Ruxolitinib for the Treatment of Chronic GVHD and Overlap Syndrome in Children and Young Adults.

BACKGROUND: Ruxolitinib, a JAK1/2 inhibitor, is used to treat chronic graft versus host disease (cGVHD) in adult allogeneic hematopoietic stem cell transplant patients, but experience in children is limited, perhaps because of lack of pediatric dosing information. In this report, we describe our pediatric and young adult dosing strategy experience in cGVHD. METHODS: Ruxolitinib was administered orally at 5 mg twice daily for children ≥25 kg or 2.5 mg twice daily if <25 kg. The dose was halved with concurrent azole administration and increased to a maximum of 10 mg twice daily if tolerated. Responses were evaluated using the 2014 NIH consensus criteria. Phosphorylation of lymphocyte STAT5 following dosing, a surrogate of JAK inhibition, was evaluated by flow cytometry. RESULTS: Twenty patients with a median age 14.6 y (range 5-26 y) received ruxolitinib for severe (n = 9) and moderate (n = 11) cGVHD. Median steroid dose was 0.5 mg/kg/d (range 0.08-1.5 mg/kg/d) at ruxolitinib initiation. Two patients with moderate cGVHD achieved a complete response (CR), while 12 patients achieved a partial response (PR) at a median of 48 d (range 17-98 d) from the first ruxolitinib dose, for an overall response rate of 70%. Eleven patients are maintaining their PRs. pSTAT5 on lymphocytes was absent or decreased (0%-6% events) in 5 evaluated patients, suggesting adequate inhibition. Three patients discontinued ruxolitinib because of neutropenia, thrombocytopenia, or elevated alanine aminotransferase. Four patients developed bacterial infections, and 3 experienced symptomatic viral infections. Two patients died from complications related to progressive severe cGVHD. CONCLUSIONS: Ruxolitinib using our dosing strategy demonstrates promise for treating cGVHD in children.

LAMP-5 is an essential inflammatory-signaling regulator and novel immunotherapy target for mixed lineage leukemia-rearranged acute leukemia.

Although great advances have been made in understanding the pathobiology of mixed lineage leukemia-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. In order to identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the in silico human surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane protein, is expressed highly and specifically in MLL-r leukemia. We found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. LAMP5 depletion significantly inhibited leukemia cell growth in vitro and in vivo. Functional studies showed that LAMP-5 is a novel modulator of innate-immune pathways in MLL-r leukemias. Downregulation of LAMP5 led to inhibition of NF-kB signaling and increased activation of type-1 interferon signaling downstream of Toll-like receptor/interleukin 1 receptor activation. These effects were attributable to the critical role of LAMP-5 in transferring the signal flux from interferon signaling endosomes to pro-inflammatory signaling endosomes. Depletion of IRF7 was able to partially rescue the cell growth inhibition upon LAMP5 downregulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r leukemia cells. Targeting surface LAMP-5 using an antibody-drug conjugate leads to significant cell viability decrease specifically in MLL-r leukemias. Overall, based on the limited expression throughout human tissues, we postulate that LAMP-5 could potentially serve as an immunotherapeutic target with a wide therapeutic window to treat MLL-r leukemias.

Hematopoietic Cell Transplantation Cures Adenosine Deaminase 2 Deficiency: Report on 30 Patients.

PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an inherited inborn error of immunity, characterized by autoinflammation (recurrent fever), vasculopathy (livedo racemosa, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency, lymphoproliferation, immune cytopenias, and bone marrow failure (BMF). Tumor necrosis factor (TNF-α) blockade is the treatment of choice for the vasculopathy, but often fails to reverse refractory cytopenia. We aimed to study the outcome of hematopoietic cell transplantation (HCT) in patients with DADA2. METHODS: We conducted a retrospective study on the outcome of HCT in patients with DADA2. The primary outcome was overall survival (OS). RESULTS: Thirty DADA2 patients from 12 countries received a total of 38 HCTs. The indications for HCT were BMF, immune cytopenia, malignancy, or immunodeficiency. Median age at HCT was 9 years (range: 2-28 years). The conditioning regimens for the final transplants were myeloablative (n = 20), reduced intensity (n = 8), or non-myeloablative (n = 2). Donors were HLA-matched related (n = 4), HLA-matched unrelated (n = 16), HLA-haploidentical (n = 2), or HLA-mismatched unrelated (n = 8). After a median follow-up of 2 years (range: 0.5-16 years), 2-year OS was 97%, and 2-year GvHD-free relapse-free survival was 73%. The hematological and immunological phenotypes resolved, and there were no new vascular events. Plasma ADA2 enzyme activity normalized in 16/17 patients tested. Six patients required more than one HCT. CONCLUSION: HCT was an effective treatment for DADA2, successfully reversing the refractory cytopenia, as well as the vasculopathy and immunodeficiency. CLINICAL IMPLICATIONS: HCT is a definitive cure for DADA2 with > 95% survival.

