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Survivin protein determination in multiple neuroblastoma lines

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling.Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53.Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination.Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629–39. ©2017 AACR.

Effect of combined RG7388 and temsirolimus on human PBMCs viability and apoptosis

Supplementary Material and Figures "clean"

Temsirolimus blocks in vivo tumor growth by suppressing mTOR activity.

The combination therapy temsirolimus and RG7388 does not affect the expression of other apoptotic/anti-apoptotic proteins

Effect of combined RG7388 and temsirolimus on viability of human PBMCs subpopulations

Effect of combined RG7388 and temsirolimus on colony-forming unit generation of CD34+ Hematopoietic Stem-Progenitor Cells

Effect of combined treatment on cell viability

p53 knockdown in neuroblastoma cells

Bioluminescence data of xenograft tumors upon treatment suspension

Purpose To determine the maximum tolerated dose (MTD) of autologous chimeric antigen receptor T cells (HER2-CART) after lymphodepletion and to describe the cellular kinetics, safety, feasibility of repeat treatment cycles, and antitumor activity of HER2-CART in patients with advanced sarcoma. Patients and Methods On this phase I study, 13 patients with progressive, metastatic HER2+ sarcoma were treated in 14 enrollments; one patient who achieved complete remission was re-enrolled because of disease recurrence. Lymphodepletion consisted of fludarabine (Flu) 25 mg/m2/dose x 5 days with or without cyclophosphamide (Cy) 30 mg/kg/dose x 2 days. The dose cohorts evaluated include 1x108 total T-cells/m2 after Flu (cohort A); 1x108 total T-cells/m2 after Flu/Cy (cohort B) and 1x108 CAR-positive T-cells/m2 after Flu/Cy (cohort C). The evaluation of safety was the primary endpoint. Secondary outcomes included assessment of cellular kinetics and antitumor activity. Results All study participants received at least one infusion of HER2-CART after lymphodepletion and were included in the analysis (median age 14 years; range 4-55 years). Seven received multiple HER2-CART infusions. CART expansion was observed after all but two of 21 total infusions given after lymphodepletion. Nine of 12 (75%) patients in cohorts A and B developed grade 1-2 CRS. Two patients in cohort C experienced dose limiting toxicity (DLT) with grade 3-4 CRS. All patients recovered from treatment-related adverse events during study follow-up. Median OS was 8.2 months (95% CI, 4.4-17 months). Antitumor activity was observed at the MTD (cohort B), with 1-year OS and PFS of 62.5% (95% CI, 22.9%-86.1%) and 33.3% (95% CI, 7.8-62.3%), respectively. Three patients remain alive, with one in long-term remission at the sixth year of follow-up. While infused CART products did not exhibit a distinct immunophenotype by sarcoma histology, we observed different trends in expression of immune checkpoints when compared by disease and by treatment response. Conclusion and Relevance: Autologous HER2-CART can be infused safely after lymphodepletion in patients with progressive sarcoma, and the preliminary evidence of antitumor activity observed supports testing their efficacy in a phase II trial. Trial Registration: ClinicalTrials.gov identifier: NCT00902044

