Novel BRAF fusions in pediatric histiocytic neoplasms define distinct therapeutic responsiveness to RAF paradox breakers

Abstract

Running Title: Novel BRAF fusions in pediatric histiocytic tumors Histiocytic neoplasms are a diverse group of clonal hematopoietic disorders that are driven by mutations activating the mitogen-activated protein kinase (MAPK) and phosphoinositide 3kinases (PI3K) pathways 1-3 . While BRAF-V600E is the most common alteration in histiocytic neoplasms, multiple alternate pathway activating mechanisms have been described, including MAP2K1, ARAF, PIK3CA, NRAS, and KRAS mutations as well as BRAF, ALK, and NTRK1 fusions 1-3 . BRAF-fusions previously reported in cases of histiocytic neoplasms 1,4-7 were found to contain the N-terminal region of another gene (often of unclear significance) joined to the BRAF kinase domain (including exons 9-18 or 11-18), resulting in the loss of the BRAF N-terminal regulatory RAS-binding regions (exons 1-8). Despite the prevalence of BRAF-alterations in histiocytic tumors, to date there have been no detailed molecular investigations comparing BRAF-fusions found in distinct sub-types of histiocytic neoplasms and only one study explored responsiveness of such BRAF-fusions to single-agent RAF-therapies 4 . To address this, we present two pediatric histiocytosis cases with divergent pathologic and clinical features, each harboring a novel BRAFfusion as identified by a clinically validated next-generation sequencing panel (Supplemental Methods and Table 1) and study their in vitro responsiveness to RAF-targeted inhibitors.2 Case 1, a 16 year-old female with a 2.5 cm rapidly growing subcutaneous thigh mass was diagnosed with a malignant histiocytic neoplasm ("M group") 8 , with a phenotype spanning histiocytic sarcoma (CD163/CD14/CD68+) and Langerhans cell sarcoma (CD1a/Langerin/S100) with a modestly elevated Ki-67 proliferation index (up to 20%) ( Figure 1A-F). Targeted RNAsequencing identified a fusion of MTAP (NM_002451.3) exons 1-7 to BRAF (NM_004333.4) exons 9-18. Resection margins were negative. The patient is disease-free three years post-resection. Case 2, a 12-year-old female with a 5.3 cm rapidly enlarging heel mass encasing and invading the calcaneus was diagnosed with a juvenile xanthogranuloma (JXG) family lesion (CD163/CD68/CD14/fascin/Factor XIIIa+) ( Figure 1G-M). Despite the lack of cytologic atypia or increased mitotic rate by H&E stain, a Ki-67 proliferation index stain revealed increased staining in lesional cells up to 40%. Targeted RNA-seq identified a fusion of MS4A6A (NM_022349.3) exons 1-6 and BRAF (NM_004333.4) exons 11-18. During staging, the patient was found to have PET-avid dissemination to lymph nodes and lung ( Figure 1N-P). While the morphologic features were consistent with a low-grade histiocytic lesion of JXG phenotype, the integration of the 1) high Ki-67 proliferation index, 2) aggressive clinical behavior with lymphatic/metastatic-like spread, and 3) novel molecular BRAF-fusion suggested an atypical JXG family neoplasm with uncertain biological behavior. The patient was treated with 12 cycles of clofarabine with clinical remission of metastatic sites and ne...