ARAF Mutations Research Articles

Genetic Landscape and Its Prognostic Impact in Children With Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare myeloid neoplasm that predominantly affects young children. To investigate genetic alterations and their correlation with clinical characteristics and prognosis in pediatric LCH. We performed targeted sequencing to detect mutations in LCH lesions from pediatric patients. A total of 30 genomic alterations in 5 genes of the MAPK pathway were identified in 187 of 223 patients (83.9%). BRAF V600E (B-Raf proto-oncogene, serine/threonine kinase) was the most common mutation (51.6%), followed by MAP2K1 (mitogen-activated protein kinase kinase 1) alterations (17.0%) and other BRAF mutations (13.0%). ARAF (A-Raf proto-oncogene, serine/threonine kinase) and KRAS (KRAS proto-oncogene, GTPase) mutations were relatively rare (2.2% and 0.9%, respectively). Additionally, FNBP1 (formin-binding protein 1)::BRAF fusion and MAP3K10 (mitogen-activated protein kinase kinase 10) mutations A17T and R823C were identified in 1 case each, with possible constitutive activation of ERK1/2 phosphorylation. BRAF V600E was more frequent in patients with risk organ involvement, while MAP2K1 mutation was more prevalent in patients with single-system LCH (P = .001). BRAF V600E was associated with craniofacial bone, skin, liver, spleen, and ear involvement (all P < .05). Patients with other BRAF mutations had a higher proportion of spinal column involvement (P = .006). Univariate analysis showed a significant difference in progression-free survival among the 4 molecular subgroups for patients treated with first-line therapy (P = .02). According to multivariate analysis, risk organ involvement was the strongest independent adverse prognostic factor (hazard ratio, 8.854; P < .001); BRAF or MAP2K1 mutation was not an independent prognostic factor. Most pediatric patients with LCH carry somatic mutations involving the MAPK pathway, correlating with clinical characteristics and outcomes for first-line chemotherapy.

Abstract A012: Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib

Abstract Acquisition of ARAF mutations upon treatment with the potent and selective RAF dimer (type II) inhibitor belvarafenib has been previously observed, but whether these alterations or others are observed in patients treated with a combination of belvarafenib and the MEK inhibitor cobimetinib is unknown. The mutational profiles from circulating tumor DNA (ctDNA) were determined using FoundationOne liquid ctDNA assay from paired baseline (BL) and end of treatment (EOT) peripheral blood samples from 79 patients with locally advanced or metastatic RAF or RAS-mutant solid tumors treated with belvarafenib and cobimetinib in the HM-RAFI-103 trial (NCT03284502). The proportion of mutations were compared between BL and EOT using a McNemar’s test (unadjusted) to identify mutations significantly enriched or lost following treatment. ARAF mutations were not observed to significantly increase following treatment with belvarafenib + cobimetinib (4% BL vs 4% EOT in all solid tumors, p = 1; 6% BL vs 6% EOT in melanoma), and in patients with pre-existing ARAF mutations, the variant allele frequency did not consistently increase following treatment. The MEK1 gene had the greatest increase in mutations following treatment, present in 5% (4/79) patients at BL vs 16% (13/79) at EOT (p = 0.02) across all solid tumors, which included 44 colorectal cancer (CRC) cases (56%), and 18 melanoma (23%). While CRC comprised the largest fraction of all solid tumors in this study, it did not contribute the greatest gain in MEK1 mutations at EOT, with 7% (3/44) of CRC patients having MEK1 mutations at BL vs 14% (6/44) at EOT (p = 0.2). The indication with the greatest number of patients developing previously undetected MEK1 mutations at EOT was melanoma, where MEK1 mutations were present in 0% (0/18) patients at BL vs 28% (5/18) at EOT (p = 0.07). The MEK1 mutations gained in melanoma, which included Q58_E62del, P124L, and C121S, occurred in the N-terminal or catalytic core domains of MEK1, were known to be of functional impact, and all but one patient developing them had ≥ 1 known to be gain of function via the Clinical Knowledge Database; one patient gained three distinct MEK1 mutations following treatment. The MEK1 C121S mutation was previously observed to appear in a melanoma patient who developed resistance to the BRAF V600 inhibitor vemurafenib, and in vitro studies demonstrated it also conferred resistance to MEK inhibition (Wagle et al., 2011). Melanoma patients in the HM-RAFI-103 trial gaining MEK1 mutations at EOT were more commonly BRAF-mutant(mt) or NRAS non-mt melanoma at BL, but sample size is small and results were non-significant (p = 0.3 and 0.3, for association with BRAFmt and NRASmt, respectively, Fisher’s exact test). All melanoma patients who developed MEK1 mutations post treatment had either a partial response (1/5) or stable disease (4/5) as best overall response. Together, these data suggest the acquisition of gain of function MEK1 mutations following treatment of solid tumors, including melanoma, with combined type II RAF dimer and MEK inhibition. Citation Format: Stephanie Hilz, Marissa Chen, Malgorzata Nowicka, Harini Chakravarthy, Maryam Moshref, Luca Gerosa, Jennifer Eng-Wong, Cassie Chou, Young S. Noh, Yoon-hee Hong, Yibing Yan. Emergence of MEK1 mutations in RAF or RAS-mutant solid tumors following treatment with a combination of selective type II RAF inhibitor belvarafenib and MEK inhibitor cobimetinib [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A012.