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder that is characterized by the inflammatory lesions with pathogenic CD1a(+)CD207(+) dendritic cells (DCs). BRAFV600E and other somatic activating MAPK gene mutations have been identified in differentiating bone marrow and blood myeloid cells, but the origin of the LCH lesion CD1a(+)CD207(+) DCs and mechanisms of lesion formation remain incompletely defined. To identify candidate LCH CD1a(+)CD207(+) DC precursor populations, gene-expression profiles of LCH lesion CD1a(+)CD207(+) DCs were first compared with established gene signatures from human myeloid cell subpopulations. Interestingly, the CD1c(+) myeloid DC (mDC) gene signature was most enriched in the LCH CD1a(+)CD207(+) DC transcriptome. Additionally, the BRAFV600E allele was not only localized to CD1a(+)CD207(−) DCs and CD1a(+)CD207(+) DCs, but it was also identified in CD1c(+) mDCs in LCH lesions. Transcriptomes of CD1a(+)CD207(−) DCs were nearly indistinguishable from CD1a(+)CD207(+) DCs (both CD1a(+)CD207(low) and CD1a(+)CD207(high) subpopulations). Transcription profiles of LCH lesion CD1a(+)CD207(+) DCs and peripheral blood CD1c(+) mDCs from healthy donors were compared to identify potential LCH DC-specific biomarkers: HLA-DQB2 expression was significantly increased in LCH lesion CD1a(+)CD207(+) DCs compared with circulating CD1c(+) mDCs from healthy donors. HLA-DQB2 antigen was identified on LCH lesion CD1a(+)CD207(−) DCs and CD1a(+)CD207(+) DCs as well as on CD1c(+)(CD1a(+)CD207(−)) mDCs, but it was not identified in any other lesion myeloid subpopulations. HLA-DQB2 expression was specific to peripheral blood of patients with BRAFV600E(+) peripheral blood mononuclear cells, and HLA-DQB2(+)CD1c(+) blood cells were highly enriched for the BRAFV600E in these patients. These data support a model in which blood CD1c(+)HLA-DQB2(+) mDCs with activated ERK migrate to lesion sites where they differentiate into pathogenic CD1a(+)CD207(+) DCs.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by pathologic immune activation in which prompt recognition and initiation of immune suppression is essential for survival. Children with HLH have many overlapping clinical features with critically ill children with sepsis and systemic inflammatory response syndrome (SIRS) in whom alternative therapies are indicated. To determine whether plasma biomarkers could differentiate HLH from other inflammatory conditions and to better define a core inflammatory signature of HLH, concentrations of inflammatory plasma proteins were compared in 40 patients with HLH to 47 pediatric patients with severe sepsis or SIRS. Fifteen of 135 analytes were significantly different in HLH plasma compared with SIRS/sepsis, including increased interferon-γ (IFN-γ)–regulated chemokines CXCL9, CXCL10, and CXCL11. Furthermore, a 2-analyte plasma protein classifier including CXCL9 and interleukin-6 was able to differentiate HLH from SIRS/sepsis. Gene expression in CD8+ T cells and activated monocytes from blood were also enriched for IFN-γ pathway signatures in peripheral blood cells from patients with HLH compared with SIRS/sepsis. This study identifies differential expression of inflammatory proteins as a diagnostic strategy to identify critically ill children with HLH, and comprehensive unbiased analysis of inflammatory plasma proteins and global gene expression demonstrates that IFN-γ signaling is uniquely elevated in HLH. In addition to demonstrating the ability of diagnostic criteria for HLH and sepsis or SIRS to identify groups with distinct inflammatory patterns, results from this study support the potential for prospective evaluation of inflammatory biomarkers to aid in diagnosis of and optimizing therapeutic strategies for children with distinctive hyperinflammatory syndromes.

Hodgkin lymphoma (HL) histopathology is characterized by rare malignant Reed&ndash, Sternberg cells among an inflammatory infiltrate. We hypothesized that characteristics of inflammation in pediatric HL lesions would be reflected by the levels of inflammatory cytokines or chemokines in pre-therapy plasma of children with HL. The study objectives were to better define the inflammatory pre-therapy plasma proteome and identify plasma biomarkers associated with extent of disease and clinical outcomes in pediatric HL. Pre-therapy plasma samples were obtained from pediatric subjects with newly diagnosed HL and healthy pediatric controls. Plasma concentrations of 135 cytokines/chemokines were measured with the Luminex platform. Associations between protein concentration and disease characteristics were determined using multivariate permutation tests with false discovery control. Fifty-six subjects with HL (mean age: 13 years, range 3&ndash, 18) and 47 controls were analyzed. The cytokine/chemokine profiles of subjects with HL were distinct from controls, and unique cytokines/chemokines were associated with high-risk disease (IL-10, TNF-&alpha, IFN-&gamma, IL-8) and slow early response (CCL13, IFN-&lambda, 1, IL-8). TNFSF10 was significantly elevated among those who ultimately relapsed and was significantly associated with worse event-free survival. These biomarkers could be incorporated into biologically based risk stratification to optimize outcomes and minimize toxicities in pediatric HL.

Mutations in members of the mitogen-activated protein kinase (MAPK) pathway are extensively studied in epithelial malignancies, with BRAF mutations being one of the most common alterations activating this pathway. However, BRAF mutations are overall quite rare in hematological malignancies. Studies over the past decade have identified high-frequency BRAF(V600E), MAP2K1, and other kinase alterations in two groups of MAPK-driven hematopoietic neoplasms: hairy cell leukemia (HCL) and the systemic histiocytoses. Despite HCL and histiocytoses sharing common molecular alterations, these are phenotypically distinct malignancies that differ in respect to clinical presentation and suspected cell of origin. The purpose of this review is to highlight the molecular advancements over the last decade in the histiocytic neoplasms and HCL and discuss the impact these insights have had on our understanding of the molecular pathophysiology, cellular origins, and therapy of these enigmatic diseases as well as perspectives for future research directions.

Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–like histiocytosis in immune-deficient mice. BRAFV600E-expressing human CB CD34+ HSPCs did not generate hairy cell leukemia in xenograft mouse models.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent MAPK pathway activation. Standard-of-care chemotherapies are inadequate for most patients with multisystem disease, and optimal strategies for relapsed and refractory disease are not defined. The mechanisms underlying development of inflammation in LCH lesions, the role of inflammation in pathogenesis, and the potential for immunotherapy are unknown. Analysis of the immune infiltrate in LCH lesions identified the most prominent immune cells as T lymphocytes. Both CD8+ and CD4+ T cells exhibited “exhausted” phenotypes with high expression of the immune checkpoint receptors. LCH DCs showed robust expression of ligands to checkpoint receptors. Intralesional CD8+ T cells showed blunted expression of Tc1/Tc2 cytokines and impaired effector function. In contrast, intralesional regulatory T cells demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1 or MAPK inhibitor reduced lesion size, but with distinct responses. Whereas MAPK inhibitor treatment resulted in reduction of the myeloid compartment, anti-PD-1 treatment was associated with reduction in the lymphoid compartment. Notably, combined treatment with MAPK inhibitor and anti-PD-1 significantly decreased both CD8+ T cells and myeloid LCH cells in a synergistic fashion. These results are consistent with a model that MAPK hyperactivation in myeloid LCH cells drives recruitment of functionally exhausted T cells within the LCH microenvironment, and they highlight combined MAPK and checkpoint inhibition as a potential therapeutic strategy.

Hogstad et al. show that the somatic BRAFV600E mutation in myeloid dendritic cell precursors in Langerhans cell histiocytosis promotes lesion formation through impaired dendritic cell migration and resistance to apoptosis, which can be rescued with targeted MAPK pathway inhibition., Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal–related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)–mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.

To the editor: Langerhans cell histiocytosis (LCH) is a hematologic disorder that presents with a wide spectrum of symptoms, ranging from focal lesions to potentially lethal multiorgan disease, affecting 4 to 8 per million children per year[1][1] and 1 to 2 per million adults per year.[2][2]

Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling. Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53. Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination. Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629–39. ©2017 AACR.

// Rikhia Chakraborty 1, 2 , Oliver A. Hampton 3, 5 , Harshal Abhyankar 1, 2 , Daniel J. Zinn 1, 2 , Amanda Grimes 1, 2 , Brooks Skull 1, 2 , Olive Eckstein 1, 2 , Nadia Mahmood 6 , David A. Wheeler 3, 5 , Dolores Lopez-Terrada 1, 4 , Tricia L. Peters 4 , John M. Hicks 4 , Tarek Elghetany 4 , Robert Krance 1, 2, 7 , Poulikos I. Poulikakos 8, 9, 10 , Miriam Merad 8, 9, 11 , Kenneth L. McClain 1, 2 , Carl E. Allen 1, 2 and Donald W. Parsons 1, 2, 3, 4, 5 1 Texas Children’s Cancer Center, Texas Children’s Hospital, Houston, TX 77030, USA 2 Department of Pediatrics, Division of Pediatric Hematology-Oncology, Baylor College of Medicine, Houston, TX 77030, USA 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA 5 Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA 6 Body and Nuclear Radiology Sections, Texas Children’s Hospital, Houston, TX 77030, USA 7 Center for Cell and Gene Therapy, Houston, TX 77030, USA 8 Department of Oncological Sciences, Icahn School of Medicine, New York, NY 10029, USA 9 Tisch Cancer Institute, Icahn School of Medicine, New York, NY 10029, USA 10 Immunology Institute, Icahn School of Medicine, New York, NY 10029, USA 11 Department of Dermatology, Icahn School of Medicine, New York, NY 10029, USA Correspondence to: Donald W. Parsons, email: dwparson@txch.org Carl E. Allen, email: ceallen@txch.org Keywords: juvenile xanthogranuloma, MAPK1 , ERK activation, histiocytic disorder, somatic mutation Received: October 13, 2016 Accepted: March 13, 2017 Published: April 29, 2017 ABSTRACT Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that is usually benign and self-limiting. We present a case of atypical, aggressive JXG harboring a novel mitogen-activated protein kinase (MAPK) pathway mutation in the MAPK1 gene, which encodes mitogen-activated protein kinase 1 or extracellular signal-regulated 2 (ERK2). Our analysis revealed that the mutation results in constitutive ERK activation that is resistant to BRAF or MEK inhibitors but susceptible to an ERK inhibitor. These data highlight the importance of identifying specific MAPK pathway alterations as part of the diagnostic workup for patients with histiocytic disorders rather than initiating empiric treatment with MEK inhibitors.

