Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid tumor that is highly prevalent in children. At present, it is believed that the uncontrolled proliferation and differentiation of the mononuclear phagocytic system caused by abnormal activation of the MAPK pathway (often caused by BRAFV600E mutation) is the main pathogenesis of the disease, and the mononuclear phagocytes carrying the pathogenic mutation can further migrate to the skin, bone and other lesions to form pathological LCH cells (CD207+CD1a+) [1,2]. Treatment with BRAF-targeted inhibitors (darafenib) significantly improves prognosis in patients with BRAFV600E mutation LCH [3,4]. However, there are still quite a few patients who have left serious sequelae, and there are still many unknowns about the pathogenesis of the disease and the mechanism of action of targeted therapy. Therefore, combined with the mutation level of the pathogenic gene and the clinical effect of targeted therapy, it is of great significance to analyze the cellular subclasses and molecular characteristics of the mononuclear phagocytic system of LCH patients with high precision, which is of great significance for further elucidating the pathogenesis of LCH, revealing the molecular basis of disease hierarchy heterogeneity in patients, and in-depth understanding of the mechanism of action of targeted therapy.
Recently, the Liu Bing Research Group of the Fifth Medical Center of the PLA General Hospital, the Zhang Rui Research Group of Beijing Children's Hospital, the Florenc Ginhoux Research Group of the Singapore Immunology Collaborative Group, and the Lan Yu Research Group of the School of Basic Medical Sciences of Jinan University collaborated in the Blood Journal online titled Transcriptomic Landscape of Circulating Mononuclear Phagocytes in Langerhans Cell Histiocytosis at Single-cell Level research paper. This study constructs for the first time a single-cell transcriptome atlas of circulating mononuclear phagocytosis in LCH patients, reveals the extensive activation of the RAS-MAPK-ERK pathway in multiple cell types within the patient's circulation, and further explores the mechanism of action of the targeted inhibitor Darafenib in the clinical treatment of brafV600E patients with mutations.
Firstly, a large cohort of 217 LCH children with first diagnosis was enrolled, and plasma free BRAFV600E (cell-free BRAFV600E, cfBRAFV600E) was detected by digital droplet polymerase chain reaction, and mutation grouping was carried out according to cfBRAFV600E load: cfBRAFV600E negative group, low mutation group and high mutation group, and it was found that there was a significant correlation between mutation load and clinical risk stratification. The proportion of circulating CD45+ Lin-HLA-DR+ mononuclear phagocyte subsets in 49 of the children was analyzed by flow cytology, and the proportion of plasmacytoid dendritic cells (pDC) in the clinically high-risk group and the cfBRAFV600E high-mutation group was significantly reduced. The above data show that the patient's peripheral blood cfBRAFV600E mutation load and pDC ratio can be used as an objective indicator of LCH disease risk stratification.
Next, the authors performed single-cell transcriptome sequencing (scRNA-seq) for 14 patients with CD45+ Lin-HLA-DR+ circulating mononuclear phagocytes in children with LCH, and found that gene expression levels associated with the RAS-MAPK-ERK pathway were widely upregulated in all mutation groups in various myeloid mononuclear phagocyte subsets. Interestingly, in NK/T cells, gene participation in RAS and MAPK-related pathways was also expressed higher in LCH patients than in the control group, while NK/T cells typically did not carry pathogenic mutations, suggesting that noncellular autonomic factors were involved in the important pathological process of extensive activation of the MAPK pathway. The above studies suggest that the BRAFV600E mutation may cause mononuclear phagocytes to already be in a state of abnormal activation of the mapk pathway characteristic of the disease in the circulation.
Subsequently, the authors performed scRNA-seq on a population of circulating mononuclear phagocytes before targeted therapy and 1 month after treatment in five refractory/relapsed BRAFV600E mutant LCH, focusing on how the circulating mononuclear phagocytosis system responds to the BRAF inhibitor darafenib. Corresponding to a good clinical response to targeted therapy, the cells of the patient after treatment showed significant downregulation of immunosuppressive effect signals (e.g., IL-1 and IL-6) and pro-inflammatory signals in addition to the downregulation of the activity of the RAS-MAPK-ERK pathway, and in addition, glycolysis and pentose phosphate pathway signal transduction were also significantly downregulated after treatment. The above findings suggest that darafenib may play a role in immunity and metabolic regulation, which provides a new perspective and theoretical basis for in-depth understanding of the mechanism of action of targeted therapy and further optimization of targeted therapy strategies.
Finally, the authors performed scRNA-seq on skin lesion cells in children with LCH, and identified four heterogeneous CD207+CD1a+ subpopulations, one of which specifically expressed and enriched RAS-MAPK-ERK-related genes and pathways. In addition, based on the integrated analysis of STRT-seq sequencing data of peripheral blood and skin lesions in this study and the published 10x sequencing data of LCH lesions, the authors found that LCH cells had the highest similarity with circulating blood cDC2/DC3 transcriptomes. Therefore, this study is the first to analyze the immune cell subsets of peripheral blood and lesion epidermis at the single-cell level, providing a basis for the etiological hypothesis of LCH cells derived from circulating precursor cells.
In summary, this study comprehensively uses immunophenotyping, clinical observation and single-cell transcriptomics to focus on the circulating mononuclear phagocytosis system and RAS-MAPK-ERK signaling pathway in children with LCH, reveal the molecular expression changes of various cell subsets and patient heterogeneity that are closely related to the development of disease, and optimize the risk stratification strategy of disease, aiming to promote LCH treatment into the era of precision and personalized medicine.
Shi Hui and Huang Tao, doctoral candidates of the Academy of Military Sciences, He Han, master's student He Han, researcher Bian Zhilei of the First Affiliated Hospital of Zhengzhou University, Cui Lei, associate researcher of Beijing Children's Hospital, and Egle Kvedaraite of Karolinska Institutet are the first authors of the paper. Liu Bing, Zhang Rui, Florent Ginhoux and Lan Yu are the corresponding authors of the paper.
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https://www.sciencedirect.com/science/article/pii/S0006497121012374
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