Phase 2 Trial of Single-Agent Cobimetinib for Adults with Histiocytic Neoplasms

Background: Histiocytic neoplasms (HN) are clonal hematopoietic disorders characterized by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway. BRAF inhibition is highly effective for patients with HN harboring the BRAFV600E mutation, and implementation of BRAF inhibitors transformed management of BRAFV600E-mutated Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH). No standard or approved therapies existed, however, for patients with HN lacking the BRAFV600E mutation. Efficacy of mitogen-activated protein kinase (MEK) inhibition for HN harboring various MAPK pathway mutations has been observed in case reports and series. We present here results from 26 patients treated in a phase 2 trial of single-agent cobimetinib for adults with HN (NCT02649972). The data were submitted to the Food and Drug Administration (FDA) in a supplemental new drug application for cobimetinib, and FDA approval was granted in October, 2022. Methods: This is an open-label phase 2 trial of cobimetinib 60mg daily for patients with (1) BRAFV600-wildtype (wt) histiocytosis or (2) BRAFV600-mutant histiocytosis intolerant or without access to BRAF inhibitor therapy. Eligible patients were age 16 or older, had histologic confirmation of HN diagnosis, ECOG 0 to 3, multi-system HN or HN refractory to conventional (i.e. chemotherapeutic or immunosuppressive) therapy or single-system HN deemed unlikely to benefit from conventional therapy, and with disease measurable by positron emission tomography (PET) Response Criteria (PRC). Patients were eligible with any tumor mutation or with no identified tumor mutation. The primary endpoint was overall response rate (complete metabolic response + partial metabolic response; ORR) by PRC. Secondary endpoints included safety (CTCAE 4.0), duration of response (DoR) and progression-free survival (PFS) as determined by modified PERCIST, overall survival (OS), and response rate by RECIST. Data cut-off was March 28, 2019 for efficacy evaluation, and safety follow-up cut-off was September 28, 2019. Results: Twenty-six patients (13 ECD, with Rosai-Dorfman disease [RDD], 4 with LCH, 2 with juvenile xanthogranuloma [JXG], 3 with mixed histiocytosis) with a median age of 50.5 were analyzed. Six patients had BRAFV600E-mutant disease, one of these with additional mutations in KRAS and NRAS. Of the remaining 20, 15 had non-BRAFV600 MAPK pathway mutations (4 KRAS [one of them with ARAF mutation as well], 6 MEK1, 1 MEK2, 1 RAF1, 1 ARAF, 2 non-V600 BRAF mutations, and 5 had no mutation identified). Twenty-six patients were evaluable for safety, 24 for PRC, 19 for RECIST. Response rates are listed in Table 1 and PRC responses are presented in Figure 1. The ORR by PRC was 83.3% (95% CI 62.62-95.26), in the PRC-evaluable population. Responses were observed across MAPK mutations, histiocytosis subtypes and tumor genotypes. By BRAFV600 status, in PRC-evaluable population, the ORR for BRAFV600E-mutant disease was 100% (95% CI 47.82-100), and for BRAFV600-wt disease was 78.9% (95% CI 54.43-93.95). Median DoR was 31.1 months (95% CI 16.8-NE) in the PRC-evaluable population. ORR by RECIST was 63.2% (95% CI 38.36-83.71), in the RECIST-evaluable population. Three patients died on study, none related to cobimetinib. Grade 3 or 4 toxicities in &amp;gt;10% of patients included decreased lymphocyte count (19%), increased blood creatine phosphokinase (12%), decreased ejection fraction (12%), hypokalemia (12%), hyponatremia (12%), acute kidney injury (12%) and dyspnea (12%). Conclusions: Cobimetinib demonstrates robust efficacy in HN irrespective of underlying MAPK pathway mutation, including BRAF-wt and BRAFV600-mutant HN. Safety of cobimetinib in this patient population was consistent with the known safety profile.

