ALBERT

Description: The Congenital Dyserythropoietic Anemia Registry (CDAR, ClinicalTrials.gov Identifier: NCT02964494) was created to investigate the natural history, biology, and molecular pathogenetic mechanisms of CDA. To date, there are 6 genes known to cause CDA (CDAN1, C15orf41, SEC23B, KIF23, KLF1, GATA1). However, 57% of patients registered in CDAR so far (17 out of 33 patients) have an unidentified genetic cause. We have utilized whole exome sequencing (WES) in family-trio design to search for novel candidate gene mutations that may be responsible for the disease. Three unrelated patients with dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay were found to have missense mutations in the gene VPS4A which encodes an ATPase that participates with the ESCRT III machinery in endosomal vesicle trafficking, centrosome localization, and the abscission step of cytokinesis. It has been shown to play an essential role in division of HeLa cells in vitro where it concentrates at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of these VPS4A variants in CDA and further investigate the role of VPS4A in erythropoiesis. Patients 1 and 3 had de novo mutations (R284W and G203A) and transfusion-dependent anemia with presence of binucleated erythroblasts in the bone marrow resembling CDA type I. Of note, the patients' erythroblasts exhibited cytoplasmic bridges (Figure 1A) rather than the nuclear chromatin bridges observed in CDA-I. Patient 2, offspring of consanguineous parents, presented with hemolytic anemia and was found to have a homozygous mutation (A28V) in a highly conserved alanine residue in the microtubule-interacting domain (MIT) of VPS4A. She had rare evidence of dyserythropoiesis with fewer than 3% binucleated erythroblasts in bone marrow studies. All three patients had significant neurodevelopmental delay with axial hypotonia and appendicular hypertonia. Flow cytometry analysis of peripheral blood from each of these patients revealed a unique cell population which is negative for RNA (by thiazole orange) but still CD71 positive suggesting that loss of VPS4A function also impacts reticulocyte maturation, likely because of defective endosomal vesicle trafficking. Using CD34+ cells in ex vivo erythropoiesis cultures, we first confirmed that VPS4A is expressed in human erythroblasts and localizes at the spindle poles and midbody during mitosis and cytokinesis in these cells. RNA isolated from reticulocytes from patients 1 and 2 was assessed for expression of VPS4A and the paralogous VPS4B. Samples from patient 1 had reduced expression of VPS4A (

Description: The Runx1/Core Binding Factor- β (CBF β) transcriptional complex is required for the establishment of hematopoiesis during development. To permit the analysis of Runx1 expression in hematopoietic cell subsets, we have recently developed a novel murine line in which the expression of both full-length Runx1 and GFP is driven by the Runx1 promoter [Runx1-internal ribosomal entry site-green fluorescent protein (Runx1-GFP) knock-in mouse; Blood 103:2522]. Analysis of these mice has revealed that Runx1 is expressed in all hematopoietic lineages with the exception of erythroid cells. During our analysis, we identified in the bone marrow of these mice a cell population that expresses GFP at levels 2–5 fold higher than any other cell type (GFPhi). These cells have low forward and side scatter properties and do not express c-kit or several lineage-associated cell surface markers (lin−). In comparison to c-kit+lin− cells, these GFPhic-kit−lin− cells possess little colony forming activity in vitro. While they lack primary CFU-S activity in contrast to c-kit+lin− cells, GFPhic-kit−lin− cells possess secondary CFU-S activity. A c-kit−lin− hematopoietic stem cell (HSC) has been described by others that contributes to long-term, but not short-term, hematopoietic reconstitution in lethally irradiated recipients and may represent progenitors of c-kit+lin− HSCs in vivo (Ortiz et al. Immunity 10:173). The GFPhi bone marrow cells that we have identified share many of the properties of those c-kit−lin− cells identified by others; consequently, they likely represent the same cell population. Our ability to isolate these cells based on differential GFP expression should enable us to highly purify these GFPhic-kit−lin− cells and further characterize their immunophenotypic and biologic properties.