Background & Aim Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the highly immunosuppressive tumor microenvironment. These adoptively transferred CAR T cells are susceptible to inhibition via the engagement of inhibitory receptors on their surface including PD1, CTLA4, LAG3 and BTLA. The recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of these co-inhibitory receptors may represent a common mechanism of T cell inhibition. Upon its recruitment and activation, SHP2 dephosphorylates key stimulatory signaling molecules, thus limiting T cell activation. We hypothesized that disrupting SHP2 expression will simultaneously offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Methods, Results & Conclusion CRISPR/Cas9 technology was used to knockout (KO) SHP2 from human T cells. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 signaling endodomain) targeting HER2. The phenotype of wild-type and edited CAR T cells was investigated using mass cytometry and confirmed with flow cytometry. Their function was tested in vitro using xcelligence (an electrical impedance-based long-term cytotoxicity assay) and in vivo in an orthotopic xenograft mouse model of GBM. Efficient SHP2 KO in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect T cell expansion, proliferation or phenotypic profile. However, SHP2KO CAR T cells eliminated LN229 cells (a GBM cell line) in vitro more efficiently compared to wild-type CAR T cells. In vivo, SHP2KO CAR T cells showed better control of engrafted LN229 tumor cells and improved the survival of treated mice in comparison to wild-type CAR T cells. In conclusion, these results suggest that SHP2 may limit the function of CAR T cells and that SHP2 deletion may improve their anti-GBM activity.

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAF(V600E). We recently discovered that the BRAF(V600E) mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAF(V600E) mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAF(V600E) mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.

Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2173.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with constitutive activation of the MAPKinase RAS-RAF-MEK-ERK cell signaling pathway. We analyzed 9 LCH cases without BRAF V600 and MAP2K1 mutations by whole exome sequencing. We identified a new somatic BRAF splicing mutation in 2 cases. Both cases were childhood single system (SS) LCH cases, with self-healing outcome of the bone lesions. This mutant consisted in a 9 base pair duplication (c.1511_1517 + 2 duplication), encoding for a predicted mutant protein with insertion of 3 amino acids (p.Arg506_Lys507insLeuLeuArg) in the N-terminal lobe of the kinase domain of BRAF. Transient expression of the c.1511_1517 + 2dup BRAF mutant in HEK293 cells enhanced MAPKinase pathway activation, and was not inhibited by vemurafenib but was inhibited by PLX8394, a second-generation BRAF inhibitor able to inhibit signaling of BRAF monomers and dimers. Future LCH molecular screening panel should include this new mutation to better define its prevalence in LCH and its restriction to autoregressive bone SS LCH. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0690-z) contains supplementary material, which is available to authorized users.

Supplemental methods and data. (DOCX 904 kb)

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207+ dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.

Objective To identify features associated with multisystem involvement and therapeutic failure in patients with skin Langerhans cell histiocytosis (LCH). Study design We reviewed medical records of 71 consecutive patients with LCH with skin involvement evaluated at Texas Children's Hospital and analyzed clinical features, laboratory results, and the presence of circulating cells with the BRAF-V600E mutation with respect to initial staging and clinical outcomes. Results Skin disease in patients older than 18 months of age at diagnosis was associated with the presence of multisystem disease (OR, 9.65; 95% CI, 1.17-79.4). Forty percent of patients referred for presumed skin-limited LCH had underlying multisystem involvement, one-half of these with risk-organ involvement. Patients with skin-limited LCH had a 3-year progression-free survival of 89% after initial therapy, and none developed multisystem disease. Patients with skin/multisystem involvement had a 3-year progression-free survival of 44% with vinblastine/prednisone therapy, and risk-organ involvement did not correlate with failure to achieve nonactive disease. Circulating cells with BRAF-V600E were detected at higher frequency in patients with multisystem involvement (8 of 11 skin/multisystem vs 1 of 13 skin-limited; P = .002). Conclusion Skin-limited LCH necessitates infrequent therapeutic intervention and has a lower risk of progression relative to skin plus multisystem LCH. The less-aggressive clinical course and lack of circulating cells with the BRAF-V600E mutation in skin-limited LCH suggest a different mechanism of disease origin compared with multisystem or risk-organ disease.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) pathway activation. Standard of care chemotherapy strategies are inadequate or overly toxic for the majority of patients with multisystem disease. The mechanisms underlying development of inflammation in LCH lesions are poorly understood, and potential for immunotherapy has not been determined. Analysis of the LCH lesion identified the most prominent immune cells as T lymphocytes, second only to pathologic CD207+/CD1a+ DCs (LCH DC). Both CD8+ and CD4+ T cells exhibited 'exhausted' phenotypes with high expression of the immune checkpoint inhibitor receptors. LCH DC cells also showed robust expression of ligands to checkpoint inhibitor receptors. Lesion CD8+ T cells exhibited blunted expression of Th1 cytokines with impaired effector function. In contrast, regulatory T cells (Tregs) isolated from LCH lesions demonstrated intact suppressive activity. Treatment of BRAFV600ECD11c LCH mice with anti-PD-1, anti-TIM-3 and MEKi decreased the lesion size: MEKi treatment resulted in reduction of the myeloid compartment; anti-PD-1 and anti-TIM-3 were associated with reduction in the lymphoid compartment. However, combined treatment with MEKi and either anti-PD-1 or anti-TIM-3 significantly decreased both CD8+ T cell and myeloid LCH cells in a synergistic fashion. These results thus indicate MAPK hyperactivation in myeloid LCH cells drives recruitment and activation of functionally exhausted T cells within the LCH microenvironment. While the results from this study did not demonstrate a significant impact of checkpoint inhibition alone on tumor burden in experimental mice, combined MAPK and checkpoint inhibition may be a potentially effective therapeutic strategy for patients with LCH. Disclosures Bollard: self: Patents & Royalties: patent on HIV T cells; NexImmune: Equity Ownership; Torque: Equity Ownership; Cellectis: Membership on an entity's Board of Directors or advisory committees; Caballetta: Equity Ownership; Mana Therapeutics: Equity Ownership.