Somatic ARAF mutations in pediatric Langerhans cell histiocytosis: clinicopathologic, genetic and functional profiling.

ARAF mutations have been identified in a limited subset of patients with Langerhans cell histiocytosis (LCH), a rare disorder characterized by abnormal proliferation of Langerhans cells. LCH is primarily instigated by mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, with BRAFV600E and MAP2K1 mutations constituting most cases. ARAF mutations in LCH highlight the heterogeneity of the disease and provide insights into its underlying molecular mechanisms. However, the occurrence of ARAF-positive LCH cases is extremely rare, with only two reported globally. Although they may be linked to a more aggressive form of LCH and a more severe clinical progression, the clinical significance and functional consequences of these mutations remain uncertain. We performed next-generation sequencing (NGS) to explore driver mutations in 148 pediatric LCH patients and recognized a series of mutations, including an identical novel somatic ARAF mutation, c.1046_1051delAGGCTT (p.Q349_F351delinsL), in four pediatric LCH patients. It was considered an ARAF hotspot mutation. All reported ARAF-positive patients worldwide exhibited characteristic pathological features of LCH, albeit with involvement across multiple systems. In vitro functional studies showed that this mutation could trigger the MAPKinase pathway and phosphorylate its downstream effectors MEK1/2 and ERK1/2 (relatively weaker than BRAFV600E). Over-activation of mutant A-Raf kinase could be inhibited by the BRAF inhibitor vemurafenib. LCH is uncommon, and ARAF mutation is even rarer. In our study, we have identified a novel hotspot somatic ARAF mutation, which has been verified through functional analysis to be an activating mutation. LCH patients with ARAF mutation typically have an unfavorable prognosis due to limited treatment experiences, although they do not exhibit a high relapse rate. To aid in the development of personalized treatment approaches and prognostic markers for LCH patients, it is recommended to conduct typical pathological and immunohistochemical examinations, as well as genetic tests utilizing a targeted gene panel or whole exome sequencing (WES), for LCH diagnosis, thereby promoting the use of inhibitor treatment strategies.

