Vemurafenib acts as a molecular on-off switch governing systemic inflammation in Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by activating mutations in the MAPK pathway, most commonly, in about 55% of cases, BRAF^V600E.^1,2  It is defined by the accumulation of CD1A⁺CD207⁺ cells in different organs.³  Patients with involvement of liver, spleen, or the hematopoietic systems have a higher risk of disease-related mortality.⁴  Importantly, involvement of these so-called risk organs is a functional definition inferred by organ dysfunction.⁵  In most cases, no or few infiltrating CD1A⁺CD207⁺ LCH cells are found in these organs,^6-9  suggesting that these pathogenic phenotypes might be caused by other cells or indirectly.

Multisystem LCH is treated with risk-adapted chemotherapy. Moreover, RAF inhibitors are increasingly being used, but usually, patients relapse on discontinuation of therapy.^10,11  Here we report a patient with risk organ–positive LCH who was successfully treated with a combination of vemurafenib and 2-chlorodeoxyadenosine and cytarabine (2-CdA/ARA-C). Furthermore, we suggest that, although the CD1A⁺CD207⁺ histiocytes are the hallmark of LCH, other BRAF-mutated cells contribute significantly to morbidity in patients with multisystem LCH by causing systemic inflammation (and hence risk organ involvement). We corroborate our hypothesis with detailed analysis of the clinical course, cytokine patterns, and BRAF^V600E levels in different blood cell types, together with the transcriptome analysis of single cells from bone marrow and peripheral blood of the patient.

Samples were obtained as part of routine diagnostic procedures. The study was approved by the ethics committee of the Medical University of Vienna, and parents gave their written and informed consent.

Peripheral blood and bone marrow cells were isolated by density gradient centrifugation and subsets were purified by fluorescence-activating cell sorting using a FACScan flow cytometer (BD FACSAria).

Droplet-digital polymerase chain reaction (PCR) for BRAF^V600E was performed as previously described¹⁰  using the Mutation Assay BRAF p.V600E c.1799T-A, Human (Bio-Rad Laboratories) and the QX200 Droplet Digital PCR System (Bio-Rad Laboratories).

Cytokines were analyzed with the Inflammation 20-Plex Human ProcartaPlex Panel (ThermoFisher) according to the manufacturers instructions. Samples were measured on a MAGPIX Instrument (Luminex Corporation).

Single-cell RNA-seq was performed using the 10x Genomics Chromium Single Cell Controller with the Chromium Single Cell 3' Kit as previously described¹²  and detailed in the supplemental Methods. The downstream analysis was performed using the Seurat package¹³  (v3.1.4) for R (v3.6.2). Cell types were annotated by mapping the cells on a reference dataset of preannotated cells using the Azimuth¹⁴  pipeline. Differential expressed genes were calculated with the model-based analysis of single-cell transcriptomics (MAST).¹⁵  Gene set enrichment analysis (GSEA)¹⁶  was calculated for Gene Ontology's Biological Process dataset.

A 2-year-old child with multisystem LCH (involvement of bone, skin, hematopoietic system, and liver) who deteriorated on treatment with prednisone and vinblastine was switched to therapy with vemurafenib. Clinically, the patient improved within days. He became more active, fever and hypoproteinemia subsided, and blood counts recovered. Because long-term effects of vemurafenib are unclear and because evidence thus far suggests that patients relapse after stopping the inhibitor,^10,11,17  we decided to start treatment with 2-chlorodeoxyadenosine (2-CdA, cladribine) and cytarabine (ARA-C).³  Vemurafenib was paused on chemotherapy days but otherwise continued (Figure 1A). The vemurafenib discontinuance immediately led to fever, decreased total protein (TP) levels, and increased C-reactive protein (CRP). However, on resumption after the chemotherapy cycle, the fever subsided within hours, TP started to increase again, and CRP levels decreased. We observed the same pattern in the second and third chemotherapy cycles (Figure 1B). Vemurafenib dose was reduced after the third cycle of 2-CdA/ARA-C and subsequently discontinued during continuation therapy. The patient has now been without vemurafenib for 19 months, continuation therapy was stopped 7 months ago according to protocol, and the patient is still in remission (supplemental Figure 2).

RAF inhibition was strongly correlated to the disease activity score¹⁸  (Figure 1D). The most striking observation was the rapid and reversible response to the inhibitor, which suggests a functional switch in cell state rather than a cytotoxic effect. Vemurafenib was particularly efficient in inhibiting parameters of risk organ involvement (eg, cytopenia, hypoproteinemia), whereas the bone lesions responded to initial chemotherapy (Figure 1C). In line, levels of CCL2, an inflammation-induced chemokine¹⁹  previously described as upregulated in patients with multisystem LCH,²⁰  strictly correlate with administration of vemurafenib (Figure 1D).

