Activating Mutations in CSF1R and Additional Receptor Tyrosine Kinases in Sporadic and Familial Histiocytic Neoplasms

Genomic analyses of histiocytic neoplasms (including Langerhans Cell Histiocytosis (LCH) and the non-LCH Erdheim-Chester Disease (ECD)) have revolutionized our understanding of these disorders as clonal hematopoietic malignancies driven by MAPK signaling and led to FDA approval of vemurafenib for BRAF^V600E-mutant ECD. Despite these advances, several questions about the pathogenesis of the histiocytoses remain unanswered. For example, the cell-of-origin of the histiocytoses is not definitively known. In addition, histiocytoses represent a spectrum of diseases, and genetic alterations across histiocytosis subtypes have not been comprehensively evaluated. Finally, although the histiocytoses most commonly occur as sporadic, non-hereditary disorders, familial clustering has been well documented and occurs most often in monozygotic twins. This has been taken to suggest a hereditary component of the disease, but germline genetic causes for histiocytoses are not known. Here we performed comprehensive genomic analyses of 218 patients with all subsets of histiocytoses, including monozygotic twins with histiocytosis. In so doing, we uncover a novel series of activating receptor tyrosine kinase (RTK) alterations in the histiocytoses, including the first example in any disease of recurrent, activating mutations in CSF1R, the RTK required for monocyte/macrophage development.

We initially performed whole exome sequencing (WES) of skin lesions, blood, and fingernails from identical (monozygotic, dichorionic), one-year-old twins with systemic juvenile xanthogranuloma (JXG). This identified an identical in-frame deletion in CSF1R (CSF1R^Y546_K551del) in the skin lesions of both children. Recent data from murine models suggest that CSF1R-expressing yolk sac derived precursors of tissue-resident macrophages may be a cell-of-origin of the histiocytoses (Mass, et al. Nature 2017). Consistent with this hypothesis, the identical CSF1R mutation was shared across the histiocytosis lesions in both twins but was absent from blood or fingernails.

We next sought to determine if similar mutations in CSF1R might exist in sporadic histiocytosis cases. We therefore sequenced 92 ECD (42%), 58 LCH (27%), 50 JXG (23%), 12 RDD (5%), and 6 histiocytic sarcoma (HS) (3%) lesions using WES, targeted DNA sequencing with a 585-gene panel, and targeted RNA-sequencing for fusions in 74 genes. This identified recurrent mutations in BRAF^V600E, MAP2K1, N/KRAS, and ARAF, as well as BRAF, NTRK1, and ALK fusions as previously described in ECD and LCH (Fig. A-C). Interestingly, CSF1R mutations were also found in 8 cases, most commonly as CSF1R^Y546_K551del, and were predominantly in JXG (10%; n=5/50). Consistent with the recurrent nature of this mutation, expression of CSF1R^Y546_K551del, but not CSF1R^WT, conferred robust cytokine-independent growth to cell lines normally dependent on cytokines (Ba/F3 and 32D cells; Fig. D). We also identified individuals with mutations in CSF3R, KIT, ALK, MET, JAK3, and CRAF, as well as a RET fusion. These studies additionally identified important differences in the spectrum of kinase alterations across histiocytoses subtypes. For example, the BRAF^V600E mutation was the most common kinase alteration in LCH and ECD but was not identified in JXG or RDD. Furthermore, BRAF fusions were predominantly seen in LCH and JXG. Meanwhile, NTRK1 and ALK fusions were mainly identified in JXG and ECD, respectively.

From a therapeutic perspective, CSF1R activating mutations sensitized cells to inhibition with the CSF1R-specific, small-molecule kinase inhibitors pexidartinib and BLZ945. In addition, in the course of this study, a patient bearing ALK-rearranged ECD required therapy, and we were able to evaluate response to the ALK inhibitor crizotinib. Crizotinib-treatment resulted in profound and sustained therapeutic improvements in this patient (Fig. E).

Overall, the above data demonstrate the occurrence of activating CSF1R and other RTK alterations in patients with histiocytic neoplasms, many of which have direct therapeutic importance (such as the first demonstration of ALK inhibitor efficacy in ALK⁺ histiocytosis). In addition, the discovery of somatically acquired CSF1R activating mutations in identical twins with histiocytosis provides the first human evidence that tissue-resident macrophages may serve as a cell-of-origin of the histiocytoses.

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