ABSTRACT:We evaluated long-term outcomes of 288 children with refractory-Langerhans cell histiocytosis (R-LCH) from 26 countries, who were prescribed off-label MAPK inhibitors (MAPKis) according to clinical indications. MAPKi indications included 148 R-risk-organ-positive (R-RO+), 67 R-risk-organ-negative (R-RO-), 13 lung destruction (lung), 9 sclerosing cholangitis (SC), 49 neurodegeneration (ND), and 2 diabetes insipidus (DI) cases. Median ages at diagnosis and MAPKi onset were 1.3 and 2.3 years, respectively, with median follow-up of 3.7 years (1166 person-years). Agents mostly prescribed as monotherapies were 184 prescriptions of vemurafenib, 115 of dabrafenib, 3 of encorafenib, 42 of cobimetinib, 45 of trametinib, and/or 1 prescription of binimetinib; followed 51 times by various chemotherapies or hematopoietic stem-cell transplantation, or 28 times by combined anti-BRAF-anti-MEK. Short-term responses (<8 weeks) ranged from 98% (R-RO+ and R-RO-), to 30% (lung) to none (ND, DI, and SC); although long-term lung and ND responses could be observed. Skin rash was the most frequent adverse event (∼55%), and 7 others included 1 case of cardiomyopathy and 6 of retinitis. Five developed MAPKi-unrelated tumors and 9 patients died. Five-year survival was 98%. After 113 patients with R-LCH discontinued MAPKi, 69 experienced disease reactivation. None of the various empirical maintenance therapies were able to prevent secondary reactivation. Among the 143 assessable patients without ND-LCH at MAPKi onset, 60 developed ND (45%, 5-year risk). MAPKis appeared to be safe and effective in children with R-RO+/RO-LCH, whereas other indications' responses were less frequent or occurred later. Further studies are needed to find effective maintenance-therapy approaches, particularly to prevent frequently observed secondary ND.

Abstract Neurodegenerative Langerhans cell histiocytosis (ND‐LCH) is a severe and debilitating complication of LCH with no effective treatment. In this study, we assessed the efficacy of MAPK inhibitors (MAPKi) in a large cohort of children with ND‐LCH. We included pediatric patients with ND‐LCH who received MAPKi between 2013 and 2025. Clinical and radiological responses were evaluated using validated and newly developed scoring systems. We included 60 patients and assessed the response to MAPKi in 54 (clinical n = 53, radiological n = 54). Of the 53 clinically evaluable patients, 44 (83%) presented with symptoms (impaired global functioning n = 39, 74%; neurological manifestations n = 38, 72%; and psychiatric disturbances n = 27, 51%). MAPKi were started at a median of 25 months (interquartile range [IQR] 5–67) from ND diagnosis. Clinical responses were observed in 29 patients (55%), while the remaining showed disease stabilization (n = 21, 39%) or progression (n = 3, 6%). Of the 54 patients with magnetic resonance imaging (MRI) follow‐up, radiological improvement was documented in 40 (74%), while 12 (22%) demonstrated stable findings, and 2 (4%) showed progression. We observed significant improvements in both clinical (P < 0.001) and MRI abnormalities (P < 0.001). None of the nine asymptomatic patients developed symptoms, while MRI lesions improved in six of them. Positive responses occurred irrespective of baseline disease severity and treatment delay. Adverse events, evaluable in 60 patients, were reported in 31 of them (52%) but were generally not severe and manageable. In conclusion, we demonstrated robust efficacy of MAPKi in ND‐LCH, with an acceptable safety profile. These findings support a promising therapeutic approach that warrants prospective validation in this challenging condition.

Supplementary Data S1. Definition of response, Description of response criteria used by sites to evaluate tumor response, Definition of extend of resection, Definition of extend of resection used by sites.