Characterizing non-small cell lung cancer driven by mutations in ARAF and CRAF family members.

e15085 Background: Cancer genome screening is becoming standard clinical practice, but the number of actionable mutations detected is limited. To expand on our previous report of the possibility that ARAF S214C and CRAF S257L located in the conserved region 2 (CR2) were actionable driver mutations, we investigated responses to targeted therapies in mouse models and the prevalence of these alterations across many cancer types represented in a large real-world database of molecularly profiled patient tumors. Methods: Conditional transgenic mice that expressed human ARAF S214C or CRAF S257L in their lungs were established and treated with sorafenib or trametinib. Activation of the MAPK pathway following sorafenib or trametinib treatment was assessed in vitro by phospho-ERK IHC. DNA sequencing (592-gene or whole exome) was performed for patient samples submitted to Caris Life Sciences (Phoenix, AZ), and the prevalence of ARAF/CRAF variants was determined for each cancer type. Results: Both ARAF S214C and CRAF S257L dimerized to activate the MAPK pathway. Although a low concentration of sorafenib promoted ERK phosphorylation, a high concentration of sorafenib inhibited ERK phosphorylation. Transgenic mice rapidly developed lung adenocarcinoma when the mutant proteins were expressed. Sorafenib treatment induced tumor necrosis in both ARAF- and CRAF-mutated tumors, and 3 months of sorafenib treatment prolonged the survival of CRAF-mutant mice. Pan-tumor analysis of real-world patient samples revealed that ARAF and CRAF mutations in CR2 were found in 0.125% and 0.234% of tumors, respectively. ARAF mutations in CR2 were found in 0.415% of cholangiocarcinoma, 0.132% of colorectal cancer, and 0.173% of non-small cell lung cancer (NSCLC). CRAF mutations in CR2 were found in 0.509% of colorectal cancer, 0.502% of gastric cancer, 0.303% of NSCLC, and 0.978% of melanoma. The most prevalent CR2 mutations were ARAF S214F and CRAF S257L. Conclusions: Our in vitro results suggest ARAF S214C and CRAF S257L mutations are rare driver mutations that may be effectively treated with RAF inhibitors and warrant further investigation. [Table: see text]

Clinicogenomic associations in childhood Langerhans cell histiocytosis: an international cohort study.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder caused by somatic genetic alterations in hematopoietic precursor cells differentiating into CD1a+/CD207+ histiocytes. LCH clinical manifestation is highly heterogeneous. BRAF and MAP2K1 mutations account for ∼80% of genetic driver alterations in neoplastic LCH cells. However, their clinical associations remain incompletely understood. Here, we present an international clinicogenomic study of childhood LCH, investigating 377 patients genotyped for at least BRAFV600E. MAPK pathway gene alterations were detected in 300 (79.6%) patients, including 191 (50.7%) with BRAFV600E, 54 with MAP2K1 mutations, 39 with BRAF exon 12 mutations, 13 with rare BRAF alterations, and 3 with ARAF or KRAS mutations. Our results confirm that BRAFV600E associates with lower age at diagnosis and higher prevalence of multisystem LCH, high-risk disease, and skin involvement. Furthermore, BRAFV600E appeared to correlate with a higher prevalence of central nervous system (CNS)-risk bone lesions. In contrast, MAP2K1 mutations associated with a higher prevalence of single-system (SS)-bone LCH, and BRAF exon 12 deletions seemed to correlate with more lung involvement. Although BRAFV600E correlated with reduced event-free survival in the overall cohort, neither BRAF nor MAP2K1 mutations associated with event-free survival when patients were stratified by disease extent. Thus, the correlation of BRAFV600E with inferior clinical outcome is (primarily) driven by its association with disease extents known for high rates of progression or relapse, including multisystem LCH. These findings advance our understanding of factors underlying the remarkable clinical heterogeneity of LCH but also question the independent prognostic value of lesional BRAFV600E status.

Rare molecular subtypes of lung cancer.