Description: Hem1 is a hematopoietic specific member of the HEM family proteins, which have been identified as components of the WAVE regulatory complex. To further characterize the role of Hem1 in hematopoietic cell function, we have generated mice deficient in Hem1 using gene targeting methodology. Hem1-deficient mice manifest several phenotypic abnormalities, including peripheral blood lymphopenia and neutrophilia, splenomegaly, premature mortality, and with variable penetrance thymic hypoplasia and alopecia. Flow cytometric analysis of peripheral blood and spleen demonstrated that Hem1−/ − mice have a marked reduction in peripheral B cells, changes that were evident in both adult and neonatal mice; the splenomegaly in Hem1−/ − mice was attributable to increased extramedullary hematopoiesis. The frequency of bone marrow B cell progenitors was also markedly reduced in Hem1−/ − mice. To assess the role of Hem1 in hematopoietic stem cell (HSC) function, competitive bone marrow transplantation assays were performed. In contrast to wild-type HSCs, Hem1-deficient HSCs had poor competitive repopulating activity in irradiated recipient mice. KSL cell analysis demonstrated no significant difference in the frequency of lin-c-kit+Sca1+ HSCs between wild-type and Hem1−/ − bone marrow, suggesting that the defective competitive repopulating activity of Hem1−/ − HSCs is attributable to defective bone marrow homing or stem cell niche interaction. Given the biochemical evidence implicating the HEM proteins as a component of the WAVE regulatory complex, we also assessed the ability of Hem1-deficient leukocytes to undergo cytoskeletal remodeling in vitro. Using a transwell assay, Hem1−/ − bone marrow storage pool neutrophils demonstrated markedly blunted chemotactic responses to formylated peptide which was attributable to defective f-actin formation. Hem1−/ − peripheral CD4+ T cells similarly manifested chemotactic defects in response to SDF-1, and showed blunted proliferation when stimulated with antibodies against CD3 and CD28. Finally, a model of Streptococcus pneumoniae infection was employed to test the role of Hem1 in the in vivo function of neutrophils. Hem1−/ − mice were dramatically more sensitive to S. pneumoniae than wild-type littermates, as manifested by the inability to eliminate S. pneumoniae organisms in vivo and higher mortality. In summary, Hem1 deficiency results in deficiencies and functional defects in multiple hematopoietic lineages due to defective signaling to the actin cytoskeleton, and importantly, Hem1 plays a critical role in innate immunity to S. pneumoniae in vivo.

Description: Background: Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm primarily of childhood, characterized by a neoplastic proliferation of Langerhans-like cells. The clinical presentation of LCH is highly variable, and while some children are cured with combination chemotherapy, many will relapse and experience irreversible morbidity. Genomic profiling of LCH has identified recurrent somatic activating mutations in BRAF and MAP2K1, all of which culminate in activation of the mitogen-activated protein kinase pathway. However, key mechanistic and clinical questions such as the curative potential of targeted therapy and the cell of origin remain unanswered. Methods: Four infants with risk-organ positive (RO+) multisystem BRAF V600E-mutant LCH and secondary hemophagocytosis were treated at our institution with the BRAF V600E-inhibitor dabrafenib after failing chemotherapy, or up front at the discretion of the treating physician. Response assessment was performed as indicated for good clinical care. Excess bone marrow obtained during routine clinical evaluation was collected under an IRB-approved protocol, and transplanted into busulfan-conditioned NOD/LtSz-SCID interleukin-2(IL2)RG-/- (NSG) or NSG-SGM3 (NSGS) mice. Donor chimerism was assessed by flow cytometry for mouse and human CD45 and lineage reconstitution was determined with standard human markers. BRAF V600E detection was performed on extracted DNA from xenografts using a TaqMan Mutation Detection RTPCR assay. Results: At presentation, all patients exhibited features of HLH (hemophagocytic lymphohistiocytosis) with fever, splenomegaly, pancytopenia, elevated markers of inflammation, hypofibrinogenemia, and hemophagocytosis