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Supplementary Figure 1. Somatic mutations detected in histiocytic neoplasms. Supplementary Figure 2. Frequencies of known activating kinase mutations in histiocytic neoplasms. Supplementary Figure 3. ARAF mutations and variants of unknown significance detected in BRAFV600E-wildtype, non-Langerhans Cell Histiocytic neoplasms. Supplementary Figure 4. Clinical and histological images of the non-Langerhans cell histiocytosis (non-LCH) lesions from index patients with kinase fusions identified by RNA-seq. Supplementary Figure 5. Gene expression analysis of histiocytic neoplasms by RNA-seq. Supplementary Table 1. Characteristics of patient samples with histiocytic neoplasms used in this study and genomic analysis utilized for each sample. Supplementary Table 2. Whole exome sequencing metrics. Supplementary Table 3. Somatic variants identified by whole exome and whole transcriptome sequencing and validated by droplet-digital PCR and/or custom-capture targeted next-generation sequencing with variant allele frequencies (VAFs). Supplementary Table 4. List of the top 1% of differentially expressed genes across histiocytic samples from mRNA sequencing. Supplementary Table 5. PCR primers with M13F2 and M13R2 tails (blue) for Sanger sequencing used in the targeted sequencing recurrence testing for MAP2K1 exons 2 and 3 and all coding regions of ARAF.

Adult-onset histiocytoses (AOH), primarily Rosai-Dorfman disease (RDD), Erdheim-Chester Disease (ECD), and adult Langerhans cell histiocytosis (ALCH), are a group of related histiocytic neoplastic disorders featuring multisystemic manifestations. The disorders are largely incurable, and are essentially chronic neoplastic diseases with a variable prognosis. Prompt diagnosis and treatment is important to prevent debilitating and even life-threatening complications. Survivorship issues abound in AOH, due to their multisystemic manifestations and the sometimes recalcitrant chronic inflammation, which can lead to other debilitating complications such as fatigue, weakness, and pain. Because these disorders are rare, few healthcare professionals are proficient in their management; therefore the aim of these guidelines is to offer guidance on how to manage patients, and how to create survivorship care plans through the efforts of an interdisciplinary team.

Erdheim-Chester disease (ECD) is a rare histiocytosis that was recently recognized as a neoplastic disorder owing to the discovery of recurrent activating MAPK (RAS-RAF-MEK-ERK) pathway mutations. Typical findings of ECD include central diabetes insipidus, restrictive pericarditis, perinephric fibrosis, and sclerotic bone lesions. The histopathologic diagnosis of ECD is often challenging due to nonspecific inflammatory and fibrotic findings on histopathologic review of tissue specimens. Additionally, the association of ECD with unusual tissue tropism and an insidious onset often results in diagnostic errors and delays. Most patients with ECD require treatment, except for a minority of patients with minimally symptomatic single-organ disease. The first ECD consensus guidelines were published in 2014 on behalf of the physicians and researchers within the Erdheim-Chester Disease Global Alliance. With the recent molecular discoveries and the approval of the first targeted therapy (vemurafenib) for BRAF-V600–mutant ECD, there is a need for updated clinical practice guidelines to optimize the diagnosis and treatment of this disease. This document presents consensus recommendations that resulted from the International Medical Symposia on ECD in 2017 and 2019. Herein, we include the guidelines for the clinical, laboratory, histologic, and radiographic evaluation of ECD patients along with treatment recommendations based on our clinical experience and review of literature in the molecular era.

