Park, Christopher Y.

Abstract The identification of mutations in BRAFV600E and other kinases in systemic histiocytoses changed our understanding of the pathophysiology of these disorders from inflammatory non-malignant conditions to clonal disorders driven by altered MAP kinase signaling. In addition, BRAFV600E mutations have recently been noted in bone marrow (BM) CD34+ cells of patients with Langerhans Cell Histiocytosis (LCH), suggesting that LCH represents a clonal disorder arising from hematopoietic stem/progenitor cells (HSPCs). However, it is unknown if HSPCs from LCH or other histiocytoses patients can functionally give rise to these disorders. Moreover, whether or not patients with histiocytosis have additional hematological disorders related to the presence of HSPC-based mutations is unclear. Here we attempted to understand the spectrum of hematological disorders in patients with histiocytoses and the cell-of-origin of histiocytic neoplasms by functionally analyzing HSPCs from histiocytoses patients. We first analyzed a multicenter cohort of 190 adult histiocytoses patients. This unexpectedly identified a high frequency of patients with a co-existing myeloid malignancy. Of the 190 patients, 9.5% (18/190) were diagnosed with Erdheim-Chester Disease (ECD) and/or LCH concomitantly with a classic MPN (3.7%; 7/190), MPN/MDS overlap (4.7%; 9/190), or MDS (1.1%; 2/190) (Figure). This clinical observation was confirmed by detection of co-existing histiocytoses-associated kinase mutations (including BRAF, MAP2K1, NRAS, or KRAS mutations) with JAK2 or CALR mutations, as well as other mutations recurrently found in myeloid malignancies (such as IDH1/2, ASXL1, and TET2 mutations (Figure)). These data therefore reveal a heretofore-unrecognized clinical overlap of adult-onset systemic histiocytoses and WHO-classified myeloid malignancies while further highlighting the possibility of an aberrant HSPC origin for histiocytic neoplasms. In most cases, the BRAF/MAP2K1/RAS mutation predominated in the histiocytic lesion while the JAK2/CALR mutation predominated in the BM/peripheral blood suggesting likely distinct clones driving each clinical condition. Next, to test the self-renewal and differentiation capacity of HSPCs from histiocytoses patients, CD34+ cells were purified from 8 patients including 6 with ECD or LCH plus a concomitant myeloid neoplasm, and 2 with ECD alone. 0.1-0.8x106 CD34+ cells from each patient were transplanted by intrafemoral injection into sublethally irradiated NOD/SCID IL2Rγ transgenic human GM-CSF, IL-3, and SCF expressing (NSG-SGM3) mice. Human engraftment was monitored by monthly flow cytometric analysis of peripheral blood until mice became moribund. Thereafter, animals were sacrificed and tissues were analyzed using flow cytometry, histology, immunohistochemistry, and sequencing analyses to detect driver mutations identified in the histiocytosis/co-occurring myeloid neoplasm. Successful engraftment of human myeloid cells was verified in grafts generated from 2/8 patients after a mean of 90 days (range: 60-120 days). This included lethal engraftment of KRAS-mutated ECD patient cells at 90 days manifested by infiltration of human hCD45+ cells co-expressing human myeloid and monocyte lineage markers hCD33 and hCD14 in the bone marrow, spleen, liver, lung, and kidney (Figure). Immunostains revealed that tissues were infiltrated by an hCD45+hCD163+hCD68+ population of foamy histiocytes, characteristic of ECD (Figure). Furthermore, genomic analysis of DNA from the engrafted animals BM and spleen revealed the same KRASG12S mutation found in the donor patients ECD lesion. Engraftment of a patient diagnosed with concomitant BRAFV600E-mutant ECD and ASXL1/TET2-mutant CMML was seen at 60 days. In this case, however, only engraftment of human CMML was detected without evidence of the BRAFV600E-mutant ECD component. This study suggests that adult histiocytic neoplasms frequently co-occur with other myeloid neoplasms. Moreover, histiocytosis disease-driver mutations are present in the CD34+ HSPCs, which we show for the first time can initiate disease that resembles human histiocytosis in a patient-derived xenograft. Importantly, however, even though the CD34+ compartment gives rise to both the histiocytic and myeloid neoplasm disease component, each disease is likely initiated by distinct precursors. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAFV600E mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAFV600E–wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAFV600E–wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. Significance: We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders. Cancer Discov; 6(2); 154–65. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 109