on bone marrow examination. All patients displayed marked and rapid improvement following initiation of dabrafenib. With a median follow up of 21 months, all patients remain in complete clinical remission, and are thriving. None of the patients experienced significant adverse effects classically associated with dabrafenib. In two patients, despite clinical disease resolution, BRAF V600E was still detectable in bone marrow by RTPCR at 23 and 26 months respectively post initiation of dabrafenib. Remarkably, the other two patients who were treated upfront with dabrafenib achieved molecular remissions (defined as undetectable BRAF V600E allele in a previously positive patient) after 11 and 12 months respectively of therapy. Among mice transplanted with patient bone marrow cells, the median survival was 9.6 weeks. A comparison arm consisting of mice transplanted with bone marrow from a low-risk LCH patient did not develop evidence of disease during the observation period. At necropsy, animals exhibited splenomegaly, thrombocytopenia and anemia. Flow cytometry of both bone marrow and spleens from mice demonstrated robust engraftment of human hematopoietic cells, and the BRAF V600E allele was detected from marrow and splenocytes in all mice. Frequent hemophagocytic forms were visible on bone marrow cytospins, and on histologic examination, bone marrow exhibited an abundance of CD1a-negative, CD163+ histiocytes, similar to findings observed in human disease. Bone marrow and splenocytes from primary NSG xenograft recipients were transplanted into secondary NSGS mice, and two out of three animals examined similarly became ill with comparable disease latency, and achieved engraftment of human cells with detectable BRAF V600E in bone marrow and splenocytes. Conclusions: For patients with LCH with hematologic involvement and secondary HLH, disease-initiating cells are present in the bone marrow and can engraft and recapitulate disease following primary and secondary transplant into immunodeficient mice. Targeted therapy with the BRAF V600E-specific inhibitor dabrafenib leads to durable, sustained remissions. Furthermore, we demonstrate that certain patients can achieve molecular remission with BRAF V600E-specific monotherapy alone. However, in at least a subset of patients with RO+ LCH, the persistence of mutated BRAF despite prolonged targeted therapy suggests that while quiescent, transformed disease-initiating cells are not eliminated by BRAF inhibition alone. Based on these results, we hypothesize that first-line therapy with BRAF inhibitors may be more likely to induce molecular remissions. Disclosures No relevant conflicts of interest to declare.

Description: Congenital dyserythropoietic anemias (CDA) are rare hereditary diseases of abnormal erythropoiesis. The CDA Registry of North America (CDAR) (NCT02964494) was opened in 2016 to investigate the natural history and molecular biology of CDA. CDA type I (CDA-I) is a recessive form of CDA characterized by macrocytic anemia, hemolysis with inadequate reticulocytosis, and iron overload. The bone marrow shows binucleated erythroblasts with chromatin bridges by light microscopy and spongy heterochromatin in erythroblasts by electron microscopy. The phenotypic heterogeneity in presentation and course of CDA-I is remarkable. Most CDA-I cases are caused by biallelic mutations in CDAN1or C15orf41, and 10-20% do not have an identifiable mutation. Non-hematological features, especially skeletal features, were historically reported in 10-20% of patients (Wickramasinghe, 1998). Due to the rarity of CDA-I and its clinical overlap with several disorders, the diagnosis is often missed or delayed by up to 17 yrs (median) (Roy, 2019). We describe in this study the characteristics and clinical course of CDA-I patients due to CDAN1 mutations enrolled in CDAR. Patients with a phenotypic diagnosis of CDA and their family members were enrolled in CDAR. Clinical and demographic data were gathered from participants at study entry and updated periodically thereafter. Participants elect to give blood, bone marrow, and DNA samples to the biorepository associated with CDAR. Participants with a phenotypic diagnosis of CDA-I and confirmed mutations in CDAN1 were included in this study. Six participants had a diagnosis of CDA-I due to biallelic