Objective To characterize the spectrum of neurologic involvement in Erdheim–Chester Disease (ECD), a treatable inflammatory neoplasm of histiocytes. Methods Sixty‐two patients with ECD were prospectively enrolled in a natural history study that facilitated collection of clinical, imaging, laboratory, neurophysiologic, and pathologic data. Results Ninety‐four percent of the patients had neurologic abnormalities on examination or imaging, and 22% had neurologic symptoms as the initial presentation of ECD. The most common neurologic findings were cognitive impairment, peripheral neuropathy, pyramidal tract signs, cranial nerve involvement, and cerebellar ataxia. Imaging revealed atrophy and demyelination along with focal lesions that were located throughout the nervous system, dura, and extra‐axial structures. The BRAF V600E variant correlated with cerebral atrophy. Brain pathology revealed lipid‐laden, phagocytic macrophages (histiocytes) accompanied by demyelination and axonal degeneration. Interpretation In patients with ECD, neurologic morbidity is common and contributes significantly to disability. Since neurologic symptoms can be the presenting feature of ECD and, given the mean delay in ECD diagnosis is 4.2 years, it is critical that neurologists consider of ECD and other histiocytosis in patients with inflammatory, infectious, or neoplastic‐appearing white matter. Furthermore, given the broad spectrum of neurologic involvement, neurologists have an important role in a team of specialists treating ECD patients.

Key Points Question What is the prevalence of hypothalamic-pituitary-thyroid dysfunction in patients with Erdheim-Chester disease (ECD)? Findings In this cross-sectional study of 61 patients with ECD, the prevalence of central and primary hypothyroidism was 9.8% and 18.0%, respectively, in patients with ECD, higher than the corresponding rates of 0.1% and 4.7%, respectively, in the community. Meaning The findings of this study suggest that clinicians should consider screening for hypothyroidism in patients with ECD., This cross-sectional study assesses rates of hypothalamus-pituitary-thyroid dysfunction in patients with Erdheim-Chester disease., Importance Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis affecting multiple organs and commonly caused by somatic pathogenic variants in BRAF V600E and mitogen-activated protein kinase genes. Clinical features of ECD result from histiocytic involvement of various tissues; while endocrine involvement in ECD occurs frequently, the prevalence of central or primary hypothyroidism has not been thoroughly investigated. Objective To assess hypothalamus-pituitary-thyroid (HPT) dysfunction in patients with ECD. Design, Setting, and Participants This cross-sectional study included 61 patients with ECD who were enrolled in a natural history study at a tertiary care center between January 2011 and December 2018. ECD was diagnosed on the basis of clinical, genetic, and histopathological features. Data were analyzed in March 2020. Exposure Diagnosis of ECD. Main Outcomes and Measures Main outcome was the prevalence of thyroid dysfunction in adults with ECD compared with community estimates. Patients underwent baseline evaluation with a thyroid function test, including thyrotropin, free thyroxine (fT4), and total thyroxine (T4), and sellar imaging with magnetic resonance imaging or computed tomography scan. The association of HPT dysfunction was assessed for differences in age, sex, body mass index, BRAF V600E status, high sensitivity C-reactive protein level, sellar imaging, and pituitary hormonal dysfunction. Results A total of 61 patients with ECD (46 [75%] men; mean [SD] age, 54.3 [10.9] years) were evaluated. Seventeen patients (28%) had hypothyroidism requiring levothyroxine therapy. The prevalence of both central and primary hypothyroidism were higher than community estimates (central hypothyroidism: 9.8% vs 0.1%; odds ratio, 109.0; 95% CI, 37.4-260.6; P

To investigate the computed tomography (CT) thoracic findings in Erdheim-Chester disease (ECD) and evaluate the association of these findings with the BRAFV600E mutation. This was a prospective study of patients with ECD (n=61, men=46) who underwent thoracic CT imaging. CT examinations were independently interpreted by two experienced radiologists. Association of imaging findings with BRAFV600E was achieved via the Chi-square or Fisher’s exact test and odds ratios (OR) with 95% confidence intervals (CI), as appropriate. Fifty-five ECD patients (90%) showed pulmonary findings, which included interlobular septal thickening (69%), pulmonary nodules (62%), airway thickening (13%) and ground glass opacities (36%). Pulmonary nodules were classified by the pattern of distribution: subpleural regions (36%), lung parenchyma (13%) and both regions (13%). Pleural and mediastinal involvement were present in 15% and 62% of cases, respectively. The most common mediastinal finding was sheathing of the right coronary artery (34%), followed by sheathing of the thoracic aorta (30%). The BRAFV600E mutation, positive in 31 patients, was associated with the frequency of sheathing of the coronary arteries (p = 0.01). Of the thoracic findings reported in this study, we found a statistically significant positive association between the BRAFV600E mutation and presence of coronary artery sheathing. • To assess the degree of thoracic involvement in ECD with CT. • BRAF V600E mutation has a high association with right coronary artery sheathing. • BRAF V600E genetic testing detects patients at high risk of developing RCA sheathing.