Oncogenes that occur in ≤5% of non-small-cell lung cancers have been defined as 'rare'; nonetheless, this frequency can correspond to a substantial number of patients diagnosed annually. Within rare oncogenes, less commonly identified alterations (such as HRAS, NRAS, RIT1, ARAF, RAF1 and MAP2K1 mutations, or ERBB family, LTK and RASGRF1 fusions) can share certain structural or oncogenic features with more commonly recognized alterations (such as KRAS, BRAF, MET and ERBB family mutations, or ALK, RET and ROS1 fusions). Over the past 5 years, a surge in the identification of rare-oncogene-driven lung cancers has challenged the boundaries of traditional clinical grade diagnostic assays and profiling algorithms. In tandem, the number of approved targeted therapies for patients with rare molecular subtypes of lung cancer has risen dramatically. Rational drug design has iteratively improved the quality of small-molecule therapeuticagents and introduced a wave of antibody-based therapeutics, expanding the list of actionable de novo and resistance alterations in lung cancer. Getting additional molecularly tailored therapeutics approved for rare-oncogene-driven lung cancers in a larger range of countries will require ongoing stakeholder cooperation. Patient advocates, health-care agencies, investigators and companies with an interest in diagnostics, therapeutics and real-world evidence have already taken steps to surmount the challenges associated with research into low-frequency drivers.

Rosai-Dorfman-Destombes disease - histiocytic disorder with inflammatory manifestation.

Rosai-Dorfman-Destombes disease (RDD) is a rare histiocytosis characterized by accumulation of activated histiocytes within affected tissues. Although the immunophenotype of this disease was described, the pathophysiology of this disease is still not sufficiently understood. Recent studies have found NRAS, KRAS, MAP2K1, and ARAF mutations in RDD lesions, raising the possibility of a clonal origin in some forms of RDD while in other cases reactive origin or association with other malignant and autoimmune disease is supposed. RDD is a widely heterogeneous entity with a range of clinical phenotypes occurring in some patients in association with autoimmune or malignant diseases. Its therapy should reflect the localization of the disease. Monotherapy with glucocorticoids is sufficient only in limited disease. In patients with advanced disease, combined nodal and extranodal forms of RDD need more intensive therapy. In older publications, antimetabolites, vinca alkaloids and prednisone were used; in recent publications, remissions after cladribine, rituximab, sirolimus, thalidomide, lenalidomide and cobimetinib were described. This text summarizes current knowledge about this rare disease and reviews the therapeutic options.