Risk organ involvement is mainly a functional definition⁵  and does not correlate with organ infiltration of CD1A⁺CD207⁺ LCH cells. This suggests that risk organ dysfunction is not caused by infiltration of the LCH cells themselves but rather a result of systemic inflammation (similar to hemophagocytic lymphohistiocytosis²¹ ). In support of this concept is the transcriptomic similarity of the CD1A⁺CD207⁺ LCH cells in single-system and multisystem LCH: the CD1A⁺CD207⁺ cells do not cluster according to disease severity.^12,22,23  In contrast, several groups have reported that the amount of BRAF^V600E in peripheral blood correlates with disease extent.^10,24 

The MAPK pathway is a central signaling pathway in many cell types, and consequently, constitutive activation of ERK signaling could influence cell behavior. We therefore hypothesized that, rather than just serving as precursor cells of the LCH cells, peripheral blood and bone marrow cells themselves may directly contribute to disease manifestations:

First, we correlated treatment to clinical manifestations (disease activity; Figure 1D). Analysis of the number of circulating BRAF^V600E-mutated cells showed that chemotherapy with vinblastine and prednisone failed to eradicate BRAF^V600E-mutated cells (Figure 1E). Furthermore, we even observed an increase in the percentage of BRAF-mutated cells during therapy with vemurafenib, although the patient showed no signs of disease activity. Thus, vemurafenib does not eradicate BRAF^V600E-positive clones, despite inducing clinical remission.^10,11,17 

Next, we determined the frequency of BRAF^V600E in different immune cell types in both bone marrow and peripheral blood at diagnosis and a timepoint during vemurafenib-induced clinical remission (but before salvage chemotherapy; Figure 2A). The mutation was present in all cellular subsets tested, although at much higher frequency in myeloid lineage cells (Figure 2B). Although BRAF^V600E has also been described in CD19⁺ B lymphocytes²⁴  and CD3⁺ lymphocytes,²⁵  this is the first description of BRAF^V600E in natural killer (NK) cells and granulocytes. Although this might be a rare finding, we found mutated granulocytes and NK cells in 2 other patients with LCH. (supplemental Figure 3). Finally, we performed single-cell RNA sequencing analysis of mononuclear cells of the bone marrow and peripheral blood at time of diagnosis and during treatment with vemurafenib. All major cell types were identified (Figure 2D), with some changes in cell number and gene expression profiles on clinical remission during treatment with vemurafenib (Figure 2C). To understand which biological mechanisms coincide with these changes, we identified genes downregulated during therapy and performed pathway enrichment analysis (Figure 2E), which showed pathways associated with inflammation, but not cell cycle or cell division–related pathways. Among the top regulated genes in the bone marrow, we observed downregulation of amphiregulin (AREG), which is expressed in inflammatory conditions,²⁶  and CXCL8 and IL1B, which both are upregulated in LCH,^20,27  illustrating broad anti-inflammatory effects of vemurafenib on immune cells in LCH beyond CD1A⁺CD207⁺ cells in this patient (Figure 2F-G).

Research on LCH has focused on CD1A⁺CD207⁺ cells, aiming to inhibit these “LCH cells” as a cure for patients with the disease. We provide a novel view on LCH by placing the spotlight on non-CD1A⁺CD207⁺ cells: BRAF-mutated cells present in peripheral blood and bone marrow. Extrapolating from the key observations in this prototypic analysis of one patient, we speculate that the CD1A⁺CD207⁺ cells are a differentiated end point that is the pathognomonic finding in LCH, but that BRAF-mutated blood cells need to be regarded as an important part of the disease. The clinically most detrimental effects of LCH are not the tissue lesions but the organ failure, caused by the BRAF^V600E-harboring cells (or possibly by cells with other MAPK pathway mutations causing LCH). It will be necessary to validate this hypothesis in a large cohort, but the careful analysis of this patient provides a blueprint for further analyses within an international framework.

The authors thank the family of the patient who gave the permission to collect data and analyze biological samples, the Biomedical Sequencing Facility at the Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM) for assistance with next-generation sequencing, Dieter Prinz and Julia Stemberger for flow cytometry support, Fikret Rifatbegovic for drawing the visual abstract, and Eva Krivec for technical support.

This work was funded by the St. Anna Kinderkrebsforschung.

Contribution: S.K.E. performed bioinformatics and statistical analysis; R.S., G.A., P.B.S., F.H., S.K.E., and C.H. generated and analyzed data; A.A., M.M., C.H., and S.K.E. collected clinical data; and C.H. and S.K.E. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Caroline Hutter, St. Anna Children’s Cancer Research Institute, Zimmermannplatz 10, 1090 Vienna, Austria; e-mail: caroline.hutter@stanna.at.