To use magnetic resonance imaging (MRI) and computed tomography (CT) to define abdominal involvement in Erdheim–Chester disease (ECD), and to investigate the association between these findings and the BRAFV600E mutation. This prospective study was performed on 61 ECD patients (46 men). The MRI and CT imaging studies were reviewed independently by two experienced radiologists. The association between BRAFV600E mutation and imaging findings was analysed using Fisher’s exact test, and odds ratios with 95% confidence intervals. Perinephric infiltration was the most common finding (67%), followed by involvement of proximal ureters (61%). In 56% of cases, infiltration extended to the renal sinuses, and in 38% caused hydronephrosis. Adrenal gland infiltration was present in 48% of patients. Infiltration of renal artery (49%) and aorta (43%) were the most common vascular findings, followed by sheathing of celiac, superior mesenteric artery (SMA) or inferior mesenteric artery (IMA) (23%). The BRAFV600E mutation was positive in 53% of patients with interpretable BRAF sequencing. There was a statistically significant association between this mutation and perinephric infiltration (p = 0.003), renal sinus involvement (p < 0.001), infiltration of proximal ureters (p < 0.001), hydronephrosis (p < 0.001), adrenal gland involvement (p < 0.001), periaortic infiltration (p = 0.03), sheathing or stenosis of renal artery (p < 0.001) and sheathing of other aortic branches (p = 0.04). Renal and vascular structures are the most commonly affected abdominal organs in ECD patients. Some of these findings have significant positive association with the BRAFV600E mutation. • Abdominal imaging plays a crucial role in management of Erdheim–Chester disease. • Significant associations exist between BRAF V600E mutation and several abdominal imaging findings. • Considering several associations, evaluating BRAFV600E mutation status is recommended in ECD patients.

Simple Summary Erdheim–Chester disease (ECD) is a rare histiocytic neoplasm that is frequently associated with hypothalamic–pituitary gland involvement leading to endocrine dysfunctions. Frequently, endocrinopathy is permanent and precedes the diagnosis of ECD and may also develop during the course of treatment. However, the exact nature and frequency of hypothalamic–pituitary involvement are unknown. We studied a natural history cohort of 61 subjects with Erdheim–Chester disease and found abnormal pituitary imaging in 47.5% of cases, associated with panhypopituitarism, diabetes insipidus, and a higher median number of pituitary endocrinopathies. These data confirm and significantly extend previous reports of centrally occurring endocrine dysfunction and highlight the need for routine imaging and systematic assessment of hypothalamic–pituitary endocrine function in patients with ECD. Abstract Purpose: We examined abnormal pituitary imaging (API) and associated endocrine dysfunction in subjects with ECD. Methods: A cross-sectional descriptive examination of a natural history cohort study diagnosed with ECD was conducted at a clinical research center. Subjects underwent baseline endocrine tests of anterior and posterior pituitary function and dedicated pituitary gland MRI scans. We determined the frequency of various pituitary imaging abnormalities in ECD and assessed its relationships with age, sex, body mass index (BMI), BRAF V600E status, high sensitivity C-reactive protein (hsCRP), erythrocyte sedimentation rate (ESR), pituitary hormone deficits and number, diabetes insipidus (DI), and panhypopituitarism. Results: Our cohort included 61 subjects with ECD [age (SD): 54.3 (10.9) y, 46 males/15 females]. API was present in 47.5% (29/61) of ECD subjects. Loss of the posterior pituitary bright spot (36.1%) followed by thickened pituitary stalk (24.6%), abnormal enhancement (18.0%), and pituitary atrophy (14.8%) were the most common abnormalities. DI and panhypopituitarism were more frequent in subjects with API without differences in age, sex distribution, hsCRP, ESR, and BRAF V600E status compared to normal pituitary imaging. Conclusions: We noted a high burden of API and endocrinopathies in ECD. API was highly associated with the presence of panhypopituitarism and DI. Therefore, a thorough assessment of hypothalamic–pituitary integrity should be considered in subjects with ECD.