CDAN1 mutations, comprising 18% (6/33) of affected CDAR participants. CDAN1 mutations were found in 75% of cases diagnosed phenotypically as CDA-I. All six participants presented early in life with a variable degree of non-immune hemolysis, and the diagnosis was confirmed within a median of 2 years from presentation. The characteristics of participants are summarized in table 1. Two had family history of stillbirth or fetal demise in older siblings due to hydrops fetalis. One participant presented prenatally with fetal anemia and started intrauterine transfusions at 24 weeks of gestation; 2 presented with severe anemia and signs of hydrops, pulmonary hypertension, transaminitis, severe hyperbilirubinemia, and thrombocytopenia at birth; and 3 presented with neonatal jaundice and moderate anemia. All participants required blood transfusions in the neonatal period. Three had spontaneous improvement and did not require transfusions after the first year of life. One remained transfusion-dependent at last follow up at the age of 4 yrs. One became transfusion-independent after starting interferon-alpha at 1 yr of age and did not need further transfusions even after discontinuation at 3 yrs of age. One had splenectomy at 11 y.o because he was misdiagnosed to have a membrane disorder but presented in adulthood with hemolytic anemia and pulmonary hypertension and was diagnosed at that time with CDA-I by genetic sequencing. All participants had one or more non-hematological manifestations, including hypertrophic skin folds, onychocryptosis, curved toenails, syndactyly, café-au-lait spots, macrocephaly, spinal fusion, scoliosis, and short stature. One participant suffered a thalamic stroke in the postnatal period, 2 had transient neonatal pulmonary hypertension in the setting of severe anemia, and one had pulmonary hypertension post-splenectomy in adulthood. Ferritin was high in all participants at last follow up, and 4 received chelation therapy. In summary, mutations in CDAN1 are the most common identified mutations in CDAR. CDA-I causes early-onset macrocytic anemia, which may present prenatally, with variable severity of hemolysis ranging from hydrops to mild neonatal jaundice and anemia. Non-hematological manifestations, mainly skeletal, nail and skin abnormalities are more common in CDA-I than previously reported, and their presence in infants with unexplained anemia should raise suspicion for the diagnosis. The availability of molecular testing has significantly accelerated the diagnosis. Management of patients with CDA-I requires multidisciplinary approach from an early age to improve outcome. Collaboration between clinicians, scientists, patients, and families is needed to advance the understanding and treatment of this rare disease. Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Kalfa:Agios: Other: local PI of clinical research trial; FORMA: Other: sponsored research agreement.

Description: The Runx1/core binding factor-β (CBFβ) transcriptional complex is required for the establishment of hematopoiesis during development. Despite its critical role during development, a detailed analysis of Runx1 expression within specific lineages and developmental stages of the adult hematopoietic system is lacking. To address this, we have developed a Runx1—green fluorescent protein (GFP) knock-in mouse. We show that Runx1 is expressed in several hematopoietic lineages, including myeloid, B-lymphoid, and T-lymphoid cells. By contrast, Runx1 is weakly expressed in early erythroid cells, and its expression is rapidly extinguished during later stages of erythropoiesis. Runx1 expression is induced during early B-cell development and is expressed at a uniform level during all subsequent stages of B-cell development. Within the thymus, Runx1 is expressed at the highest level in CD4-CD8- double-negative thymocytes. In peripheral T cells, Runx1 is differentially expressed, with CD4+ T cells expressing 2- to 3-fold higher levels of Runx1 than CD8+ cells. Taken together, these findings indicate that although widely expressed in the hematopoietic system, the expression of Runx1 is regulated in a cell type— and maturation stage—specific manner. In addition, the Runx1-IRES-GFP knock-in mouse strain should prove valuable for investigation of Runx1 function in adult hematopoiesis.