10011 Background: Long-term off-label use of MAP kinase inhibitors (MAPKi) to treat, refractory childhood Langerhans cell histiocytosis (LCH) was evaluated within the European consortium for histiocytosis network ( www.echo-histio.net ). Methods: 277 patients from 26 countries treated with MAPKi were classified according to the clinical indication: refractory risk organ positive/negative (RO+/RO-), isolated lung destruction (Lung), sclerosing cholangitis (SC), neurodegeneration (ND), and diabetes insipidus (DI). 252 patients had received one or several lines of chemotherapies prior to MAPKi: VBL/steroids (n = 243) then 2CdA/AraC (n = 48), 2CdA alone (n = 52), clofarabine (n = 5), VCR/AraC (n = 70) before being considered refractory. The 25 treated front line by MAPKi were newborn with aggressive disease (n = 7), or had chronic manifestations like ND, SC or DI. BRAF V600E was detected in 95% of the cases. Results: Median age at diagnosis was 1.3 years. MAPKi indication was RO+ (n = 138); RO– (n = 72); Lung (n = 7); SC (n = 9), ND (n = 45), DI (n = 2). Median age at MAPKi onset was 2.3 years, with median follow-up of 3.5 years (IQR 1.6-5.9). Vemurafenib (n = 177), Dabrafenib (n = 105), Encorafenib (n = 3), Cobimetinib (n = 41), Tramatinib (n = 41), and Binimetinib (n = 1) were prescribed mainly in monotherapy, sometimes (n = 44) with various chemotherapies or HSCT (n = 5). The short-term response (before wk 8) varied from 98% in RO+ and RO-, to 30% in Lung to a null response in ND, DI and SC, although some long-term response (after 6 months) was observed in Lung and ND. Skin rash was the most frequent adverse event (AE), affecting 55% of patients. Other AEs were observed in 7 (cardiomyopathy n = 1, retinitis n = 6). Five tumors or malignancies were observed not related to MAPKi; only in patients heavily treated by 2CdA, AraC or Clofarabine. Six deaths were observed; 5-year survival was 98%. MAPKi discontinuation for 111 patients led to LCH 66 reactivations. None of the various empirical maintenance therapies used was able to prevent secondary reactivation. Among the 133 assessable patients free of ND at MAPKi initiation, ND was observed in 52 with a 5-year risk of 55%. In some cases, ND was reversible after MAPKi dose adaptation. Conclusions: MAPKi appeared quick, safe and effective in children with refractory LCH while the response to Lung, SC, DI and ND was limited or delayed. Further studies are needed to find effective maintenance therapy. ND should be monitored in the follow up of patients treated by MAPKi.

Supplementary Figure S2. A) Hazard ratio for progression or death according to clinical characteristics. B) Hazard ratio for death according to clinical characteristics. LGG: Low-grade glioma, HGG: High-grade glioma

Supplementary Table S4. Other molecular alterations reported.

Supplementary Figure S1. Diagram of population patients.

10006 Background: rEECur, the first randomised controlled trial (RCT) in RR-ES, has previously defined high dose ifosfamide (IFOS) as the most effective regimen in this setting compared to gemcitabine-docetaxel, irinotecan-temozolomide and topotecan-cyclophosphamide. Platinum drugs show activity in RR-ES and are frequently given with etoposide in this setting. Methods: Patients aged at least 2 years with RR-ES were randomised to 3-week cycles of either IFOS 15 g/m 2 by continuous intravenous (IV) infusion over 5 days or IV carboplatin 400 mg/m 2 day 1 and etoposide 120 mg/m 2 days 1 to 3 (CE). Primary outcome was event-free survival time (EFS). Secondary outcomes included overall survival time (OS), toxicity and quality of life (QoL). A probability-based Bayesian approach was used. Results: 139 patients recruited between 22/03/21 and 28/05/24, were randomised 1:1 to IFOS (n = 69) or CE (70). Median age was 18 years (range 3-59). Patients had refractory disease (14%), 1 st recurrence (81%), > 1 st recurrence (6%). Sites of progression were primary site only (21%), pleuropulmonary metastases only (24%), and other or combined metastatic disease (55%). More CE patients had baseline GFR < 90 ml/min/1.73m 2 (41% versus 23%). Median follow-up (reverse Kaplan-Meier) was 18 months (mos). Median EFS was 5.1 mos (95% CI 3.1, 6.3) for IFOS and 3.5 mos (95% CI 2.5, 6.1) for CE. Median OS was 14.4 mos (95% CI 11.5, 20.7) for IFOS and 19.0 mos (95% CI 11.2, 24.6) for CE. Given the observed data the posterior probabilities that EFS and OS were better after IFOS than after CE (ie Pr[true hazard ratio > 1 | data]) were 87% and 55% respectively. Grade 3+ adverse events present in > 5% of patients randomised to IFOS (left hand values) compared with CE were febrile neutropenia (30% v 10%), anaemia (9% v 9%) and thrombocytopenia (3% v 7%). Acute kidney injury was present in 2% v 0% and encephalopathy in 5% v 0%. There were no measurable differences in QoL. Conclusions: There was insufficient evidence of efficacy with CE compared to IFOS to continue recruitment to phase III in this first RCT of a platinum-etoposide combination in RR-ES. IFOS remains the most effective regimen in this disease setting. The trial remains open, comparing IFOS with and without the tyrosine kinase inhibitor lenvatinib. Funded by Cancer Research UK (C22436/A28028, CTUQQR-Dec22/100006, A28474). Clinical trial information: ISRCTN36453794 .