Erdheim-Chester Disease (ECD) is a rare, potentially fatal, multi-organ myeloid neoplasm occurring mainly in adults. The diagnosis is established by clinical, radiologic, and histologic findings; ECD tumors contain foamy macrophages that are CD68+, CD163+, CD1a-, and frequently S100-. The purpose of this report is to describe the clinical and molecular variability of ECD. Sixty consecutive ECD patients (45 males, 15 females) were prospectively evaluated at the NIH Clinical Center between 2011 and 2015. Comprehensive imaging and laboratory studies were performed, and tissues were examined for BRAF V600E and MAPK pathway mutations. Mean age at first manifestations of ECD was 46 years; a diagnosis was established, on average, 4.2 years after initial presentation. Bone was the most common tissue affected, with osteosclerosis in 95% of patients. Other manifestations observed in one-third to two-thirds of patients include cardiac mass and periaortic involvement, diabetes insipidus, retro-orbital infiltration, retroperitoneal, lung, CNS, skin and xanthelasma, usually in combination. Methods of detection included imaging studies of various modalities. Mutation in BRAF V600E was detected in 51% of 57 biopsies. One patient had an ARAF D228V mutation, and one had an activating ALK fusion. Treatments included interferon alpha, imatinib, anakinra, cladribine, vemurafenib and dabrafenib with trametinib; eleven patients received no therapy. The diagnosis of ECD is elusive because of the rarity and varied presentations of the disorder. Identification of BRAF and other MAPK pathway mutations in biopsies improves ECD diagnosis, allows for development of targeted treatments, and demonstrates that ECD is a neoplastic disorder.

Background: Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis affecting multiple organs, including the endocrine system. While endocrine involvement in ECD is well characterized, infiltration of the hypothalamic-pituitary-thyroid axis may cause either primary or central hypothyroidism that is often underdiagnosed. The prevalence of hypothyroidism and the occurrence of isolated central hypothyroidism in ECD has not been thoroughly investigated. Methods: A prospective cohort study of biopsy-confirmed cases of ECD was conducted at the National Institutes of Health. Clinical, radiographic, and biochemical characteristics were assessed. All subjects underwent baseline evaluation with a thyroid function test, including TSH, free thyroxine (fT4) and total thyroxine (T4).Results: Sixty-one subjects with ECD (46 males, 54.3 ± 10.8 years) were evaluated. Sixteen subjects (26%) hadprimary hypothyroidism and were receiving thyroid hormone supplementation before enrollment, with a mean TSH 2.00 ±1.63 mcIU/mL (normal 0.27-4.20 mcIU/mL), fT4 1.52 ±1.51 ng/dL (normal 0.9-1.7 ng/dL), and T4 7.42 ±2.15 mcg/dL (normal 4.5-11.7 mcg/dL). The prevalence of primary hypothyroidism was higher than general population estimates (26% vs. 3.7%, P

Background: Erdheim Chester disease (ECD) is a rare histiocytic neoplasm associated with hypothalamic and pituitary infiltration and dysfunction. We determined the abnormal pituitary imaging (API) phenotypes in subjects with ECD and analyzed their associated endocrine dysfunctions. Methods: This was a cross-sectional examination of a natural history cohort study of 61 subjects with ECD performed at a tertiary care clinical research center. The diagnosis of ECD was based on clinical, molecular, and histopathological features. Enrolled subjects underwent baseline endocrine tests of anterior and posterior pituitary function in addition to pituitary imaging. The following variables were analyzed- age, sex, body mass index (BMI), BRAF V600E, hsCRP, ESR, pituitary hormone deficit number, diabetes insipidus (DI), and panhypopituitarism. Fisher’s exact test or t-test/Wilcoxon tests compared patients with and without API. Results: Sixty-one subjects with ECD (age ±SD: 54.3 ±10.9, 46 (75.4%) males) were studied. The prevalence of API was 32.8% (n=20), who were younger than those with normal imaging (50.3 ±10.5 vs 56.3 ±10.7 yrs, p=0.042). The most common pituitary imaging abnormalities included thickened pituitary stalk (18.03%, n=11/61), followed by pituitary encasement, small pituitary and abnormal morphology (6.55%, n= 4/61 for each). A higher prevalence of DI (45.0% vs 9.8%, p=0.003) and panhypopituitarism (45.0% vs 4.9%, p