Developmental Adaptive Immune Defects Associated with STAT5B Deficiency in Three Young Siblings.

Patients with rare homozygous mutations in signal transducer and activator of transcription 5B (STAT5B) develop immunodeficiency resulting in chronic eczema, chronic infections, autoimmunity, and chronic lung disease. STAT5B-deficient patients are typically diagnosed in the teenage years, limiting our understanding of the development of associated phenotypic immune abnormalities. We report the first detailed chronological account of post-natal immune dysfunction associated with STAT5B deficiency in humans. Annual immunophenotyping of three siblings carrying a novel homozygous nonsense mutation in STAT5B was carried out over 4 years between the ages of 7 months to 8 years. All three siblings demonstrated consistent B cell hyperactivity including elevated IgE levels and autoantibody production, associated with diagnoses of atopy and autoimmunity. Total T cell levels in each sibling remained normal, with regulatory T cells decreasing in the oldest sibling. Interestingly, a skewing toward memory T cells was identified, with the greatest changes in CD8+ effector memory T cells. These results suggest an importance of STAT5B in B cell function and naïve versus memory T cell survival. Progressive dysregulation of FOXP3+ regulatory T cells and CD8+ memory T cell subsets reveal a crucial role of STAT5B in T cell homeostasis. The early diagnosis and focused immune evaluations of these three young STAT5B-deficient siblings support an important role of STAT5B in adaptive immune development and function.

Experience with a Reduced Toxicity Allogeneic Transplant Regimen for Non-CGD Primary Immune Deficiencies Requiring Myeloablation.

PURPOSE: A need exists for reduced toxicity conditioning regimens that offer less toxicity while maintaining myeloablation, especially for primary immune deficiencies where myeloablation or high donor myeloid chimerism is required to achieve cure. We adapted a busulfan and fludarabine regimen by Gungor et al. for children and young adults undergoing allogeneic HCT for non-CGD primary immune deficiencies requiring myeloablation or high donor myeloid chimerism, and herein report our experience. METHODS: We retrospectively reviewed records of 41 consecutive patients who underwent allogeneic HCT for Wiskott-Aldrich syndrome (n = 12), primary HLH/XLP (n = 10), CD40L deficiency (n = 7), or other (n = 12) primary immune deficiencies with a conditioning regimen containing pharmacokinetic-guided busulfan dosing which achieved a cumulative AUC between 57 and 74 mg/L × h (65-80% of conventional myeloablative exposure), along with fludarabine and alemtuzumab or anti-thymocyte globulin at 3 transplant centers between 2014 and 2019. RESULTS: Forty-one patients underwent a first (n = 33) or second (n = 8) allogeneic HCT. Median age was 2.3 years (range, 0.3 years-19.8 years). All but one patient (97.5%) achieved neutrophil recovery at a median of 14 days (range, 11-34 days). One patient developed sinusoidal obstruction syndrome and two patients developed diffuse alveolar hemorrhage. Four patients developed grades II-IV acute GVHD. Three patients developed chronic GVHD. One-year overall survival was 90% (95% confidence interval [CI] 81-99%) and event-free survival was 83% (95% CI 71-94%). CONCLUSIONS: Our experience suggests that a reduced toxicity busulfan-fludarabine regimen offers low toxicity, low incidence of grades 2-4 GVHD, durable myeloid engraftment, and excellent survival, and may be considered for a variety of primary immune deficiencies where myeloablative HCT is desired.

Publisher Correction: MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia.

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

High-risk LCH in infants is serially transplantable in a xenograft model but responds durably to targeted therapy.

Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm characterized by a clonal proliferation of Langerhans-like cells. Genomic profiling has identified recurrent somatic activating mutations in the mitogen-activated protein kinase pathway, which are targetable by small-molecule inhibitors. However, key questions such as the curative potential of targeted therapy and the cell of origin remain unanswered. In this study, we describe clinical outcomes of a series of pediatric patients with multisystem BRAF V600E-mutant LCH, as well as the results of accompanying murine xenograft experiments. Four infants with LCH (range, 7-11 months at diagnosis) and secondary hemophagocytic lymphohistiocytosis were referred to our institution and subsequently treated with the BRAF V600E-specific inhibitor dabrafenib. All patients achieved complete clinical responses by 8 weeks of therapy, with remissions lasting a median of 36 months (range, 27-42 months). One infant successfully discontinued therapy long-term upon achieving a molecular response by real-time quantitative polymerase chain reaction (RT-qPCR). We further characterized the disease-propagating cell population in a subset of these patients by transplanting whole bone marrow into immunodeficient mice. Xenografted animals exhibited decreased survival with hematologic abnormalities, splenomegaly, and histiocytic infiltrates in the bone marrow resembling human disease. This process could also be secondarily transplanted, resulting in a comparable disease latency with similar histologic findings. These data further support the presence of a disease-initiating cell in the bone marrow compartment. We demonstrate that despite aggressive disease behavior in a xenograft model, these patients can achieve sustained clinical remissions with targeted monotherapy, with a select subset achieving molecular responses by RT-qPCR.

Limitations of HLH-2004 criteria in distinguishing malignancy-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by dysregulated immune activation.  Primary HLH involves hereditary deficits in cytotoxic lymphocytes while secondary HLH is triggered by extrinsic factors. The HLH-2004 criteria are widely used for clinical diagnosis, yet their specificity for HLH or their ability to differentiate primary from secondary disease is unclear, potentially leading to inappropriate treatment. We describe several cases where fulfillment of HLH-2004 criteria obscured the diagnoses of underlying malignancies and delayed curative management. These issues are remedied without waiting for genetic testing results through an alternative diagnostic approach using flow cytometry-based immunologic assays and a thorough investigation for malignancy.

Hematopoietic stem cell transplantation rescues the hematological, immunological, and vascular phenotype in DADA2.

Deficiency of adenosine deaminase 2 (DADA2) is caused by biallelic deleterious mutations in CECR1 DADA2 results in variable autoinflammation and vasculopathy (recurrent fevers, livedo reticularis, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency and bone marrow failure. Tumor necrosis factor-α blockade is the treatment of choice for the autoinflammation and vascular manifestations. Hematopoietic stem cell transplantation (HSCT) represents a potential definitive treatment. We present a cohort of 14 patients from 6 countries who received HSCT for DADA2. Indication for HSCT was bone marrow dysfunction or immunodeficiency. Six of 14 patients had vasculitis pre-HSCT. The median age at HSCT was 7.5 years. Conditioning regimens were myeloablative (9) and reduced intensity (5). Donors were HLA-matched sibling (n = 1), HLA-matched unrelated (n = 9), HLA-mismatched unrelated (n = 3), and HLA haploidentical sibling (n = 1). All patients are alive and well with no new vascular events and resolution of hematological and immunological phenotype at a median follow-up of 18 months (range, 5 months to 13 years). Plasma ADA2 enzyme activity normalized in those tested post-HSCT (7/7), as early as day +14 (myeloid engraftment). Post-HSCT hematological autoimmunity (cytopenias) was reported in 4 patients, acute graft-versus-host disease grade 1 in 2, grade 2 in 3, and grade 3-4 in 1, and moderate chronic graft-versus-host disease in 1 patient. In conclusion, in 14 patients, HSCT was an effective and definitive treatment of DADA2.

Ruxolitinib as Salvage Therapy in Steroid-Refractory Acute Graft-versus-Host Disease in Pediatric Hematopoietic Stem Cell Transplant Patients.