Abstract 5775: Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis

Abstract Background: The RAF family includes three members, ARAF, BRAF, and RAF1/CRAF. These kinases function downstream of RAS as part of the MAPK (RAF-MEK-ERK) signaling cascade. The BRAF gene is mutated in ∼7% of cancers, whereas ARAF and RAF1 mutations are rare. Gene fusions in BRAF or RAF1 fusions, which activate the MAPK pathway, can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. Here we focused on the prevalence of BRAF and RAF1 fusions in a multi-cancer cohort for patients (pts) whom might benefit from MEK/RAF inhibitors in clinical development. Methods: Genomic profiling was performed on samples from 29,102 lung cancer pts, 676 melanoma pts, 392 colorectal cancer pts, 1,463 gastric cancer pts, 1,014 sarcoma pts, 1,079 liver cancer pts, 215 gallbladder carcinoma pts, 297 ovarian cancer pts, 46 tongue cancer pts, 62 thymic carcinoma pts, 6 hemangioma pts, and 890 pancreatic cancer pts using next-generation sequencing (NGS), which covers all exons of BRAF and RAF1. The fusion patterns were analyzed. Results: A total of 5 RAF1 and 114 BRAF fusions were identified in approximately 0.34% (120/35,242) of the patients in this Chinese multi-cancer cohort. The positive rates of RAF1 fusions were 0.0034% (1/29,102) in lung cancer pts, 0.44% (3/676) in melanoma pts, and 0.10% (1/1,014) in sarcoma pts. The fusion partners of RAF1 were LYRM4 in lung cancer, LMCD1 and USP20 in melanoma, and FMR1 in sarcoma. RAF1 fusions involving the intact in-frame RAF1 kinase domain were LYRM4-RAF1, LMCD1-RAF1, and USP20-RAF1, which might be targeted with MEK/RAF inhibitors. The BRAF fusion-positive rates were 0.27% (80/29,102) in lung cancer pts, 1.18% (8/676) in melanoma pts, 2.81% (11/392) in colorectal cancer pts, 0.41% (6/1,463) in gastric cancer pts, 0.20% (2/1,014) in sarcoma pts, 0.19% (2/1,079) in liver cancer pts, 0.47% (1/215) in gallbladder carcinoma pts, 0.34% (1/297) in ovarian cancer pts, 2.17% (1/46) in tongue cancer pts, 1.61% (1/62) in thymic carcinoma pts, 16.67% (1/6) in hemangioma pts, and 0.11% (1/890) in pancreatic cancer pts. The most common 5’partners of BRAF were TRIM24 (4 cases) and SND1 (3 cases). Whereas the most common 3’partners of BRAF were BAIAP2L1 (5 cases) and TRIM24 (4 cases). Furthermore, 57 BRAF fusions contained intact BRAF kinase domain and nearly half of BRAF fusion-positive patients might be sensitive to MEK inhibitor. The most frequent location of the breakpoints occurred in intron 8 of the BRAF gene, followed by intron 7. However, the sensitivity of these fusions to MEK/RAF inhibitors in different cancers needs to be further studied. Conclusions: This study revealed the molecular features of BRAF and RAF1 rearrangements in Chinese cancer patients, which could help to identify therapeutic targets and personalize therapy management, leading to improved patient outcomes. Citation Format: Tao Li, Xin Zhang, Yanling Niu, Tonghui Ma. Landscape of BRAF and RAF1 fusions identified by next-generation sequencing in a Chinese multi-cancer retrospective analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5775.

MAP3K8 and ALK alterations in pediatric patients with atypical Spitz tumors and Spitz melanoma.

e21569 Background: Pediatric atypical Spitz tumors and Spitz melanoma (AST/SM) are characterized by structural variants (rearrangements/truncations) of various kinase genes more commonly involving MAP3K8 and ALK. The clinical features associated with these genomic abnormalities have not been well characterized. Methods: We reviewed the clinical characteristics and outcomes of children and adolescents with AST/SM who were enrolled on our institutional comprehensive registry for melanocytic lesions MACMEL (Cancer 127:3825, 2021) and abstracted clinical and outcome data for patients with MAP3K8, ALK, and other less common genomic aberrations. Results: Table shows the characteristics and outcomes of patients with AST/SM who had MAP3K8, ALK, and other genomic aberrations. There were 49 patients of whom 21 had MAP3K8 rearrangements/ truncations (n = 11;22%) or ALK rearrangements (n = 10;20%). The remaining 28 patients had fusions involving ARAF (1), ROS1 (2), NTRK1 (3), ERBB2 (1) or rare mutations (ATM = 1) or copy number changes (n = 14) or no identifiable genomic abnormality (n = 6). Female sex was more common in all three groups (32 female and 17 male). The median age for the entire cohort was 7.5 years. Most lesions arose in the extremities (n = 32; 65%). About half had stage III disease. 40 patients underwent sentinel node biopsy (SLNB), 25 had at least one positive node (7/9 MAP3K8, 5/8 ALK, 13/23 other), Forty-four patients had FISH analysis for CDKN2A and homozygous loss was seen in 9/11 MAP3K8-rearranged, 2/6 ALK-rearranged, and 11/27 other tumors. No TERT promoter mutation was detected in any of the 45 tumors that were analyzed. Two patients experienced local recurrence, one with a MAP3K8-STX fusion and one with a FLOT2-ARAF fusion and activating RAC1 mutation. All patients are alive and currently disease free (median follow up of 2.33 years). There were no statistically significant associations between gene mutations and tumor site, gender, or stage. Conclusions: Approximately 40% of patients with AST/SM harbored ALK or MAP3K8 alterations in our pediatric melanoma registry. We found no significant association between the individual genes and any specific clinical features in this limited cohort.[Table: see text]