Introduction: Pathogenic Hodgkin Reed-Sternberg (HRS) cells constitute approximately 1% of Hodgkin lymphoma (HL) tumor cells. Studies characterizing genomic lesions and gene expression of HRS gene cells have been limited due to technical challenges of studying these rare cells, and the majority of existing data has focused on adult HL. We therefore developed a multi-parameter flow sorting strategy to isolate viable cells from pediatric HL tumors and to define the transcriptomes of HRS cells and infiltrating lymphocytes in order to inform underlying mechanisms of HL pathogenesis and also create an opportunity to identify cell-specific biomarkers to predict disease risk and response to therapy. Methods : Flow cytometry was used to sort HRS cells, CD4+ T cells, CD8+ T cells, and CD20+/30+B cells from pediatric subjects' HL lesions and control tonsils. Purity was confirmed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Affymetrix GeneChip HTA 2.0 was used to assess the gene expression profiles (GEPs) for 16 HRS primary tumor cell samples, 14 HL CD4+ and CD8+ T cell samples, 6 control tonsillar CD20+, CD30+, CD4+, and CD8+ cell samples, and 6 HL cell lines. Unsupervised hierarchical clustering and principal component analysis (PCA) were used to determine relatedness, and Cibersort was performed to confirm the phenotype of the sorted cell types. GEPs of HRS, HL CD4+, and HL CD8+ cells were compared to respective controls using a univariate t-test. Significance was determined using a multivariate permutation test with the confidence level of FDR assessment at 80 percent and the maximum allowed proportion of false-positive proteins at 0.1. Gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA) were performed to analyze DEGs. Results: Effectiveness of the sorting strategy of HRS cells was confirmed by quantitative RT-PCR and IHC that demonstrated significant enrichment of CD30expression and CD30+ cells in the sorted HRS cell fraction. GEP comparisons were performed for 13 HL samples with matched HRS/CD4+/CD8+ cells: HRS vs. control tonsil CD20+/CD30+ (1934 and 3846 DEGs, respectively), HL CD4+ vs. control CD4+ (635 DEGs), HL CD8+ vs. control CD8+ (2 DEGs). We carried out a transcriptomic analysis of HRS cells, and a set of multifunctional genes were more than 2-fold downregulated (P < .001), involved in telomere maintenance and packaging (TERF2, RFC3, DNA2 and a group of HIST1) when compared to healthy lymph node CD30+ cells. A set of genes related to cytokine/chemokine dysregulation was also upregulated in HRS cells, including IL6, CCL18, and CXCL9. IPA and GSEA of specific HRS genes were also performed and demonstrated pathways associated with HL pathogenesis, including NFĸB activation and T cell exhaustion. Over-expression of genes associated with T cell pathways was demonstrated in HRS cells. While this may be a result of T cell rosetting and contamination, it may also reflect innate T cell signature within HRS cells, as HRS cells clustered separately from T cells in both unsupervised hierarchical clustering and PCA. Cibersort analysis of HRS cells revealed a heterogeneous phenotype that may reflect aberrant differentiation. In comparing clinical characteristics within HRS cells, TCEAL1 was elevated in slow vs. rapid early responders and 3 DEGs were identified when comparing EBV+/- samples. Within HL CD8 cells, KLF2 was elevated in EBV- samples. Conclusions: This study was the first to successfully isolate highly purified HRS cell populations from whole HL lesions in a pediatric HL cohort. Transcriptomic analysis of pediatric HRS cells identified mechanisms previously associated with HL pathogenesis, and also identified potential novel mechanisms, including telomere maintenance. Additional analyses demonstrated significant heterogeneity of HRS trasncriptomes across specimens that may reflect distinct differentiation pathways and differences in HRS-immune cell interactions. Finally, this study identified increased expression of some genes associated with EBV status and response to therapy. Future studies in an expanded cohort will validate these findings, compare pediatric and adult GEPs, and test these cell-specific biomarkers into the current risk stratification strategies of prospective clinical trials. Disclosures No relevant conflicts of interest to declare.