Description: CDAR (ClinicalTrials.gov Identifier: NCT02964494), a registry for patients with Congenital Dyserythropoietic Anemia (CDA) in North America, has been created with the goal to provide a longitudinal database and associated biorepository to facilitate natural history studies and research on the molecular pathways involved in the pathogenesis of CDAs. A 1 y.o. female patient with non-immune hemolytic anemia with suboptimal reticulocytosis, requiring frequent transfusions, and with the pathologic diagnosis of CDA was enrolled in CDAR. Her father had a similar phenotypical presentation in early childhood and underwent splenectomy at 3 years of age. Since then, he has rarely required transfusions but he continues to have a mild anemia at baseline with characteristics of hemolysis and with suboptimal reticulocytosis; at the time of enrollment, he had hemoglobin of 9.3 g/dL with absolute reticulocyte count of 115 x 106 cells/µl. Next Generation sequencing and deletion/duplication assay for the known CDA-associated genes (CDAN1, C15ORF41, SEC23B, KIF23, GATA1) identified no mutations. Whole-exome sequencing for the patient and her parents (family-trio design) revealed a novel PRDX2 missense variant (c.154C〉T; p.Pro52Ser) present in heterozygous state in both proband and her father; no mutation in this gene was present in the asymptomatic mother. In silico prediction programs suggest that this variant is probably damaging and deleterious, causing a non-conservative substitution of a phylogenetically highly-conserved amino acid (down to Baker's yeast), and located in an enzymatically active protein domain, adjacent to the active Cys51, with the potential to change its conformation. Peroxiredoxin II is highly expressed during terminal erythropoiesis and is one of the most abundant proteins after hemoglobin in erythroblasts and mature erythrocytes. It is an antioxidant enzyme that reduces the reactive oxygen species (ROS), like hydrogen peroxide and alkyl hydroperoxides readily produced within the erythroid cells due to the presence of heme iron and oxygen. In addition, PRDX2 has been implicated in intracellular signaling, cellular proliferation and differentiation, and as a regulator of iron homeostasis. PRDX2-/- mice were found to have hemolytic anemia with evidence of oxidative damage of the erythrocyte proteins resulting to decreased red blood cell (RBC) survival. The aim of this work is to validate the pathogenetic role of the PRDX2 variant found in this family as the molecular cause of this dominantly-inherited CDA and further investigate the role of PRDX2 in human terminal erythropoiesis. Central review of the patient's bone marrow aspirate and biopsy slides, according to the CDAR protocol, revealed erythroid hyperplasia with dyserythropoiesis, including megaloblastoid changes, nuclear lobation and fragmentation, and binucleated erythroblasts (less than 10%), compatible with atypical CDA. There were rare erythroids with cytoplasmic bridging but no nuclear bridges. Review of the peripheral blood smear showed significant poikilocytosis, mild polychromasia, and the presence of blister and ghost cells reminiscent of G6PD deficiency, pointing to RBC damage by oxidative stress. Induced pluripotent stem cells (iPSCs) and EBV-immortalized lymphocytes were generated from the patients' peripheral blood mononuclear cells after informed consent per CDAR protocol, to allow further in vitro studies of the peroxiredoxin II-deficiency. Flow cytometry confirmed significantly increased ROS in the patients' derived versus control EBV-immortalized lymphocytes as well as in the reticulocytes and mature erythrocytes of the proband and her father, indicating that their PRDX2 variant is causing loss-of-function of the enzyme and increased oxidative stress. Further work is ongoing to explore the mechanisms of pathogenicity of peroxiredoxin II deficiency towards human dyserythropoiesis and decreased erythrocyte lifespan. To our knowledge, this is the first case of anemia described in humans associated with PRDX2 mutation implicating this gene as a novel candidate gene for atypical, dominantly-inherited CDA. Disclosures No relevant conflicts of interest to declare.