Diffuse midline glioma (DMG; a subtype of pediatric high-grade glioma) is a fatal disease in children, due to the localization in critical structures of the central nervous system, its invasive nature, and limited treatment options. Molecularly, DMG with loss of histone 3 K27 trimethylation (mostly through the typical K27M-mutation in histone 3) have been relatively well characterized, however, no unambiguous Achilles’ heel for targeted therapeutic approaches could be identified to date. This study integrates detailed molecular characteristics of pediatric DMGs with clinical data in a large, international cohort in order to contribute to a better understanding necessary for further development of therapeutic approaches. A total of 162 DMG tumors were analyzed within the INFORM registry from 01/2015 to 11/2023 using comprehensive molecular profiling (including exome, whole-genome and RNA next-generation sequencing approaches, complemented with DNA methylation analysis). Molecular results were correlated with clinical data of the respective patients including the treatment regimen applied and patients’ outcomes. This well-defined cohort of histone 3 K27-altered DMG according to the current WHO classification showed typical molecular alterations for this entity, with differences in frequencies in specific subgroups. The presence of TP53 mutation and the absence of MAPK pathway alteration in the tumors were associated with worse outcomes. In a substantial proportion of patients, genetic alterations serving as targets for potential therapeutic approaches could be identified. This large, international, prospective DMG cohort combines comprehensive molecular characterization of the tumors with registry-level clinical data, thereby contributing to a better understanding of the underlying tumor biology, potential prognostic and predictive markers and the potential impact of targeted therapies.

Supplementary Table S5. Method of NTRK fusion detection as reported by participating centers.

Supplementary Table S2. Responses based on clinical assessment for each line of therapy. Non-targeted therapy and TRKi given concurrently in a specific line were excluded.

Supplementary Table S3. Survival outcome.

Langerhans cell histiocytosis (LCH) is a clonal hematopoietic disorder defined by tumorous lesions containing CD1a(+)/CD207(+) cells. Two severe complications of LCH are systemic hyperinflammation and progressive neurodegeneration. The scarcity of primary samples and lack of appropriate models limit our mechanistic understanding of LCH pathogenesis and affect patient care. We generated a human in vitro model for LCH using induced pluripotent stem cells (iPSCs) harboring the BRAFV600E mutation, the most common genetic driver of LCH. We show that BRAF(V600E/WT) iPSCs display myelomonocytic skewing during hematopoiesis and spontaneously differentiate into CD1a(+)/CD207(+) cells that are similar to lesional LCH cells and are derived from a CD14(+) progenitor. We show that BRAFV600E modulates the expression of key transcription factors regulating monocytic differentiation and leads to an upregulation of proinflammatory molecules and LCH marker genes early during myeloid differentiation. In vitro drug testing revealed that BRAFV600E-induced transcriptomic changes are reverted upon treatment with mitogen-activated protein kinase (MAPK) pathway inhibitors (MAPKis). Importantly, MAPKis do not affect myeloid progenitors but reduce only the mature CD14(+) cell population. Furthermore, iPSC-derived neurons (iNeurons) cocultured with BRAF(V600E/WT) iPSC-derived microglia-like cells, differentiated from iPSC-derived CD34(+) progenitors, exhibit signs of neurodegeneration with neuronal damage and release of neurofilament light chain. In summary, the iPSC-based model described here provides a platform to investigate the effects of BRAFV600E in different hematopoietic cell types and provides a tool to compare and identify novel approaches for the treatment of BRAFV600E-driven diseases.