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) activation. Investigations into LCH have historically been challenged by the small percentage of pathologic CD207+ DCs. No cell lines with morphology or function representing LCH lesion CD207+ DCs currently exist. We utilized four strategies to generate cell line(s) mimicking differentiated LCH pathogenic cells. First, CD207+ cells were isolated from the lesion of an LCH patient and cultured in a cytokine cocktail. The cells maintained CD1a and CD207 expression for two weeks, after which there were significant changes in cell morphology and progression to cell death. When a similar approach was implemented to isolate and culture skin CD207+ cells, the cells were viable for only three days, supporting the potential role for somatic activating MAPK mutations in LCH lesion DCs to prolong viability. CD207+ cells were then isolated from HLA-DR+ (lineage negative) cells from the lymphoid stroma of healthy tonsils (tCD207+). The tCD207+ cells were transduced with a lentivirus encoding human telomerase reverse transcriptase (hTERT). These cells, also lacking MAPK activating mutations, died two weeks post-transduction. A fourth approach has been more successful in which tCD207+ cells were cultured in a cytokine cocktail which provided MAPK pathway stimulation. The cells retained CD207 expression and survived in culture for over two weeks. The cells were then immortalized using a lentivirus encoding HOXA9. Immortalized cells maintained CD207 expression. Allele specific MAPK pathway mutations (BRAF and MAP2K1) are being generated by class II CRISPR/Cpf1 genome editing as Cpf1 has been shown to have robust activity to induce specific disruption of only mutant, but not wild-type, BRAF allele. The phenotypic and genomic characteristics of the immortalized cells expressing the different MAPK pathway mutations will be assessed by RHG-banding cytogenetic analysis, fluorescence in situ hybridization, gene expression analysis, and immuno‐cytochemistry and results will be compared to cells isolated from LCH lesions to confirm whether the established cell line may be a viable in vitro mimic of the LCH lesion DC. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), having a (9;22) chromosomal translocation. The BCR/ABL fusion protein is the hallmark of Ph+ leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases, particularly by altering the natural history of chronic phase (CP) CML and preventing the previously inexorable progression to terminal blast crisis (BC). However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. The most important mechanism of resistance against TKIs is the selection of leukemic clones driven by BCR/ABL harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). The "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of Ponatinib, a multi-target kinase inhibitor. CML and Ph+ ALL, rarely present at diagnosis with a BCR/ABL harboring a resistance mutation to TKI. Resistant clones may be present and only detectable by highly sensitive methods. We have previously shown that the resistance mutations may influence the biology of BCR/ABL and its transformation potential. We therefore hypothesized that the presence of mutations such as the T315I select for a "dormant cell population" which manifests following initial treatments with TKI inhibitors and treatment failure. The aim of this study was to determine whether the ''gatekeeper'' mutation T315I is able to confer biological features to BCR/ABL influencing its leukemogenic potential. We investigated the influence of T315I on the biology of BCR/ABL in CML and Ph+ ALL. We used Ph+ ALL patient derived long term culture (PDLTCs), factor dependent Ba/F3 cells and syngeneic mouse model of BCR/ABL induced CML-like disease. These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. We observed significantly slower proliferation of Ba/F3 cells and PDLTCs expressing BCR/ABL-T315I compared to the native BCR/ABL. This was further confirmed by undertaking mitotic index calculations and colony formation assays on both cell types. Furthermore, the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). We undertook functional studies to determine the putative signaling pathway and found that Ras/Erk1/2 pathway was activated inT315I positive cells. This study may assist towards therapy decisions in patients with CML/Ph+ ALL with a T315I mutation. Disclosures No relevant conflicts of interest to declare.