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis characterized by uncontrolled inflammation resulting in fibro-inflammatory damage of multiple organs, including the hypothalamic-pituitary-adrenal (HPA) axis. Glucocorticoid insufficiency may occur and is associated with an increased burden of disease and an increased risk of adrenal crisis. Sixty-one ECD subjects (46 males, age 54.3 ± 10.8 years) were evaluated; 56.1% (32/57) harbored a somatic activating BRAF V600E variant. Adrenal gland and sellar or suprasellar involvement was present in 18/57 (31.6%) and 9/57 (15.8%) subjects on computed tomography or magnetic resonance imaging, respectively. The BRAF-positive group was more likely to have adrenal gland involvement (56.2% vs. 20.0%; odds ratio (OR) 5.8, 95% confidence interval (CI) 1.5-17.1, P=0.008), with comparable rates for sellar or suprasellar involvement (28.1% vs. 20.0%; OR 1.6, 95% CI 0.5-5.4, P=0.5) and low random morning cortisol values (37.5% vs. 32.0%; OR 1.3, 95% CI 0.4-3.8, P=0.7). High-sensitivity C-reactive protein was significantly higher in the BRAF V600E-positive group with adrenal involvement when compared to the BRAF V600E-negative group without adrenal involvement (mean concentration: 40 mg/dL vs. 5 mg/dL, P=0.046). HPA axis in ECD tends to involve the adrenal glands in BRAF V600E-positive individuals, without influencing the rates of low cortisol levels, although there is a poor biochemical-radiological concordance in ECD. In this cohort, most subjects with HPA axis involvement did not require glucocorticoid or mineralocorticoid therapy. This study systematically demonstrates an association between the somatic activating BRAF V600E variant and the presence of radiographic and biochemical HPA axis involvement in ECD subjects. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01417520

Limited information is available regarding interstitial lung disease (ILD) in Erdheim&ndash, Chester disease (ECD), a rare multisystemic non-Langerhans cell histiocytosis. Sixty-two biopsy-confirmed ECD patients were divided into those with no ILD (19.5%), minimal ILD (32%), mild ILD (29%), and moderate/severe ILD (19.5%), based on computed tomography (CT) findings. Dyspnea affected at least half of the patients with mild or moderate/severe ILD. Diffusion capacity was significantly reduced in ECD patients with minimal ILD. Disease severity was inversely correlated with pulmonary function measurements, no correlation with BRAF V600E mutation status was seen. Reticulations and ground-glass opacities were the predominant findings on CT images. Automated CT scores were significantly higher in patients with moderate/severe ILD, compared to those in other groups. Immunostaining of lung biopsies was consistent with ECD. Histopathology findings included subpleural and septal fibrosis, with areas of interspersed normal lung, diffuse interstitial fibrosis, histiocytes with foamy cytoplasm embedded in fibrosis, lymphoid aggregates, and focal type II alveolar cell hyperplasia. In conclusion, ILD of varying severity may affect a high proportion of ECD patients. Histopathology features of ILD in ECD can mimic interstitial fibrosis patterns observed in idiopathic ILD.