PURPOSE:Tropomyosin receptor kinase (TRK) fusions are detected in less than 2% of central nervous system tumors. There are limited data on the clinical course of affected patients. EXPERIMENTAL DESIGN:We conducted an international retrospective cohort study of patients with TRK fusion-driven central nervous system tumors. RESULTS:A total of 119 patients were identified. The median age at the time of diagnosis was 4.5 years. The majority were reported to have a histology consistent with a diagnosis of high-grade glioma (HGG; 57.1%) followed by low-grade glioma (LGG; 27.7%). Pediatric patients had a better prognosis, with a median overall survival of 185.5 months compared with 24.8 months in adults (P < 0.0001). Patients with LGG also had a better outcome when compared with HGG (P = 0.0012). The objective response was 68.8% with larotrectinib compared with 38.1% for nontargeted treatment. CONCLUSIONS:Children with LGG had a favorable outcome compared with adult glioma and HGG. TRK inhibitors seem to improve tumor control.

Background: Langerhans cell histiocytosis (LCH) is a rare hematological neoplasia of myeloid origin driven by activating mutations in the mitogen-activated protein kinase pathway, most commonly BRAFV600E. The clinical presentation is heterogeneous ranging from single-system (SS) to multi-system (MS) disease, with or without involvement of risk organs (RO), i.e. liver, spleen, bone marrow. Circulating cell free (ccf-) BRAFV600E has been proposed as a prognostic marker, however molecular-based risk stratification is still an unmet need in patients with LCH. Purpose: To assess the prognostic significance of BRAFV600E in different sources of peripheral blood at baseline (before therapy) and at 6 weeks of treatment according to LCH-IV in children with BRAF-mutated LCH. Methods: Peripheral BRAFV600E was measured in five laboratories of the European Consortium for Histiocytosis (Florence-IT, Newcastle-UK, Paris-FR, Amsterdam-NL, Vienna-AU). DNA from circulating-cell-free (ccf, n=307), peripheral blood mononuclear cells (PBMC, n=186), total leukocytes (n=21) or whole blood (n=36) was screened by digital droplet or allele-specific PCR. BRAFV600E levels at baseline and at week 6, in all sources, were correlated to clinical outcome, defined as event (reactivation/treatment intensification) using Mann-Whitney U test. Survival proportions were calculated using the Kaplan-Meier method and Log-rank (Mantel-Cox) test was used to calculate the significance of the difference between survival curves. Pearson correlation coefficient was used to measure the strength of linear associations between ccf and PBMC BRAFV600E. Results: A total of 259 patients were enrolled. Median age was 29 (IQR=13-79) months; disease extension was represented by 49% SS, 27% MS-RO- and 24% MS-RO+. Data at baseline and week 6 of 259 and 121 patients, respectively, were analyzed. At baseline, BRAFV600E levels were highly correlated between ccf and PBMC-DNA in MS-RO- (r=0.97) and MS-RO+ (r=0.87) but less so in single-system (SS) patients (r=0.53). At baseline, higher BRAFV600E levels were detected in ccf- versus PBMC-DNA (median 0.093% vs 0.006%, p=0.0003). BRAFV600E levels were significantly associated with disease stage, with a median of 0% in SS, 0.11% in MS-RO-, 1.69% in MS RO+, in ccf-DNA (p<0.0001); 0% in SS, 0.01% in MS RO-, 0.5% in MS RO+, in PBMC-DNA (p<0.0001). Higher BRAFV600E levels were observed among patients undergoing events within two years, with a median of 0.07% vs 0% in SS (p=0.004), 0.39% vs 0.014% in MS-RO- (p=0.0007), 2.1% vs 0.36% in MS RO+ (p=0.04), in ccf-DNA and 0.006% vs 0% in SS (p=0.003), 0.07% vs 0.002% in MS RO- (p=0.0002), 0.70% vs 0.05% in MS RO+ (p=0.82), in PBMC-DNA, respectively. In all risk groups, it was possible to define a threshold of BRAFV600E positivity that was associated with shorter 2-year event free survival (EFS). Survival analysis showed significant differences among patients on the basis of the selected cut-offs. At baseline, we identified for ccf-DNA a threshold of 0.1% in SS (p=0.0007) and MS RO- (p=0.0004) patients; 1% in MS RO+ patients (p=0.0182); for PBMC, we established a threshold of 0.01% in SS (p=0.0099) and MS RO- (p<0.0001) patients; 1% in MS RO+ patients (p=0.1414). At week 6, longer 2-year EFS was associated with reduction of >50% of ccf-BRAFV600E (p=0.0002) and clearance of PBMC-BRAFV600E (p=0.059). Conclusion: Measurement of BRAFV600E, in ccf- and PBMC-DNA, both offer potential prognostic value in children with LCH. This multicenter international study provides the basis for molecular-based risk stratification at baseline and at early time points of treatment in different disease groups. Incorporation of this approach in next prospective therapeutical trials may confirm its role in guiding treatment decisions.