Abstract 36: ARAF mutations confer resistance to RAF dimer inhibitors in NRAS and BRAF mutant melanoma

Abstract Belvarafenib, a potent and selective RAF dimer inhibitor, exhibits clinical activity in BRAFV600E and NRAS mutant melanoma patients. Here, through generation of belvarafenib resistant NRAS mutant melanoma cells and through analysis of circulating tumor DNA from patients treated with belvarafenib, we identified novel recurrent mutations in ARAF, within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer and kinase activity dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of RAF dimer inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF driven resistance and suggests a rational combination for clinical use. Taken together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors. Citation Format: Shiva Malek. ARAF mutations confer resistance to RAF dimer inhibitors in NRAS and BRAF mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 36.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

ARAF Mutations Limit Response to RAF Dimer Inhibition.

Belvarafenib is active in BRAF- and NRAS-mutant tumors, but acquired ARAF mutations drive resistance.

Severe Lymphatic Disorder Resolved With MEK Inhibition in a Patient With Noonan Syndrome and SOS1 Mutation.

Noonan syndrome is a multiorgan system disorder mediated by genetic defects along the RASknown as RASopathies. It is the second most common syndromic cause of congenital heart disease and, in ∼20% of the cases, is associated with severe lymphatic disorders, including chylothorax and protein-losing enteropathy. Recently, we reported on the use of mitogen-activated protein kinase inhibition in a patient with an ARAF mutation and severe lymphatic disorder leading to an abrupt improvement in symptoms and complete remodeling of the central lymphatic system. Here, we present a patient with Noonan syndrome and severe lymphatic abnormality, leading to transfusion-dependent upper gastrointestinal bleeding and protein-losing enteropathy. The patient stopped responding to medical therapy and underwent several lymphatic interventional procedures, which led only to a temporary improvement in symptoms. Because of a lack of other treatment options, an expanded access approval was obtained, and the patient initiated treatment by mitogen-activated protein kinase inhibition using trametinib. This led to resolution of her symptoms, with complete normalization of her electrolyte levels, hemoglobin, and albumin within 3 months of starting the drug. Similar to the previously reported case, she also had complete and generalized remodeling of her lymphatic system. In patients with RAS pathway defects complicated by a severe lymphatic disorder, inhibition of the RAS-MAPK pathway should be considered as a possible treatment option in patients who failed conventional treatment and might be a first-line treatment in the future.

ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

BRAF V600E Mutation: A Significant Biomarker for Prediction of Disease Relapse in Pediatric Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disease presenting with usually a localized disease but sometimes a widespread aggressive disorder especially in children. Among the somatic mutations in RAF-MEK-ERK pathway, especially BRAF mutation has been detected so far in LCH. We aimed in this study to investigate the prognostic significance of the mutations of target genes playing a role in the RAF-MEK-ERK pathway in pediatric LCH. Mutation analyses were performed on tumor DNA extracted from formalin-fixed paraffin-embedded biopsy specimens of 38 pediatric LCH cases using a direct sequencing technique for BRAF, ARAF, MAP2K1, and MAP3K1 genes. The mutational status was correlated statistically with survival, clinical progression (disease relapse), and the established clinical prognostic parameters of LCH such as age, gender, localization, multisystem disease, central nervous system risk lesions, and risk organ or special-site involvement. BRAF V600E mutation was detected in 14 cases (36.8%), whereas ARAF mutation was found in only 1 case. No mutations were identified for MAP2K1 and MAP3K1 genes. The association of BRAF V600E mutation was significant in children with multisystem disease, younger age (<2 years), skin, and special organ involvement. BRAF V600E mutation was an independent predictive parameter for disease relapse. We therefore conclude that BRAF V600E mutation may be a significant marker for predicting disease progression in LCH and a candidate for targeted therapy for children with disease relapse and multisystem disease.