Description: Despite significant advances in AML therapy over the past decades, 30-40% of all young patients with AML will suffer a relapse, after which achieving long-term disease-free survival remains challenging. In pediatric and young adult patients with AML, curative-intent treatment at relapse involves intensive induction therapy to achieve CR followed by consolidation with HSCT. Historically, initial response rates to salvage therapy were limited. Recently, Children's Oncology Group presented results from a Phase II study of CPX-351 (liposomal cytarabine:daunorubicin at a synergistic ratio) in pediatric patients with AML in first relapse demonstrating an impressive CR/CRp/CRi rate of 81.3%. Separately, we presented data from our first-in-pediatrics CPX-351 Phase I trial showing 48% M1 marrow response in a population consisting of children with multiply relapsed and refractory (R/R) AML patients. We also obtained single cell RNA sequencing on study patients' peripheral blasts before, during, and after CPX-351 treatment (i.e.: Days 0, 1 and 5). Preliminary data showed that CPX-351 differentially impacts p53 targets within leukemic blast clusters over time, with an enrichment for genes regulating apoptosis (e.g.: FAS, BAX) in most clusters. This suggests that these blasts may be primed for apoptosis following CPX-351 treatment. Anti-apoptosis signaling in leukemias is an established mechanism of chemoresistance. Venetoclax is an orally available, small molecule inhibitor of anti-apoptotic protein BCL-2, which is frequently overexpressed in leukemias. Based on published preclinical data and single cell RNA sequencing data from our CPX-351 trial, we developed a Phase I study to investigate the combination of CPX-351 and venetoclax in the treatment of young patients with R/R acute leukemias. The V2 trial is a single-institution Phase I pilot study (NCT03826992) designed to test the safety and tolerability of combining venetoclax with CPX-351 in patients ages 1-39 years for the treatment of R/R acute leukemias. Inclusion diagnoses are based on preclinical sensitivities and include AML, MPAL, or AUL in first or greater relapse, or ETP-ALL, MLL-rearranged ALL, or T-ALL in second or greater relapse. Patients must have measurable disease. Exclusion criteria include CNS status 3, bone marrow failure syndromes, and prior anthracycline and mediastinal radiation exposures above acceptable cardiotoxicity risk thresholds. Subjects receive a single course of study therapy consisting of daily venetoclax and CPX-351 at the FDA approved dose for adults on Days 1, 3, and 5 (Fig. 1) with the goal of CRMRD- to allow for off-study HSCT. The venetoclax dose exploration phase begins with a starting dose of 400 mg (or the allometrically-scaled equivalent) for 21 days with a 3-day ramp to target dose. The dose exploration phase proceeds on a rolling 6 design and will require 4-12 patients to determine RP2D. Provided a tolerable RP2D is found, a dose expansion phase will open. Primary endpoints are determination of RP2D and description of the safety profile including time to hematologic recovery. Secondary endpoints are estimations of CR/CRp/CRi +/- MRD- within the context of a phase I study, and evaluation of cancer therapy-related cardiac dysfunction (CTRCD) rates. Correlative studies include evaluation of venetoclax PK with concomitant CPX-351 using modeling & simulation with a combined limited sampling and pediatric opportunistic PK sampling (POPS) strategy, and exploration of the correlations between BCL-2 family expression and pediatric tumor genetics with treatment response. Figure 1 Disclosures O'Brien: BTG: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; AbbVie: Research Funding; Amgen: Research Funding; BMS: Research Funding. Phillips:Novartis: Membership on an entity's Board of Directors or advisory committees. Vinks:Myriad Genetics: Consultancy, Patents & Royalties. Perentesis:Kurome Therapeutics: Consultancy. Absalon:Jazz Pharmaceuticals: Other: CPX-351 for clinical trial . OffLabel Disclosure: This clinical trial involves the off-label use of CPX-351 and venetoclax for relapsed/refractory pediatric and young adult leukemias.

Description: BACKGROUND: The histiocytic disorders include several heterogeneous diseases including Langerhans cell histiocytosis (LCH,) juvenile xanthogranuloma, Erdheim-Chester disease (ECD,) and Rosai-Dorfman disease. These conditions have variable clinical courses and can be refractory to contemporary therapy, resulting in end-organ damage or even death. The etiopathogenesis of LCH and the other histiocytoses remained unclear for decades, until the identification of recurring BRAF V600E mutations, and more recently mutations in the downstream gene MAP2K1 (encoding the enzyme MEK) in LCH and ECD. Given the high risk of recurrence and the unpredictable response to therapy in some patients, we sought to characterize the genomic landscape of histiocytic lesions in patients in real-time. Our principal goal was to select alternative treatments for patients with inadequate response to standard therapies. As a secondary goal, we aimed to further characterize the biological effects of MAP2K1 mutations found in LCH and ECD, and determine their susceptibility to targeted therapies. METHODS AND RESULTS: We used a hybrid capture-based sequencing platform to molecularly profile eighty-five patient samples from patients with one of the above diagnoses. Fifteen patient samples (18%) harbored the BRAF V600E point mutation, and four LCH patients carried a novel 5 or 6 amino-acid in-frame deletion (indel) in BRAF (N486_P490del or N486_T491〉K.) Eleven patient samples (13%) harbored activating mutations in MAP2K1. Additional recurrently altered genes included NRAS, KRAS and CDKN2A/B. Transcriptomic profiling also identified several patients with recurrent ALK gene fusions, previously described in other malignancies and recently also identified in histiocytosis not-otherwise-specified. One patient with multisystem LCH with CNS involvement was found to have a BRAF indel. She declined systemic chemotherapy, but agreed to treatment with a targeted agent. Based on the likelihood of resistance to BRAF V600E-specific inhibitors, we started treatment with the MEK inhibitor Trametinib resulting in resolution of disease-associated lymphadenopathy within days, and improvement of CNS symptoms as well. She remains in remission 4 months after the initiation of treatment. In two children, multi-system refractory LCH progressed to secondary HLH (hemophagocytic lymphohistiocytosis). Both demonstrated the presence of BRAF-V600E and their disease promptly responded to the BRAF inhibitor Dabrafenib. We then characterized the biological behavior of the MAP2K1 mutations using retroviral transduction in order to stably express these mutations in NIH/3T3 and BaF/3 cells. We demonstrate that these mutations all result in constitutive activation at baseline, as evidenced by increased phosphorylation of the target ERK. These mutant forms of MAP2K1 also express sustained activation of ERK in response to EGF stimulation. Additionally, we tested clinically available MEK inhibitors against mutant forms of MAP2K1, and show that all result in a dose-dependent decrease in phospho-ERK levels in vitro, supporting our hypothesis that MEK inhibition is a valid therapeutic approach in the histiocytic neoplasms. Finally, we demonstrate with an animal model that MAP2K1 is sufficient to induce disease. Using the cre-lox recombinase system in transgenic mice, we selectively express an activated form of MEK in CD11c-positive cells, which is largely restricted to the dendritic cell/macrophage lineage. These mice developed normally, but by a median of 17 weeks of age, mice became moribund and on necropsy exhibited hepatosplenomegaly with extensive infiltration of spleen, liver and lungs with CD68+ macrophages. DISCUSSION: Genomic profiling identified mutations in majority of patients, including a novel BRAF indel. We further show that these mutations result in activation of the MAP kinase pathway, and that activated MAP2K1 is capable of transforming hematopoietic cells resulting in a multisystem histiocytic disorder in mice. Finally, we demonstrate that available MEK inhibitors efficiently block disease-associated mutations. We propose that all patients with histiocytic neoplasms undergo comprehensive genomic profiling in order to identify potential causal mutations, and clinical trials for histiocytoses include MEK inhibitors in relapsed/refractory disease or even as upfront therapy. Disclosures Ali: Foundation Medicine: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Stevens:Foundation Medicine Inc.: Employment, Equity Ownership. Ross:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership.

Description: Follicular lymphoma is a rare lymphoid malignancy in pediatric patients and consequently remains poorly characterized, particularly with respect to its immunophenotype and molecular pathogenesis. A total of 23 pediatric patients with follicular lymphoma were identified, with a median age of 11 years and a male-to-female ratio of 2.3:1. Of the 19 patients for whom presenting clinical features were available, 15 patients had stage I, 1 had stage II, and 3 had stage III or IV disease. All tumors had a follicular architecture, and 74% of cases had grade 2 or 3 histologic features. All patients expressed CD20 and bcl-6, and 80% were positive for CD10. Bcl-2 expression was detected in only 5 of 16 cases. Consistent with this finding, bcl-2 gene rearrangements were detected in only 2 of 16 cases by polymerase chain reaction. These patients were treated primarily with cyclophosphamide, doxorubicin, vincristine, and prednisone–based chemotherapy; 4 patients also received involved-field irradiation. Of the 13 patients with available clinical follow-up, all but 2 achieved durable clinical remission. Importantly, all 4 patients with tumors diffusely positive for bcl-2 either presented with stage III/IV disease or had disease refractory to therapy, whereas patients with bcl-2–negative tumors uniformly had stage I disease, achieved complete remission, and experienced no relapses. These findings indicate that, in contrast to adult follicular lymphomas, dysregulated bcl-2 expression does not play a significant pathogenetic role in most pediatric follicular lymphomas. However, bcl-2 expression in pediatric follicular lymphoma identifies a subset of patients in whom disease is often disseminated at clinical presentation and is more refractory to combination chemotherapy.