Abstract Langerhans cell histiocytosis (LCH) is a rare inflammatory hematological neoplasm, characterized by the accumulation of LCH cells in various tissues and organs. These LCH cells originate from myeloid progenitors that carry a MAPK-pathway mutation. Once these mutated cells enter the tissue from the periphery, they differentiate into so-called LCH cells. In addition to LCH cells, the tumor microenvironment (TME) consists of a wide variety of immune cells. However, the interactions between these cells within the TME and their contribution to disease progression remain poorly understood. In this study, we focus on understanding the role of lymphocytes in LCH lesions. We used single-cell RNA sequencing to comprehensively profile the immune populations in LCH lesions on the transcriptomic level, which will be supplemented with spatial profiling. Our analysis identified a wide variety of infiltrating immune cells within LCH lesions with high cellular heterogeneity among patients. LCH- and T cells are the most prevalent cell types, suggesting a key role in lesion development and maintenance. Furthermore, the immune landscape includes several dendritic cell subsets – such as plasmacytoid DCs (pDCs), cDC1, and mature regulatory dendritic cells (mregDCs) – alongside tumor-associated macrophages (TAMs), monocytes, NK cells, B cells, and plasmablasts. Among T cells, regulatory T cells (Tregs) are particularly abundant and appear to be heavily influenced by LCH cells and other myeloid cells via the TNF-TNFR2 and CD86-CTLA4 pathways. Additionally, LCH cells express high levels of TGFβ – a known regulator of T cell activity – and MMP9 – which might hamper cytotoxic T cell infiltration. This suggests that LCH cells actively contribute to creating an immunosuppressive environment. Surprisingly, pDCs seem to be a major immune regulator within lesions and are likely to interact with T cells via TNFSF9 and Granzyme B. These insights into the immunosuppressive environment within LCH lesions highlight the critical role of Tregs as central regulators within the tumor microenvironment. The implications of these findings will be further explored and validated through in-vitro experiments and high-plex spatial protein profiling. Ultimately, our work may pave the way for developing targeted therapies aimed at modulating the TME, potentially enhancing anti-tumor immunity, and offering new strategies for treating LCH. Citation Format: Wouter van Midden, Raphaela Schwentner, Sebastian Eder, Philipp Ben Soussia-Weiss, Giulio Abagnale, Caroline Hutter. Exploring the composition of and interaction of the tumor microenvironment in Langerhans cell histiocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C017.

ZusammenfassungDie Langerhanszell Histiozytose (LCH) ist eine seltene neoplastische Erkrankung, die vor allem im Kindes- und Jugendalter auftritt. Die Erkrankung kann prinzipiell jedes Organ befallen, weswegen sehr unterschiedliche klinische Erscheinungsbilder möglich sind. Der klinische Verlauf der LCH reicht von einer Spontanheilung bis hin zu einem rasch progredienten tödlichen Verlauf. Die Ausbreitungsdiagnostik bestimmt das Vorgehen. Manche Patienten qualifizieren sich für eine watch-and-wait Strategie, während andere einer Chemotherapie mit den Standardmedikamenten Vinblastin und Prednison bedürfen. Durch die Identifizierung von Mutationen im MAPK-Signalweg wächst das Interesse an zielgerichteten Medikamenten wie den BRAF-Inhibitoren. Chronisch-rezidivierende Verläufe und Spätschäden sind ein weiteres Problem der Erkrankung und stehen im Mittelpunkt derzeitiger Forschungsaktivitäten.

Key Points • Oncogenic mutation subtype appears an important driver of heterogeneity in clinical presentation of pediatric LCH.• Lesional BRAFV600E status is not a significant prognostic factor for event-free survival independent from disease extent.