HistoryA 42-year old Caucasian man was referred to theNationalInstitutes ofHealth(NIH) for evaluation.Hissymptoms began approximately 10 years before re-ferral with pituitary dysfunction that manifested asdiabetes insipidus, secondary hypogonadism, andhyperprolactinemia, treated with desmopressin andtestosterone gel. Eight years later, he began to expe-rience vertigo, dizziness, diplopia, and difficultyfocusingandtracking.Thiswasfollowedbyprogress-ive left-sided weakness, poor coordination, and bal-ancedifficulties.Magneticresonanceimagingwithoutcontrast revealed multiple nonspecific white-matterlesions. Subsequent evaluation failed to identify anunderlyinginfectionorinflammatoryorigin.Systemiccorticosteroid therapy was initiated with minimalimprovement. Corticosteroids were tapered off 2monthslaterandthepatientnotednewasymptomaticskin lesions on his bilateral upper and lower extrem-ities, leading to reinitiation of systemic steroids andintroduction of interferon alfa. He denied fevers,chills, weight loss, or difficulty breathing.Physical examinationThe patient was a well-appearing Caucasian man.Cutaneous examination was notable for approxi-mately twenty 2- to 3-mm, firm, reddish-brown pap-ules scattered on the trunk and extremities, withgreater density on the front of his thighs (Fig 1).Diascopy revealed a yellowishhue. Ophthalmologicexamination was notable for nystagmus, saccadicpursuit,andmilddysmetria.Neurologicexaminationrevealed unsteady tandem gait, abnormal finger-to-nose pointing, and left-sided dysdiadochokinesia.Left arm strength was diminished (4/5). The oralmucosa was unremarkable. No lymphadenopathywas appreciated.HistopathologyA punch skin biopsy specimen of a papule on theleft thigh was obtained, and histology revealed amoderately dense dermal infiltrate of epithelioidhistiocytes characterized by rounded to slightlyelongated nuclei with inconspicuous nucleoli andmoderatetoabundanteosinophiliccytoplasm(Fig2,A). CD68 and CD163 immunochemistry highlightedthe lesional histiocytes (Fig 2, B). Lesional cells alsoshowedstrong,diffusestainingforfactorXIIIa(Fig2,C). CD1a and S100 immunostains were negative.Significant diagnostic studiesRadiographs of the upper and lower extremitiesrevealed sclerotic lesions involving the metaphyseal

Medical genetics and genomic medicine in the Dominican Republic: challenges and opportunities.

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

Erdheim-Chester disease is an uncommon non-Langerhans-cell histiocytosis, due to excessive production of histiocytes deposited in various organs and tissues in the human body. FDG PET was performed in a 68-year-old man with documented active Erdheim-Chester disease to evaluate the extent of the disease. The patient was previously treated with high-dose subcutaneous Interferon α2b, 1,000,000 units 3 times a week, but treatment was interrupted approximately 5 weeks before evaluation at the National Institutes of Health because of adverse effects of the medication. FDG PET/CT showed lesions were imaged in brain, heart, mediastinum, abdomen, and skeleton.

Erdheim-Chester disease (ECD) is a rare, non-Langerhans histiocytosis. Recent findings suggest that ECD is a clonal disorder, marked by recurrent BRAFV600E mutations in >50% of patients, in which chronic uncontrolled inflammation is an important mediator of disease pathogenesis. Although ∼500 to 550 cases have been described in the literature to date, increased physician awareness has driven a dramatic increase in ECD diagnoses over the last decade. ECD frequently involves multiple organ systems and has historically lacked effective therapies. Given the protean clinical manifestations and the lack of a consensus-derived approach for the management of ECD, we provide here the first multidisciplinary consensus guidelines for the clinical management of ECD. These recommendations were outlined at the First International Medical Symposium for ECD, comprised of a comprehensive group of international academicians with expertise in the pathophysiology and therapy of ECD. Detailed recommendations on the initial clinical, laboratory, and radiographic assessment of ECD patients are presented in addition to treatment recommendations based on critical appraisal of the literature and clinical experience. These formalized consensus descriptions will hopefully facilitate ongoing and future research efforts in this disorder.

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.

