ALBERT

Description: Introduction Burkitt Lymphoma (BL) is a highly aggressive hematological malignancy that originates from germinal center B-cells, is characterized by IG/MYC translocation, and affects both children and adults. Clinical and biological differences have been noted between endemic BL, which occurs in equatorial Africa and is virtually always associated with Epstein-Barr virus (EBV) infection, and sporadic BL that is infrequently associated with EBV. BL is highly curable using an aggressive chemotherapy regimen, but the intensive supportive care required to manage the toxicities of this treatment precludes its use for most patients with endemic BL, resulting in poorer survival for those patients. Although previous genetic studies have identified recurrent mutations in BL (including alterations in ID3 and its downstream targets TCF3 and CCND3), endemic BL has not been as well characterized and the extent of differences between sporadic and endemic BL (and their potential relation to pathogenesis and response to therapy) is not yet clear. Therefore, more extensive genomic characterization of both sporadic and endemic BL is needed to provide insight into tumor biology and to identify novel therapeutic targets that can be utilized to provide less toxic treatments. Methods We interrogated frozen tumor and matched normal blood samples from a cohort of 30 pediatric BL cases, 12 of which were collected from Uganda and 18 from Texas Children's Cancer Center (Houston, TX). Whole exome sequencing (WES) and copy number analysis were performed on the Illumina platform using the OmniExpress array and VCrome 2.1 WES capture reagent and analyzed utilizing the Baylor College of Medicine Human Genome Sequencing Center bioinformatic pipeline. A median of 135x average coverage and 〉97% of targeted bases with at least 20x coverage was observed for WES. Results WES revealed a median of 46 nonsilent somatic mutations per case for endemic BL (range 19-207), and 32 per case for sporadic BL (range 13-119). Evidence of the EBV genome was detected in all endemic BL samples and 3/18 (17%) of sporadic BL cases. Mutations were found in genes known to be frequently mutated in BL, including MYC in 7/12 (58%) of endemic cases and 12/18 (67%) of sporadic cases and TP53 in 5/12 (42%) and 9/18 (50%), respectively. Of note, mutations in DDX3X (7/12 [58%] endemic, 9/18 [50%] sporadic) and FOXO1 (5/12 [42%] endemic, 7/18 [39%] sporadic) were identified frequently in our cohort. As previously described, the ID3 pathway was more frequently targeted by mutations in sporadic BL: ID3 in 8/18 (44%) sporadic cases vs 3/12 (25%) endemic, TCF3 in 3/18 (17%) vs. 1/12 (8%), and CCND3 in 7/18 (39%) vs 2/12 (17%), respectively. Mutations in the SWI/SNF chromatin-remodeling genes ARID1A and SMARCA4 have been reported to occur in BL in a mutually exclusive fashion. In our cohort, mutations in SMARCA4 were exclusive to sporadic cases (9/18, 50%) and not found in endemic tumors (P = 0.01). Conversely, ARID1A mutations were much more frequent in endemic cases (7/12, 58%) as compared to sporadic ones (3/18, 17%) (P 〈 0.05). Only one sporadic case was found to have both genes mutated. Copy number analysis did not reveal recurrent focal copy number deletions. Amplification of 13q31.2 - q32.2 was detected in sporadic cases (4/18, 22%) but not in endemic cases, while focal amplification of 7p14.1 (3/10, 30%) and 14q11.2 (4/10, 40%) were exclusive to endemic tumors. Conclusions These findings provide novel insight into the landscapes of genomic alterations in pediatric endemic and sporadic BL. Our data confirm the recurrence of mutated genes previously associated with BL and highlight differences between endemic and sporadic BL, most notably, the exclusivity of SMARCA4 mutations in sporadic BL cases in this cohort. The recurrence of mutations in ARID1A and SMARCA4 emphasizes the critical role of these SWI/SNF proteins in BL. More extensive molecular studies (whole genome and transcriptome sequencing) of this cohort are ongoing and may reveal additional differences between endemic and sporadic BL. Additional studies will be required to more precisely assess the frequency of these alterations in BL and their link to clinical features of the disease, as well as the biological relevance of the BL genes identified through these genomic analyses. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Background: Langerhans Cell Histiocytosis (LCH) is an inflammatory myeloid neoplasia. Activating somatic mutations in MAPK pathway genes have been identified in almost all cases of LCH with BRAF-V600E as the most common somatic mutation, occurring in approximately 60% of lesions. Enforced expression of BRAF-V600E in myeloid precursors recapitulates an LCH-like phenotype in mice, supporting a central pathogenic role for MAPK activation in LCH. Given the near universal occurrence and central pathogenic role of activating MAPK pathway gene mutations in LCH, targeted inhibition of the MAPK pathway is a rationale therapeutic strategy to explore in patients with relapsed disease. Design/Methods: Medical records from 20 pediatric patients with LCH (systemic and/or LCH-associated neurodegeneration) from 12 institutions were systematically reviewed; 2 patients had disease which was mixed LCH with juvenile xanthogranuloma (JXG) and 2 patients had secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS). All patients had failed at least one prior systemic therapy and had a proven MAPK pathway somatic mutation. Response assessment was based on applicable criteria for each individual including PET (metabolic) criteria, bone marrow evaluation, brain MRI, clinical history and physical exam. Results: All patients in this series were less than 21 years old (median age at start of therapy 6.9 years; range: 0.4-20.7 years) with median disease duration of 4 years prior to start of MAPK inhibitor (range 0.07-18.4 years). Twelve patients had LCH-ND diagnosed clinically and/or by radiographic imaging, while the remaining 8 patients had systemic disease with no LCH-ND (7 with high risk organ involvement). In terms of best response, 3 patients (15%) achieved a complete response (CR) and eleven patients (55%) achieved a partial response (PR), while 3 patients (15%) only achieved stable disease (SD), and 1 patient (5%) died early on in therapy due to progressive disease (PD) complicated by secondary HLH/MAS. Of the 12 patients who had LCH-ND, none achieved a CR, but 10 (83%) had a best response of PR by either clinical or radiographic assessment. Median progression free survival (PFS) was 11.8 months (range 2-36 months), while median time to disease progression or recurrence was 4.6 months (range 1-46 months). Four of the 20 patients (20%) had a Grade 3 or 4 toxicity; 2 of these patients required dose modification in order to successfully resume therapy. Seven patients had measurable peripheral blood mononuclear cells (PBMC) or bone marrow cells with detectable BRAF-V600E mutation just prior to start of MAPK inhibitor with subsequent assessments obtained during therapy. Conclusions: MAPK pathway inhibitors may be a relatively safe salvage therapy for refractory systemic LCH and LCH-ND but the efficacy and durability of responses with this strategy remains to be delineated. Children with relapsed/refractory high-risk LCH who were at the highest risk of death from disease generally benefited from this strategy. Patients with longstanding neurodegenerative disease seem to have the least benefit from MAPK inhibitors. However, patients with relatively early onset neurodegenerative disease, especially without clinical manifestations, seem to have the greatest benefit. While the presence of persistent BRAF-V600E+ circulating cells appears to be associated with risk of relapse and type of LCH organ involvement, quantifiable changes in these levels did not consistently correlate with clinical disease activity or response (in contrast to those treated with chemotherapy). We hypothesize that inhibition of the MAPK pathway may confer clinical benefit by blocking differentiation and proliferation of lesions, but may not have cytotoxic effect on precursor cells. This is supported by the observation that MAPK regulates lesion progression by inhibiting CCR7 expression, thereby blocking emigration of LCH cells from the lesion. Persistence of BRAF-V600E+ mononuclear cells in blood and bone marrow even in patients with impressive clinical responses may underlie the high rates of eventual progression/relapse with this type of treatment. Future prospective trials of MAPK pathway inhibitors for patients with refractory LCH possibly in combination with chemotherapy will be needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by lesions including pathogenic CD207+ dendritic cells among an inflammatory infiltrate, and has a median age at diagnosis of 30 months. Approximately 50% of children with LCH relapse, and 40% experience a second relapse event within two years. Sequencing studies have identified activating somatic mutations in MAPK pathway genes in ~85% of LCH lesions. Notably, LCH cases who are carriers of BRAFV600E+ experience a 2-fold increased risk of relapse. However, the role of inherited genetic effects in LCH relapse remains unknown. Therefore, we conducted a genome-wide association study (GWAS) to characterize the role of inherited genetic variants on risk of LCH relapse. Methods: LCH cases (n=117) for this discovery GWAS were recruited from Texas Children's Hospital, of which 52 patients experienced a relapse event and 65 patients did not. Genotyping was performed on the Illumina Omni5 Quad BeadChip. We tested the association between common variants (minor allele frequency 〉5%) and LCH relapse risk in PLINK. A genome-wide threshold of significance was applied at P-value

Description: Introduction: Hematopoietic stem cell transplantation (HSCT) for patients with hemophagocytic lymphohistiocytosis (HLH) following myeloablative conditioning regimens (MAC) is associated with high rates of non-relapse mortality. A previously reported prospective, phase 2 multi-center trial (RICHI) using using RIC strategy of fludarabine, melphalan and alemtuzumab (day -14) for HLH and primary immune deficiency syndromes (PIDS) demonstrated improved mortality rates but fewer than half the patients with HLH (41%) successfully engrafted without secondary graft failure, need for donor lymphocyte infusion (DLI) or second transplant. Incorporation of thiotepa during conditioning has been to shown to be safe and improve engraftment. We report the results of a retrospective analysis of nine consecutive patient treated with the inclusion of thiotepa into the RICHI backbone (RICHI+TT). Methods: Patients received a single additional dose of thiotepa 10mg/kg on day -3 added to the fludarabine/melphalan/alemtuzumab backbone (RICHI+TT) with the same graft-versus-host disease prophylaxis of methyprednisolone through day +28 and cyclosporine through day 180. To determine sustained engraftment, we used the same parameters the RICHI study defined as 〉 5 % donor chimerism without any intervention and alive at 1 year post-transplant. Results: Our cohort consisted of 8 males and 1 female with a median age of 7 years (range 1-18 years). Seven patients had HLH with proven pathogenic genetic mutations (biallelic PRF1 - 2, UNC13D - 2, STXBP1- 1, RAB27A-1, STAT3 gain of function-1), while the other 2 patients had HLH without identified pathogenic mutations (1- chronic active EBV, 1- juvenile idiopathic arthritis with refractory macrophage activation syndrome).The majority of patients received a bone marrow product (n = 8), one patient received a peripheral blood stem cell product; 6 patients received a graft from a matched related donor , two from a mismatched unrelated donor, and one from a matched unrelated donor. All patients engrafted at a median of 15 days post-transplant (8 patients at 100% donor chimera; 1 patient at 99% donor chimera at initial engraftment). Six of the 9 patients were evaluable to assess donor chimerism at 1 year as per study definitions with a median follow up of 875 days (range: 366 -1000 days). All 6 patients had 〉 5% donor chimerism and were alive at 1 year. Five of the 6 evaluable patients met criteria for sustained donor engraftment without need for intervention and all maintained 100% donor chimerism at last follow-up (Table 1). Only one of the six patients had evidence of falling donor chimerism; this stabilized at 40% donor chimerism after DLI. No patients had primary or secondary graft failure. Three patients were not evaluable for long-term assessment due to death prior to 1 year. Six of the 9 patients described here are alive and disease-free with stable long-term engraftment. The incorporation of Thiotepa to the RICHI backbone improved on previously reported sustained donor engraftment (Table 2). Conclusions: The RICHI+TT approach had better long-term donor engraftment with a decreased need for DLI or second transplant without increased rates of