We describe our retrospective clinical experience with ruxolitinib for steroid-refractory acute graft-versus-host disease (GVHD) in pediatric allogeneic hematopoietic stem cell transplant (HSCT) patients. Ruxolitinib was administered orally at 5 mg twice daily for children ≥ 25 kg or 2.5 mg twice daily if <25 kg. We excluded patients who received new immune suppressive agents within 2 weeks before initiation of ruxolitinib from response analysis. Patients were called a treatment failure if ruxolitinib was stopped before completion of 4 weeks of therapy because of adverse effects and not because of progression of acute GVHD. Thirteen patients received ruxolitinib, and 11 patients were assessable for response. One patient achieved a complete response, 4 had a partial response, and 2 had no response at 4 weeks after the first ruxolitinib dose. Four patients were treatment failures. Overall response rate was 45%. Adverse effects (n = 13) included grades 3 to 4 elevated alanine transaminase (n = 7), grades 3 to 4 neutropenia (n = 5), and grade 4 thrombocytopenia (n = 3). Infectious complications in patients included for response analysis (n = 11) were Epstein-Barr viremia (n = 2), adenovirus (n = 2), BK (n = 3), bacterial infections (n = 6), and fungal infections (n = 1). Seven of 13 patients were alive at a median follow-up of 401 days (range, 219 to 969) after HSCT. We observed a high rate of reversible adverse effects in children with steroid-refractory acute GVHD and a fair overall response of ruxolitinib as a salvage therapeutic agent. Further pharmacokinetic studies are needed to determine the best-tolerated dose of ruxolitinib that will achieve efficacy without significant adverse effects.

Real-time genomic profiling of histiocytoses identifies early-kinase domain BRAF alterations while improving treatment outcomes.

Many patients with histiocytic disorders such as Langerhans cell histiocytosis (LCH) or Erdheim-Chester disease (ECD) have treatment-refractory disease or suffer recurrences. Recent findings of gene mutations in histiocytoses have generated options for targeted therapies. We sought to determine the utility of prospective sequencing of select genes to further characterize mutations and identify targeted therapies for patients with histiocytoses. Biopsies of 72 patients with a variety of histiocytoses underwent comprehensive genomic profiling with targeted DNA and RNA sequencing. Fifteen patients (21%) carried the known BRAF V600E mutation, and 11 patients (15%) carried various mutations in MAP2K1, which we confirm induce constitutive activation of extracellular signal-regulated kinase (ERK) and were sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK, the product of MAP2K1). We also identified recurring ALK rearrangements, and 4 LCH patients with an uncommon in-frame deletion in BRAF (N486_P490del or N486_T491>K), resulting in constitutive activation of ERK with resistance to V600E-specific inhibitors. We subsequently describe clinical cases where patients with aggressive multisystem LCH experience dramatic and sustained responses to monotherapy with either dabrafenib or trametinib. These findings support our conclusion that comprehensive genomic profiling should be regularly applied to these disorders at diagnosis, and can positively impact clinical care.

Incidence and Outcomes of Central Nervous System Hemophagocytic Lymphohistiocytosis Relapse after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation.

Hemophagocytic lymphohistiocytosis (HLH) is an immune regulatory disorder that commonly presents with central nervous system (CNS) involvement. The only cure for genetic HLH is hematopoietic stem cell transplantation (HSCT), typically treated with reduced-intensity conditioning (RIC) regimens. We sought to estimate the incidence of CNS relapse after RIC HSCT, determine risk factors, and evaluate outcomes. We performed a retrospective chart review of 94 consecutive children and young adults with primary HLH who received RIC HSCT. CNS relapse within 1 year after transplantation was diagnosed by review of clinical symptoms, cerebral spinal fluid (CSF), and radiologic findings. Four (4.25%) patients developed symptoms of possible CNS HLH after HSCT and 3 patients were diagnosed. Eight patients underwent screening lumbar puncture because of history of active CNS disease at the onset of the conditioning regimen and 4 had evidence of continued disease. The overall incidence of CNS relapse and continued CNS disease after RIC HSCT was 8%. All patients with CNS disease after HSCT responded to CNS-directed therapy. Whole blood donor chimerism at the time of CNS relapse was low at 1% to 34%, but it remained high at 88% to 100% for patients with continued CNS disease. Overall survival for patients with CNS relapse was 50%, compared with 75% for patients without CNS disease (P = .079). Our data suggest that a low level of donor chimerism or active CNS disease at the time of transplantation increase the risk of CNS HLH after HSCT. Surveillance CSF evaluation after allogeneic RIC HSCT should be considered in patients with risk factors and CNS-directed treatment should be initiated if appropriate.

A xenograft model of macrophage activation syndrome amenable to anti-CD33 and anti-IL-6R treatment.

Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.