Systemic histiocytic disorders, including Langerhans cell histiocytosis (LCH) and the non-Langerhans cell histiocytic disorder Erdheim-Chester Disease (ECD) are clinically heterogeneous diseases whose underlying etiology has long been obscure. Recent identification of BRAF V600E mutations in ~50% of LCH and ECD patients, as well as mutations in MAP2K1 in ~25% of BRAF-wild type LCH patients has refined our understanding of these disorders as clonal malignancies driven by constitutive MAP kinase signaling. However, the compendium of mutations co-occurring with BRAF V600E and MAP2K1 mutations in ECD/LCH have yet to be defined since unbiased genomic sequencing studies have not been performed in ECD. Moreover, the molecular bases of the phenotypic differences between LCH and ECD have remained elusive. Therefore, we performed whole exome sequencing (WES) of adult and pediatric LCH and ECD patients to further elucidate our understanding of the molecular bases of these disorders. Nineteen histiocytic disorder cases including fresh-frozen tissue biopsies from 10 adult (2 LCH and 8 ECD) and 8 pediatric patients (all LCH), along with paired normal tissue from blood were evaluated by WES (1 adult ECD patient had synchronous biopsies from 2 different anatomic sites- these 2 samples are treated independently in this analysis). Percentage of tumor involvement ranged from 25%-50% based on review of histologic sections by board-certified hematopathologists. High quality sequence variants unique to the tumor DNA including SNVs and indels were identified for subsequent analyses. All candidate missense genetic variants were evaluated by 3 in silico analysis methods (PROVEAN; SIFT; PolyPhen-2) for predicted mutational effects on protein function. For missense variants, only those determined to be damaging to protein function in 2 or more in silico methods were retained for further evaluation. Mutations passing these analytical steps were further evaluated for biological significance using several web-based, freely available pathway analysis databases. We first examined mutations in the MAP kinase pathways identified by the WES of LCH and ECD patients (Figure). Of LCH cases, 40% harbored BRAF V600E mutations while 30% had mutations in MAP2K1. Three patients carried in-frame deletion mutations in the negative regulatory domain of MAP2K1. Interestingly, mutations in the JNK and p38 MAP kinase pathways were identified in 20% of LCH patients, one of which was BRAF/MAP2K1 wild type (Figure). Of the ECD patients, 44% were BRAF V600E mutant, and we identified one BRAF V600E-wild type ECD patient as having a MAP2K1 mutation (MAP2K1 K57N) and another as having an NRAS (NRAS Q61R) mutation. As with LCH patients, WES analysis again noted mutations in p38 and JNK MAP kinase pathway members in BRAF/NRAS/MAP2K1-wild type ECD patients further highlighting involvement of all 3 MAP kinase pathways in ECD/LCH pathogenesis. We next evaluated mutated genes not known to be directly involved in MAP kinase signaling in order to understand how non-MAP kinase signaling pathways might contribute to LCH/ECD pathogenesis. Recurrent mutations in members of the Notch, WNT/β-catenin, NF-κB, and FGF signaling pathways were identified (Figure). Interestingly, loss-of-function mutations in Notch pathway mediators identified in this study occurred only (i) in MAML3 and MAMLD1 and (ii) in LCH (60% of patients) but not ECD. These findings, combined with recent identification of the monocytic-origin of LCH and the discovery that loss of Notch signaling results in monocytic clonal disorders, highlights a potential role for Notch signaling in the pathogenesis of LCH. Likewise, identification of a DNMT3A mutation in a BRAF V600E-mutant ECD patient and the association of Dnmt3a loss with clonal dominance of Dnmt3a-deficient hematopoietic cells suggests a potential role for disordered epigenetic regulation in ECD. This WES study of somatic mutations in LCH and ECD confirms previously reported frequencies of BRAF V600E and MAP2K1 mutations in LCH, identifies MAP2K1 mutations in ECD for the first time, and reveals additional candidate mutations in MAPK signaling that are mutually exclusive of BRAF/MAP2K1 mutations. Furthermore, this first WES study of ECD reveals somatic mutations in multiple genes that regulate diverse cellular processes co-occurring with recurrent mutations in MAPK signaling pathways. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.