non-relapse mortality. Prospective studies are needed to determine the optimal treatment strategies for patients with HLH who require HSCT for cure. Disclosures Heslop: Cell Medica: Research Funding; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Allovir: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Despite indistinguishable histology and the common feature of Birbeck granules in lesion biopsies, clinical presentation of patients with Langerhans Cell Histiocytosis (LCH) is highly variable, from single lesion cured by curretage, to multi-system disease requiring aggressive chemotherapy or stem cell transplant. Risk stratification for Langerhans Cell Histiocytosis has historically assigned clinical risk groups based on anatomic location and extent of LCH lesions, which is the basis for dose and duration of chemotherpy on recent Histiocyte Society trials. In this study, we test the hypothesis that distinct subgroups of patients with LCH may be identified by relative levels of circulating biomarkers. Methods Pre-therapy plasma was collected on 97 patients with LCH (82 Pediatric: 17 High-Risk, 23 Multisystem/Multifocal “Non-risk”, 42 Single Lesion “Non-risk”; 15 Adult: 5 High-Risk, 5 Multisystem/Multifocal “Non-risk”, 5 Single Lesion “Non-risk”) and 49 control subjects (32 Pediatric, 17 Adult). Quantitative levels of plasma proteins (158 analytes) was determined by multiplex analysis with Millipore MagPix kits and the Luminex plate reader. Data were analyzed with both unsupervised and supervised methodologies. Results Consensus clustering with non-negative matrix factorization (NMF) clusters identified three groups which were analyzed along with clinical categories. Significant clinical variables included age (adult samples clustered in NMF group 1) and LCH risk category (High-Risk LCH samples clustered in NMF group 3). Samples from patients with the BRAF-V600Emutation or relapse within 1 year did not cluster into any NMF group with signifiance. Additionally, supervised analysis identified specific molecules that were significantly differentially expressed between different clinical categories after multiple testing correction (FDR

Description: Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

Description: Purpose: Tumor necrosis factor alpha (TNF-alpha) is produced in Langerhans cell histiocytosis (LCH) lesions and is elevated in active high-risk vs low-risk LCH vs control patient blood, where TNF-alpha has been reported at 12 pg/ml in juvenile rheumatoid arthritis (JRA). Anti-TNF therapeutic etanercept is used in TNF-mediated diseases, such as JRA, and was successfully used in one case of LCH. We therefore conducted phase II study for etanercept therapeutic efficacy for refractory or relapsed LCH patients. Methods: Luminex platform was used to assess patient blood TNF-alpha level. This phase II study was approved by the Baylor College of Medicine IRB with research support from Amgen/Immunex. Eligibility included LCH patients with progressive disease post-initial treatment and ≥1 salvage therapy. Etanercept was injected 0.4mg/kg subcutaneously twice/week for 12 weeks. Therapeutic response were evaluated at 4 and 8 weeks. Results: TNF-alpha is elevated in blood LCH high-risk (average 45pg/ml) vs low-risk (average 13pg/ml) vs control (average 13pg/ml). Five LCH patients (1.6-42 years) with multi-system involvement, 2 with high-risk disease were enrolled. A median of 5 doses of etanercept were administered (range 1-14). One high-risk patient died 18 days after the first dose from disease progression. At week 4 evaluation, one patient had stable disease and 3 progressed and all subjects progressed by week 8. Ultimately 0/5 patients had disease improvement with etanercept. Conclusion: Etanercept therapy, at JRA-effective dose, in this trial did not improve LCH disease. It is possible that this may not be an effective strategy. Alternatively, it is also possible that drug dose or distribution was suboptimal. While anti-TNF therapies, including etanercept, are effective for treatment of RA, etanercept is not effective for granulomatous diseases such as Crohn's, sarcoidosis or Wegner's disease. Additional studies exploring potential efficacy of higher dose or alternative forms of TNF-alpha inhibition in LCH may be warranted. Disclosures No relevant conflicts of interest to declare.