An ShRNA Screen Identifies MEIS1 as a Driver of Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are a major source of mortality in neurofibromatosis type 1 (NF1) patients. To identify MPNST driver genes, we performed a lentiviral short hairpin (sh) RNA screen, targeting all 130 genes up-regulated in neurofibroma and MPNSTs versus normal human nerve Schwann cells. NF1 mutant cells show activation of RAS/MAPK signaling, so a counter-screen in RAS mutant carcinoma cells was performed to exclude common RAS-pathway driven genes. We identified 7 genes specific for survival of MPSNT cells, including MEIS1. MEIS1 was frequently amplified or hypomethylated in human MPSNTs, correlating with elevated MEIS1 gene expression. In MPNST cells and in a genetically engineered mouse model, MEIS1 expression in developing nerve glial cells was necessary for MPNST growth. Mechanistically, MEIS1 drives MPNST cell growth via the transcription factor ID1, thereby suppressing expression of the cell cycle inhibitor p27(Kip) and maintaining cell survival.

Runx1 Deficiency Decreases Ribosome Biogenesis and Confers Stress Resistance to Hematopoietic Stem and Progenitor Cells.

The transcription factor RUNX1 is frequently mutated in myelodysplastic syndrome and leukemia. RUNX1 mutations can be early events, creating preleukemic stem cells that expand in the bone marrow. Here we show, counterintuitively, that Runx1-deficient hematopoietic stem and progenitor cells (HSPCs) have a slow growth, low biosynthetic, small cell phenotype and markedly reduced ribosome biogenesis (Ribi). The reduced Ribi involved decreased levels of rRNA and many mRNAs encoding ribosome proteins. Runx1 appears to directly regulate Ribi; Runx1 is enriched on the promoters of genes encoding ribosome proteins and binds the rDNA repeats. Runx1-deficient HSPCs have lower p53 levels, reduced apoptosis, an attenuated unfolded protein response, and accordingly are resistant to genotoxic and ER stress. The low biosynthetic activity and corresponding stress resistance provides a selective advantage to Runx1-deficient HSPCs, allowing them to expand in the bone marrow and outcompete normal HSPCs.

Experience with Alemtuzumab, Fludarabine, and Melphalan Reduced-Intensity Conditioning Hematopoietic Cell Transplantation in Patients with Nonmalignant Diseases Reveals Good Outcomes and That the Risk of Mixed Chimerism Depends on Underlying Disease, Stem Cell Source, and Alemtuzumab Regimen.

Alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens are increasingly used for the hematopoietic cell transplantation (HCT) of pediatric and young adult patients with nonmalignant diseases. Early experience suggests that these regimens are associated with good survival but a high incidence of mixed chimerism, which we have previously shown to be influenced by the alemtuzumab schedule. We hypothesized that the underlying diagnosis and donor graft source would also affect the development of mixed chimerism and that the majority of patients would survive RIC HCT without graft loss. To examine this, we conducted a retrospective study of 206 patients with metabolic diseases, non-Fanconi anemia marrow failure disorders, and primary immune deficiencies who underwent 210 consecutive RIC HCT procedures at Cincinnati Children's Hospital. Ninety-seven percent of the patients engrafted. Mixed donor and recipient chimerism developed in 46% of patients. Patients with marrow failure had a low risk of mixed chimerism (hazard ratio [HR], .208; 95% confidence interval [CI], .061 to .709; P = .012). The risk of mixed chimerism was high in patients who received a cord blood graft (HR, 3.122; 95% CI, 1.236 to 7.888; P = .016). As expected, patients who received a proximal or higher dose per kilogram of alemtuzumab schedule also experienced higher rates of mixed chimerism (all HR > 2, all P < .05). At the time of last follow-up (median, 654 days; range, 13 to 3337), over 75% of patients had greater than 90% whole blood donor chimerism. A second transplantation was performed in 5% of patients. Three-year survival without retransplantation was 84% (95% CI, 71% to 98%) for patients who underwent transplantation with an HLA-matched sibling donor. Survival without retransplantation was negatively affected by lack of a matched related donor, increasing age, and development of grades III and IV acute graft-versus-host disease. We conclude that alemtuzumab, fludarabine, and melphalan RIC HCT offers good results for many patients and that the risk of developing mixed chimerism is influenced by underlying diagnosis, graft source, and alemtuzumab dosing.