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207+ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in ∼75% of LCH cases, including recurrent BRAF-V600E and MAP2K1 mutations. To elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole-exome sequencing (WES, n = 6), targeted BRAF sequencing (n = 19), and/or whole-transcriptome sequencing (RNA-seq, n = 6) on 24 LCH patient samples lacking BRAF-V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the β3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A-BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207+ cells from lesions. ERK activation was resistant to BRAF-V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for patients with LCH.

Langerhans cell histiocytosis is a disorder characterized by lesions that include CD207+ dendritic cells along with an inflammatory infiltrate. Langerhans cell histiocytosis has a highly variable clinical presentation, ranging from a single lesion to potentially fatal disseminated disease. The uncertainty as to whether Langerhans cell histiocytosis is a reactive or a neoplastic disease has resulted in a long-standing debate on this question, and the limited understanding of the pathogenesis of the disease has impeded clinical improvement for patients. The current standard of care for multisystem Langerhans cell histiocytosis, empirically derived chemotherapy with vinblastine and prednisone, cures fewer than 50% of patients, and optimal therapies for relapse and neurodegenerative disease remain uncertain. Recent research advances support a model in which Langerhans cell histiocytosis arises due to pathologic activation of the mitogen-activated protein kinase (MAPK) pathway in myeloid precursors. Redefinition of Langerhans cell histiocytosis as a myeloid neoplastic disorder driven by hyperactive ERK supports the potential of chemotherapy with efficacy against immature myeloid cells, as well as mutation-specific targeted therapy.

By controlling and limiting inflammatory conditions, naturally occurring regulatory T cells (Tregs), defined as circulating CD4(+)CD25(bright)FoxP3(+) cells, play critical roles in maintaining tolerance and preventing autoimmunity and thus have tremendous potential for adoptive immunotherapy. Because they represent a scanty subset of the CD4(+) T-lymphocyte subset, several approaches have been developed to isolate and expand ex vivo polyclonal Tregs. However, one limitation of the functional analyses performed on these cultured Tregs is the incomplete characterization of their tissue-trafficking properties. As this aspect provides crucial information for their therapeutic effects, we have here explored the chemokine receptor expression profile and function of Tregs cultured ex vivo with validated expansion protocols. Our data show that ex vivo cultured Tregs retained the expression of CCR7 but dramatically downregulated CCR5 as compared with freshly isolated Tregs. The differential chemokine receptors expression pattern corroborated with their respective steady state messenger RNA expression and also with their migration toward specific chemokines. Our analyses suggest that ex vivo cultured Tregs may display impaired or suboptimal migration to the inflamed tissues releasing RANTES and MIP-1α chemokines.

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.

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 Results: High-quality genotype data for 583,173 germline variants were tested for an association with LCH relapse risk in the discovery GWAS stage. We identified a variant in high linkage disequilibrium with a cluster of loci on Chromosome 9 that surpassed our threshold of suggestive significance (non-coding RNA LOC100506532 rs2182640; P-value=6.98x10-6). This intronic variant was associated with a decreased risk of LCH relapse that remained statistically significant after adjusting for age at diagnosis, sex, and the top two PCs (aOR: 0.16; 95% CI: 0.07-0.35). While this non-coding RNA gene is largely uncharacterized, non-coding RNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Results from validation of this risk locus in an independent replication set are forthcoming. Conclusions: In this genome-wide assessment of inherited genetic variation and LCH relapse, we identified a genomic region that may be associated with LCH relapse. It is unclear which variant in the LOC100506532 cluster is a potentially causal allele. One of these variants may be a proxy for the causal locus, or may act through an effect on other genes in the same region or at distal sites. Notably RXRA, a retinoic acid receptor gene, is located in close proximity (~400kb) to LOC100506532 and regulates gene expression in various biologic processes. Risk variants within this gene have been identified to modulate disease-specific survival in melanoma, another BRAF-driven malignancy. While we are in the process of validating the association between LOC100506532 rs2182640 and LCH relapse risk in an independent replication set, our discovery GWAS results provide initial evidence to suggest that inherited risk factors influence LCH disease severity. Disclosures No relevant conflicts of interest to declare.

The physiologic control of cytokine receptor activation is primarily mediated by reciprocal activation of receptor-associated protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Here, we show that immediately following ligand-dependent activation, IL-4 receptor induces an intracellular calcium flux via IRS-PI3K-PLC-γ pathway which, in turn, induces PKC-dependent activation of NAD(P)H oxidase (NOX)5 that generates reactive oxygen species (ROS). IL-4 also induces NOX1-mediated ROS production via IRS-PI3K-RAC1 pathway. ROS, in turn, promote IL-4 receptor activation by oxidatively inactivating PTP1B that physically associates with and deactivates IL-4 receptor. However, ROS are not required for the initiation of IL-4 receptor activation. ROS generated by activated EPO-, TNF-α- or IL-3 receptor also promote IL-4 signaling. These data reveal that inactivation of receptor-associated PTP-activity by cytokine-generated ROS is a physiologic mechanism for the amplification of cytokine receptor activation in both cis and trans, unfolding a novel means of cytokine signaling cross-talk.

BRAF-V600E expression is identified in hematopoietic progenitor and precursor myeloid dendritic cells in patients with high-risk LCH, and enforced expression of BRAF-V600E in CD11c+ cells recapitulates a high-risk LCH-like phenotype in mice., Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207+ dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207+ DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c+ and CD14+ fractions and in bone marrow (BM) CD34+ hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207+ DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.

Background: Langerhans Cell Histiocytosis (LCH) is characterized by inflammatory lesions with pathologic CD207+ dendritic cells (DCs) with constitutively activated ERK. LCH lesions are associated with a local cytokine storm and exhibit a heterogeneous collection of inflammatory infiltrates (eosinophils, neutrophils and lymphocytes). The mechanisms behind tumor formation, both accumulation of pathologic CD207+ DCs as well as recruitment of inflammatory infiltrate, remain to be defined. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF-V600Eand MAP2K1 mutations. However, the inflammatory infiltrate within LCH lesions is composed of cells without any BRAF mutation or ERK activation. We hypothesize that the pathologic inflammation in LCH is orchestrated by interactions between the pathologic MAPK-activated DCs and normal immune cells. Defining the nature and function of infiltrating T cells in LCH will not only provide insights into mechanisms of pathogenesis and potential therapeutic targets, but may also inform novel physiologic mechanisms of DC-mediated T cell function. Methods: Clonality was investigated by exploring T cell receptor beta (TCRB) rearrangement in LCH patients. Quantitative analysis of different immune cell populations in the tumor microenvironment in 25 LCH patients was performed using spanning tree progression of density normalized events (SPADE). Results: Our initial experiments identified clonal populations among LCH lesion-infiltrating CD3+ T cells. The complementarity-determining region 3 (CDR3) of the TCRBV07 gene demonstrated marked monoclonal expansion, suggesting a potential involvement of antigen selection in the pathogenesis of LCH. SPADE analysis of infiltrating CD3+ T cells revealed high expression of T cell immunoglobulin mucin (TIM-3), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to healthy controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in LCH patients. In addition, in a cohort of LCH patients harboring wild-type BRAF, but not the BRAF-V600E mutation, a distinct subset of infiltrating CD3+ T cells were detected expressing both CD4 and CD8α receptors at high levels (double-positive, DP). Preliminary analysis showed high levels of IFNγ, but not TNF-α, after stimulation of these DP T cells. Conclusions: The results from this study support a model in which LCH lesion DCs with activated MAPK signaling select and recruit activated T cells with potential for clonal expansion to LCH lesions. Multiple markers of T cell exhaustion were identified on infiltrating LCH lesion T cells. While the significance of double-positive T cells in LCH is uncertain, similar cells have been reported in the target organs of several auto-immune conditions and hematological and solid malignancies, consistent with double-positive T cells in sites of immune exhaustion. The function of this double-positive T cell population remains to be defined, but we postulate that these cells may participate in tumor immune responses in LCH due to their potential of having broad tumor reactivity. While the CD207+ DCs in LCH lesions is histologically uniform across the disease spectrum, the composition of the immune microenvironment is variable. The variability of the DP T cell population in BRAF-V600E vs BRAF wild-type DCs study suggests that specific mechanisms of MAPK activation may have some distinct impact on T cell recruitment and activation. While recent discoveries have enhanced understanding of the origin and aberrant MAPK activation of the pathologic DCs, very little is known about the role of lymphocytes or the inflammatory microenvironment. Characterizing the populations, function, and interactions of the LCH lesion immune cells may provide novel therapeutic opportunities, such as checkpoint inhibitors. Further, identifying the mechanisms that underlie the "cytokine storm" of LCH lesion may inform physiologic interactions between MAPK-activated DCs and effector immune cells. Disclosures No relevant conflicts of interest to declare.