Diagnosis and therapeutic regimen
Histopathological examination confirmed that the patients had LCH, as indicated by positive immunostaining for CD1a and Langerin (CD207) in the lesion tissues. Patients were divided into single-system (SS) or multisystem (MS) LCH according to the number of organs or systems involved and classified as risk organ (RO) positive (liver, spleen, and/or hematological system) or negative according to the extent of LCH[1].
Patients were treated with a systemic chemotherapy regimen (CCHG-LCH 2019 protocol, www.chictr.org.cn, identifier: ChiCTR1900025783), which was based on the LCH III and LCH-S-2005 protocols10. Briefly, the first-line treatment was vindesine-steroid combination therapy, beginning with one or two six-week courses of intensive initial induction therapy followed by maintenance therapy. The BRAF inhibitor dabrafenib was given to patients who carried the BRAF -V600E mutation and had recurrent/refractory LCH disease. The details of the treatment are shown in the Supplementary Methods.
Mass spectrometry-based plasma proteomics analysis
We collected diagnostic plasma samples from 11 patients, including 5 with MS RO+, 6 with SS-LCH and 5 healthy controls, for proteomic analysis (Supplementary Table S1). Date-independent acquisition (DIA) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was performed. The differentially expressed proteins (DEPs) between MS RO+ LCH and SS LCH were explored. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also conducted to reveal the functions of the DEPs in biological processes.
Single-cell RNA-seq (sncRNA) analysis
We performed a bioinformatics analysis using a single-cell RNA sequencing (scRNA-seq) dataset of PBMCs from our previous study11. Briefly, we divided the samples into two groups: the MS RO+ LCH group (n = 7) and the SS LCH group (n = 3). Differentially expressed genes (DEGs) were also analyzed.
ELISA for plasma sCSF1R in LCH
For patients receiving first-line therapy, plasma samples were collected at two time points: at the time of diagnosis and at week 6 (after the first initial induction therapy). For patients receiving dabrafenib treatment, serial blood samples were collected at every evaluation time point (baseline, one month, and every three months later) during follow-up. The levels of sCSF1R were tested using an automated microplate reader (Thermo, USA) at 450 nm according to the instructions of the Human CSF1R ELISA Kit (Boster, China).