Frequency of MAP2K1, TP53, and U2AF1 Mutations in BRAF-mutated Langerhans Cell Histiocytosis: Further Characterizing the Genomic Landscape of LCH.

Langerhans cell histiocytosis is a proliferative disorder of neoplastic Langerhans cells with activating mutations in the Erk signaling pathway. TP53 and U2AF1 mutations have been implicated in other myelomonocytic malignancies and we hypothesized that mutations in these genes may cosegregate in LCH patients according to BRAF mutation status. Towards this end, we collected cases with a pathologic diagnosis of Langerhans cell histiocytosis from Stanford University Hospital. We analyzed the status of known pathogenic alleles in BRAF, ARAF, TP53, U2AF1, and MAP2K1 on formalin-fixed, paraffin-embedded tissue by direct sequencing. A total of 41 cases (71%) had a BRAFV600E allele detected by sequencing. MAP2K1 mutations were also detected in 5 cases: 3 of 17 (18%) cases with wild-type BRAF and 2 of 41 (5%) cases with BRAFV600E mutations (P=0.14). No cases contained the previously reported ARAF mutation, Q347_A348del. All 10 cases with TP53 mutations contained mutant BRAFV600E allele (P=0.021). Of the 11 cases with U2AF1 mutated, 9 of 41 cases co-occurred with BRAFV600E mutations (P=0.31) and 2 of 17 with wild-type BRAF. Interestingly, we do not find that somatic activating MAP2K1 mutations are mutually exclusive with BRAFV600E mutations as has been reported previously. Instead, our data suggests that MAP2K1 mutations may be present along with BRAF either at diagnosis or may be acquired during disease progression. Furthermore, we demonstrated that likely deleterious TP53 mutations correlate with BRAF mutational status and may play a role in the underlying pathogenesis.

Three Cases of Erdheim-Chester Disease With Intraocular Manifestations: Imaging and Histopathology Findings of a Rare Entity

To report intraocular manifestations of Erdheim-Chester Disease (ECD) with multimodal imaging. A retrospective observational case series. This was a multicenter case series of 3 patients with confirmed tissue diagnosis of ECD that showed intraocular manifestations and were imaged at baseline and follow-up visits. Intraocular manifestations are rarely observed in association with ECD. Intraocular manifestations of ECD seen on multimodal imaging include histiocytic choroidal infiltration causing choroidal lesions, complicated by recurrent serous retinal detachment (SRD). Short-term resolution of SRD was observed with ocular therapies including intravitreal injections ofanti-vascular endothelial growth factor or verteporfin photodynamic therapy in combination with systemic chemotherapy therapies and oral corticosteroids; however, recurrences were common. Chorioretinal biopsy confirmed the diagnosis of ECD in 1 case, with the presence of histiocytic infiltration, fibrosis, and characteristic immunohistologic staining. In another case, with a novel ARAF positive mutation, treatment with sorafenib showed regression of the choroidal lesions and resolution of the SRD on multimodal imaging. These lesions were previously resistant to other forms of therapy. Rare intraocular manifestations of ECD confirmed on histopathology can be imaged with multimodal imaging. We report 3 cases, including 1 case diagnosed through histology from chorioretinal biopsy and another case associated with a novel ARAF mutation responsive to targeted therapy with sorafenib. The identification of novel somatic mutation associated with ECD enabled treatment with a new-targeted systemic agent. Multimodal imaging in these cases can also be used to monitor response to therapy.

Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms.

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 BRAF(V600E) 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 BRAF(V600E)-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 BRAF(V600E)-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. 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.

Abstract NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations

Abstract NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations