients underwent UCBT; all were treated at the first dose level (10(5) cells/kg). At the 10(5) cells/kg dose level two subjects experienced grade 3 intestinal GVHD, thus meeting stopping criteria. For three subjects, neutrophil engraftment was early (12, 17, and 20 days), while one subject experienced primary graft failure. We observed early donor T cell trafficking and found that expanded T cells produced supraphysiologic levels of cytokines relevant to engraftment and to lymphoid differentiation and function. Taken together, these preliminary data suggest rapid engraftment in recipients of a single UCBT combined with relatively low doses of activated T cells, though potentially complicated by severe GVHD.

Blockade of lymphocyte chemotaxis in visceral graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic stem-cell transplantation (HSCT). The chemokine receptor CCR5 appears to play a role in alloreactivity. We tested whether CCR5 blockade would be safe and limit GVHD in humans. METHODS: We tested the in vitro effect of the CCR5 antagonist maraviroc on lymphocyte function and chemotaxis. We then enrolled 38 high-risk patients in a single-group phase 1 and 2 study of reduced-intensity allogeneic HSCT that combined maraviroc with standard GVHD prophylaxis. RESULTS: Maraviroc inhibited CCR5 internalization and lymphocyte chemotaxis in vitro without impairing T-cell function or formation of hematopoietic-cell colonies. In 35 patients who could be evaluated, the cumulative incidence rate (±SE) of grade II to IV acute GVHD was low at 14.7±6.2% on day 100 and 23.6±7.4% on day 180. Acute liver and gut GVHD were not observed before day 100 and remained uncommon before day 180, resulting in a low cumulative incidence of grade III or IV GVHD on day 180 (5.9±4.1%). The 1-year rate of death that was not preceded by disease relapse was 11.7±5.6% without excessive rates of relapse or infection. Serum from patients receiving maraviroc prevented CCR5 internalization by CCL5 and blocked T-cell chemotaxis in vitro, providing evidence of antichemotactic activity. CONCLUSIONS: In this study, inhibition of lymphocyte trafficking was a specific and potentially effective new strategy to prevent visceral acute GVHD. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00948753.).

NF-Y is necessary for hematopoietic stem cell proliferation and survival.

HSC function depends on the tight control of proliferation and the balance between self-renewal and differentiation. Here, we report that the trimeric transcription factor NF-Y is critical for the survival of cycling, but not quiescent HSCs. With the use of a conditional knockout mouse model, we demonstrate that NF-Ya deletion creates an accumulation of HSCs in G(2)/M and prompts apoptosis, causing hematopoietic failure and death of the animal. These defects are accompanied by the dysregulation of multiple genes that influence cell cycle control (cyclin b1 and p21), apoptosis (Bcl-2), and self-renewal (HoxB4, Notch1, Bmi-1) and are independent of p53. Our results identify NF-Y as a pivotal upstream participant in a regulatory network necessary for the preservation of cycling HSCs.

Long-term functional engraftment of mesenchymal progenitor cells in a mouse model of accelerated aging.

Age-related osteoporosis is characterized by a decrease in bone-forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere-based Wrn(-/-) Terc(-/-) model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell-autonomous mechanism in senile bone loss, transplantation of young wild-type whole bone marrow into Wrn(-/-) Terc(-/-) mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn(-/-) Terc(-/-) mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging.

Notch signaling is a critical regulator of allogeneic CD4+ T-cell responses mediating graft-versus-host disease.

Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is caused by donor T cells that mediate host tissue injury through multiple inflammatory mechanisms. Blockade of individual effector molecules has limited efficacy in controlling GVHD. Here, we report that Notch signaling is a potent regulator of T-cell activation, differentiation, and function during acute GVHD. Inhibition of canonical Notch signaling in donor T cells markedly reduced GVHD severity and mortality in mouse models of allogeneic HSCT. Although Notch-deprived T cells proliferated and expanded in response to alloantigens in vivo, their ability to produce interleukin-2 and inflammatory cytokines was defective, and both CD4(+) and CD8(+) T cells failed to up-regulate selected effector molecules. Notch inhibition decreased the accumulation of alloreactive T cells in the intestine, a key GVHD target organ. However, Notch-deprived alloreactive CD4(+) T cells retained significant cytotoxic potential and antileukemic activity, leading to improved overall survival of the recipients. These results identify Notch as a novel essential regulator of pathogenic CD4(+) T-cell responses during acute GVHD and suggest that Notch signaling in T cells should be investigated as a therapeutic target after allogeneic HSCT.

TAT-mediated transduction of NF-Ya peptide induces the ex vivo proliferation and engraftment potential of human hematopoietic progenitor cells.

Retroviral overexpression of NF-Ya, the regulatory subunit of the transcription factor NF-Y, activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer, more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4, a target gene of NF-Ya, using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction, the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover, TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated, transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy.

Host-reactive CD8+ memory stem cells in graft-versus-host disease.

Graft-versus-host disease (GVHD) is caused by alloreactive donor T cells that trigger host tissue injury. GVHD develops over weeks or months, but how this immune response is maintained over time is unknown. In mouse models of human GVHD, we identify a new subset of postmitotic CD44(lo)CD62L(hi)CD8(+) T cells that generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8(+) T cells, while self-renewing. These cells express Sca-1, CD122 and Bcl-2, and induce GVHD upon transfer into secondary recipients. The postmitotic CD44(lo)CD62L(hi)CD8(+) T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigens and dendritic cells and require interleukin-15. Thus, their long life, ability to self-renew and multipotentiality define these cells as candidate memory stem cells. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including GVHD.

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells.

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.

Identification of stem cell transcriptional programs normally expressed in embryonic and neural stem cells in alloreactive CD8+ T cells mediating graft-versus-host disease.

A hallmark of graft-versus-host-disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation, is the cytopathic injury of host tissues mediated by persistent alloreactive effector T cells (T(E)). However, the mechanisms that regulate the persistence of alloreactive T(E) during GVHD remain largely unknown. Using mouse GVHD models, we demonstrate that alloreactive CD8(+) T(E) rapidly diminished in vivo when adoptively transferred into irradiated secondary congenic recipient mice. In contrast, although alloreactive CD8(+) T(E) underwent massive apoptosis upon chronic exposure to alloantigens, they proliferated in vivo in secondary allogeneic recipients, persisted, and caused severe GVHD. Thus, the continuous proliferation of alloreactive CD8(+) T(E), which is mediated by alloantigenic stimuli rather than homeostatic factors, is critical to maintaining their persistence. Gene expression profile analysis revealed that although alloreactive CD8(+) T(E) increased the expression of genes associated with cell death, they activated a group of stem cell genes normally expressed in embryonic and neural stem cells. Most of these stem cell genes are associated with cell cycle regulation, DNA replication, chromatin modification, and transcription. One of these genes, Ezh2, which encodes a chromatin modifying enzyme, was abundantly expressed in CD8(+) T(E). Silencing Ezh2 significantly reduced the proliferation of alloantigen-activated CD8(+) T cells. Thus, these findings identify that a group of stem cell genes could play important roles in sustaining terminally differentiated alloreactive CD8(+) T(E) and may be therapeutic targets for controlling GVHD.

Lestaurtinib (CEP701) is a JAK2 inhibitor that suppresses JAK2/STAT5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders.

Recent studies have demonstrated that patients with myeloproliferative disorders (MPDs) frequently have acquired activating mutations in the JAK2 tyrosine kinase. A multikinase screen determined that lestaurtinib (formerly known as CEP-701) inhibits wild type JAK2 kinase activity with a concentration that inhibits response by 50% (IC(50)) of 1 nM in vitro. We hypothesized that lestaurtinib would inhibit mutant JAK2 kinase activity and suppress the growth of cells from patients with MPDs. We found that lestaurtinib inhibits the growth of HEL92.1.7 cells, which are dependent on mutant JAK2 activity for growth in vitro and in xenograft models. Erythroid cells expanded from primary CD34(+) cells from patients with MPDs were inhibited by lestaurtinib at concentrations of 100 nM or more in 15 of 18 subjects, with concomitant inhibition of phosphorylation of STAT5 and other downstream effectors of JAK2. By contrast, growth of erythroid cells derived from 3 healthy controls was not significantly inhibited. These results demonstrate that lestaurtinib, in clinically achievable concentrations, inhibits proliferation and JAK2/STAT5 signaling in cells from patients with MPDs, and therefore holds promise as a therapeutic agent for patients with these disorders.

c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.

Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.

A new approach to the blocking of alloreactive T cell-mediated graft-versus-host disease by in vivo administration of anti-CXCR3 neutralizing antibody.

Chemokines and chemokine receptors play critical roles in directing the migration of alloreactive donor T cells into graft-vs-host disease (GVHD) target organs. However, blockade of GVHD by antagonist Ab against chemokine receptors remains an elusive goal. Using a mouse model of human GVHD, we demonstrate that in vivo administration of anti-CXCR3 Ab for 21 days (long-term), but not for 7 days (short-term), inhibits alloreactive CD8(+) T cell-mediated GVHD. During a graft-vs-host reaction, infused donor CD8(+) T cells generate two subsets of potent inducers of GVHD: CXCR3(+)CD8(+) and CXCR3(-)CD8(+) T cells. Compared with CXCR3(+)CD8(+) T cells, CXCR3(-)CD8(+) T cells produce less granzyme B, Fas ligand, IFN-gamma, and TNF-alpha. Interestingly, stimulation with either dendritic cells or IL-2 induces a dynamic conversion between CXCR3(+)CD8(+) and CXCR3(-)CD8(+) T cells. Short-term anti-CXCR3 Ab treatment inhibits only CXCR3(+)CD8(+) T cell-mediated GVHD, but not the disease induced by CXCR3(-)CD8(+) T cells. Prolonged in vivo administration of anti-CXCR3 Ab significantly reduces the infiltration of alloreactive CD8(+) T cells into GVHD target organs and inhibits GVHD mediated by either CXCR3(+)CD8(+) or CXCR3(-)CD8(+) T cells. Thus, we have established a novel and effective approach with the potential to give rise to new clinical methods for preventing and treating GVHD after allogeneic hematopoietic stem cell transplantation.

Decreased Naïve T-cell Production Leading to Cytokine Storm as Cause of Increased COVID-19 Severity with Comorbidities.

Aging, type 2 diabetes, and male gender are major risk factors leading to increased COVID-19 morbidity and mortality. Thymic production and the export of naïve T cells decrease with aging through the effects of androgens in males and in type 2 diabetes. Furthermore, with aging, recovery of naïve T-cell populations after bone marrow transplantation is delayed and associated with an increased risk of chronic graft vs. host disease. Severe COVID-19 and SARS infections are notable for severe T-cell depletion. In COVID-19, there is unique suppression of interferon signaling by infected respiratory tract cells with intact cytokine signaling. A decreased naïve T-cell response likely contributes to an excessive inflammatory response and increases the odds of a cytokine storm. Treatments that improve naïve T-cell production may prove to be vital COVID-19 therapies, especially for these high-risk groups.

Preterminal host dendritic cells in irradiated mice prime CD8+ T cell-mediated acute graft-versus-host disease.

To understand the relationship between host antigen-presenting cells (APCs) and donor T cells in initiating graft-versus-host disease (GVHD), we followed the fate of host dendritic cells (DCs) in irradiated C57BL/6 (B6) recipient mice and the interaction of these cells with minor histocompatibility antigen- (miHA-) mismatched CD8+ T cells from C3H.SW donors. Host CD11c+ DCs were rapidly activated and aggregated in the T cell areas of the spleen within 6 hours of lethal irradiation. By 5 days after irradiation, <1% of host DCs were detectable, but the activated donor CD8+ T cells had already undergone as many as seven divisions. Thus, proliferation of donor CD8+ T cells preceded the disappearance of host DCs. When C3H.SW donor CD8+ T cells were primed in vivo in irradiated B6 mice or ex vivo by host CD11c+ DCs for 24-36 hours, they were able to proliferate and differentiate into IFN-gamma-producing cells in beta(2)-microglobulin-deficient (beta(2)m(-/-)) B6 recipients and to mediate acute GVHD in beta(2)m(-/-) --> B6 chimeric mice. These results indicate that, although host DCs disappear rapidly after allogeneic bone marrow transplantation, they prime donor T cells before their disappearance and play a critical role in triggering donor CD8+ T cell-mediated GVHD.

Hematopoietic stem-cell contribution to ectopic skeletogenesis.

BACKGROUND: Fibrodysplasia ossificans progressiva is a rare genetic disorder of ectopic skeletogenesis associated with dysregulation of bone morphogenetic protein (BMP) signaling. Hematopoietic cells have been implicated in the ectopic skeletogenesis of fibrodysplasia ossificans progressiva, and their replacement has been postulated as a possible cure. However, the definitive contribution of hematopoietic cells to the pathogenesis of ectopic skeletogenesis remains obscure. METHODS: We employed both careful clinical observation and in vivo murine transplantation studies to more precisely determine the contribution of hematopoietic cells to ectopic skeletogenesis. We identified a patient with fibrodysplasia ossificans progressiva who had undergone bone marrow transplantation for the treatment of intercurrent aplastic anemia twenty-five years earlier and investigated whether the clinical course of the fibrodysplasia ossificans progressiva had been influenced by bone marrow replacement or immunosuppression, or both. In complementary studies, we transplanted hematopoietic stem cells from constitutively expressing LacZ transgenic mice to identify the contribution of hematopoietic cells to BMP4-induced heterotopic ossification, a histopathologic model of fibrodysplasia ossificans progressiva. RESULTS: We found that replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia. In complementary murine transplantation studies, we found that cells of hematopoietic origin contributed to the early inflammatory and late marrow-repopulating stages of BMP4-induced heterotopic ossification but were not represented in the fibroproliferative, chondrogenic, or osteogenic stages of heterotopic ossification. Interestingly, both recombinant human BMP4 induction in an animal model and the dysregulated BMP signaling pathway in a patient with fibrodysplasia ossificans progressiva were sufficient to recruit at least two populations of cells, one of hematopoietic origin and at least one of non-hematopoietic origin, that contribute to the formation of an ectopic skeleton. CONCLUSIONS: Taken together, these findings demonstrate that bone marrow transplantation did not cure fibrodysplasia ossificans progressiva in the patient in this study, most likely because the hematopoietic cell population is not the site, or at least not the dominant site, of the intrinsic dysregulation of the BMP signaling pathway in fibrodysplasia ossificans progressiva. However, following transplantation of bone marrow from a presumably normal donor, immunosuppression of the immune system appeared to ameliorate activation of ectopic skeletogenesis in a genetically susceptible host. Thus, cells of hematopoietic origin may contribute to the formation of an ectopic skeleton, although they are not sufficient to initiate the process alone.

Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations.

BACKGROUND: Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. METHODS: The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient's electronic medical record. RESULTS: NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. CONCLUSIONS: Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases.