BRAF Gene Fusions Research Articles

Histopathologic, genomic, transcriptomic, and functional characteristics of eight melanocytic tumors with BRAF fusions showing stronger MAPK pathway activation compared to BRAF V600E tumors.

Activating BRAF gene alterations are central to melanocytic tumor pathogenesis. A small, emerging subset of melanocytic tumors driven by BRAF fusions has distinct therapeutic implications and has been described to have Spitzoid morphology patterns. However, such morphological patterns do not encompass all cases, and little is known about the functional molecular events. We conducted a retrospective search through our molecular archives to identify melanocytic tumors with BRAF fusions. We reviewed clinical, histopathological, and genomic features. We further explored transcriptomic and protein-level findings. Histopathologic patterns varied, with many cases without a distinctive pattern. We identified novel and diverse BRAF gene fusion partners. Differential transcriptomic analysis between low-risk BRAF fusion tumors and reference BRAF V600E tumors showed no differentially expressed genes. However, quantitatively stronger MAPK pathway activation of BRAF fusion tumors over BRAF V600E tumors was demonstrated by statistically significant stronger staining of p-ERK immunohistochemistry. Gene-specific RNA analysis shows comparable BRAF transcript levels between the two groups. The quantitatively stronger activation of the MAPK pathway of BRAF fusion tumors, instead of qualitatively different transcriptomes, may account for the morphology difference from conventional BRAF V600E tumors. BRAF fusions likely act through dysregulated protein function rather than RNA upregulation related to the characteristics of the fusion partners.

Oncocytic intraductal carcinoma of parotid gland with a novel AGK::BRAF gene fusion.

Oncocytic intraductal carcinoma of parotid gland with a novel AGK::BRAF gene fusion.

Primary CNS histiocytic sarcoma: Two case reports highlighting a novel MIGA2::BRAF gene fusion and genome-wide DNA methylation profiling results.

Primary CNS histiocytic sarcoma: Two case reports highlighting a novel MIGA2::BRAF gene fusion and genome-wide DNA methylation profiling results.

Relevance of detection of RAF fusion transcripts in pan-negative melanoma in routine practice.

Pan-negative melanomas account for 30% of melanomas. In case of immunotherapy failure, therapeutic options are limited. Oncogene fusions represent a target of interest in many solid cancers. In melanoma, the frequency of oncogene fusion is not well documented and not routinely investigated. We conducted a single-center retrospective study. The objective was to determine the frequency of oncogene fusion detected by RNA sequencing, in patients with advanced or metastatic pan-negative melanoma. In parallel, an extended molecular alteration search was performed using extended targeted next-generation sequencing. We identified 59 patients with advanced pan-negative melanoma between January 2021 and January 2023. It was a cutaneous melanoma in 71.1% of the cases, a mucous melanoma in 15.2% of the cases. We identified nine patients with a RAF fusion, including seven BRAF gene fusion and two RAF1 fusion. Of the other molecular alterations, NF1 mutation was the most frequent molecular alteration identified. Among the nine patients with RAF fusions, all the patients initially received treatment with anti-PD1 ± anti-CTLA4 immunotherapy. After immunotherapy failure, five patients benefited from second-line targeted therapy (two with BRAF and MEK inhibitors combination, three MEK inhibitors alone). The response rate was 20%. In a population of pan-negative melanoma, we detected 15.2% of RAF fusion. Fusion detection allowed the introduction of a second line of targeted therapy, in the absence of a validated therapeutic option in 55.5% of cases. This study suggests the relevance of detecting RAF fusion in a selected population.

Disseminated Xanthogranuloma with BRAF Gene Fusion and Profound Response to MEK Inhibitor Therapy

Background: Histiocytic neoplasms represent a diverse group of clonal hematopoietic disorders, varying in severity from xanthogranuloma to multisystemic Erdheim-Chester Disease (ECD). Both Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (nLCH) are characterized by activating mutations in the MAPK pathway, primarily involving BRAF point mutations. In this report, we present a compelling case of disseminated xanthogranuloma with a unique BRAF fusion, which exhibited a remarkable therapeutic response to MEK inhibition. This finding adds to our understanding of the molecular heterogeneity within histiocytic neoplasms and highlights the potential significance of targeted therapies in managing these rare but clinically significant disorders Clinical Observation: In this clinical observation, we present the case of a 71-year-old female who sought medical attention due to a 3-year history of progressive cutaneous lesions, accompanied by hoarseness and blurry vision. A biopsy of a lesion revealed characteristic features consistent with xanthogranuloma. Further examination through direct laryngoscopy unveiled lesions on the vocal cords and tracheal masses, which exhibited identical histologic findings as the skin biopsies. A comprehensive analysis involving massively parallel DNA sequencing revealed the presence of a GAB2-BRAF fusion, along with a tumor mutational burden of 1 mutation/megabase (Mb). Immunohistochemical testing of the endobronchial biopsy demonstrated a combined positive score of 10% for PDL-1 expression, suggesting the potential for immune checkpoint blockade. Given the implicated role of MAPK pathway activation due to the identified fusion, a therapeutic approach utilizing the MEK inhibitor Trametinib was initiated. Encouragingly, the patient exhibited a favorable response with a reduction in tumor burden, near-resolution of hoarseness, and improved vision. However, this treatment was accompanied by MEK inhibitor-associated cardiotoxicity, leading to the discontinuation of Trametinib. Regrettably, following the cessation of Trametinib, the patient's lesions rapidly progressed, and her disease worsened beyond the pre-treatment state. Taking into account the 10% PDL-1 positivity, immunotherapy with Pembrolizumab was commenced, resulting in partial improvement. Nonetheless, the patient developed immunotherapy-related cardiotoxicity. In light of the continued disease progression off therapy, the MEK inhibitor Cobimetinib was introduced. The patient responded rapidly to this therapeutic intervention; however, during a one-week break in Cobimetinib treatment, the disease worsened back to the baseline. Consequently, Cobimetinib was discontinued, and a continuously dosed MEK inhibitor, Binimetinib, was cautiously administered at a lower dose to mitigate the risk of cardiotoxicity. As of the time of this report, the patient remains on Binimetinib, achieving complete clinical remission, and there is no evidence of further cardiotoxicity. This clinical observation underscores the challenges and complexities in managing advanced xanthogranuloma with unique molecular alterations and highlights the importance of closely monitoring and tailoring therapies to optimize patient outcomes. Conclusion: To our knowledge, this is the first clinical trial documenting MEK inhibition for a patient with xanthoma disseminatum harboring a BRAF fusion. Seeking to target downstream of BRAF, MEK inhibition demonstrated a dramatic response, with rapid relapse upon cessation, supporting a central oncogenic role for BRAF fusion. Further studies should explore alterations in the RAS-BRAF-ERK pathway in nLCH and consider clinical trials with MEK inhibitors. Our clinical experience suggests continuous MEK inhibitor exposure may be necessary for effective disease control. These findings advance our understanding of therapeutic options in histiocytic neoplasms, paving the way for more precise and impactful treatments.

Clinical relevance of gene mutations and rearrangements in advanced differentiated thyroid cancer

Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making. We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development. A significant correlation was found between RAI-R development and genetic alterations (P= 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P= 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P= 0.006). Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.

Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma.

Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in threenon-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.

Braf fusion genes in a large cohort of thyroid carcinomas

Braf fusion genes in a large cohort of thyroid carcinomas

The analysis of BRAF fusions characteristics in Chinese solid tumor patients.

e15073 Background: BRAF encodes a RAF kinase, which signal downstream of RAS and activate the MAPK pathway, and has emerged as a major oncogenic driver and a potential therapy target. BRAF V600E base substitution is an approved target for the BRAF inhibitors in solid tumors. BRAF gene fusions represent a different mechanism of BRAF activation. BRAF fusions have shown responses to MEK inhibitors and non-specific RAF inhibitors (eg, sorafenib) in Melanoma (NCCN). We sought to analysis of BRAF fusions characteristics in Chinese solid tumor patients to explore the possibility of therapeutic intervention in solid tumors. Methods: We comprehensively characterized BRAF fusions from next-generation sequencing (NGS) data from May 2019 to January 2023 in 80083 various solid tumors. Considering the characteristics of the receptor tyrosine kinase, we focused on the BRAF fusions which contain the kinase domain. Results: A total of 164 unique cases (70.1% (115/164) glioma and 29.9% (49/164) non-glioma with BRAF fusions) were identified. The non-glioma cohort consisted up mainly lung tumors (49%, 24/49), colorectal tumors (18.4%, 9/49), pancreatic tumors (10.2%,5/49), melanoma (9.2%, 4/49) and biliary tumors (4.1%, 2/49). In the glioma cohort, the median age of the 115 patients was 12 with a range of 0–66; 43 (37.4%) of the patients were female and 72 (62.6%) were male. On the other hand, the median age was 64 with a range of 33–82; 23 (46.9%) of the patients were female and 26 (53.10%) were male in the non-glioma cohort. In the glioma cohort, 82.4% of the partners were KIAA1549. However, the most common fusion partners were AGK (14.04%), TRIM24(14.0%), SND1(12.28%), TPK1(8.77%) in the non-glioma cohort. These BRAF fusions comprised 49 unique 5′ fusion partners, of which 51% (25) were known and 49.0% (24) were novel. 14 novel partners were TMEM176B, HMBOX1, SEMA3E, LOC441204, PDE4D, DENND2A, STIM1, TNS3, PRRC2B, BCR, MET, COL4A3BP, TPM4, ZBTB16 and 10 novel partners were intergenic region. 130 patients with BRAF fusions could be evaluated for concurrent mutations. Of interest is that no KRAS and BRAF V600E mutations were identified in the 130 cases. Conclusions: BRAF fusions were detected in 0.2% (164/80083) of Chinese solid tumor patients. 70.1% of BRAF fusion positive cases were diagnosed with glioma and 29.9% were diagnosed with non-glioma. 14 novel BRAF fusion partners were discovered. BRAF fusion is an important driver genomic alteration and occur in a mutually exclusive pattern with other activating mutations in the MAP kinase signaling pathway. BRAF fusion targeted therapies should be further explored in solid tumors.

Abstract 3322: Diagnosis and monitoring of pediatric cancer patients using low-pass whole genome and targeted sequencing of cell-free DNA

Abstract Cell-free DNA (cfDNA) assays are transforming cancer care by enabling minimally invasive diagnosis, minimal residual disease (MRD) monitoring and by guiding risk stratification and therapy. However, progress in pediatric cancers is limited due to a) limited amounts of cfDNA b) differences in the cancer driver landscape - higher prevalence of copy number alterations (CNA) and fusions with few point mutations and c) small sample sizes. Here, we addressed some of these challenges by performing low-pass whole genome (LPWGS) and/or targeted sequencing (TS) of cfDNA on a cohort of 125 pediatric solid (n=78) and brain (n=47) tumor patients enrolled either at diagnosis, after therapy or relapse. Plasma was collected from patients with sarcoma, renal, hepatic and germ-cell tumors; cerebrospinal fluid (CSF) during surgery from children with low and high grade gliomas, ependymomas, craniopharyngiomas and embryonal tumors; and aqueous humor for retinoblastoma. WGS libraries were constructed with the xGen Prism DNA Library Kit using 5ng of input cfDNA and sequenced (Illumina NextSeq 500) to a mean coverage of 4.1x. CNAs were evaluated using ichorCNA and compared with clinical microarray results from primary tumors. Selected cases with previously clinically reported BRAF, EWSR1 and FOXO1 canonical gene fusions and driver mutations from our OncoKids assay were analyzed using TS (Twist Bioscience) sequenced to a mean coverage of 582x. In treatment naive cases that had an aberration detected in the primary tumor, we observed an overall positivity rate in 67% (72/107) of cases using cfDNA. For a subset of 45 cases with at least 10% circulating-tumor (ct) DNA, we compared LPWGS results with microarray data from the primary tumor and identified 88/92 (96%) of the clinically reported events suggesting its utility as a diagnostic tool. In adult cancers, proportion of short (<150bp) to long fragments (S/L ratio) using LPWGS was shown to correlate with ctDNA concentration and consequently detectability. Therefore, we categorized tumor types as low-burden if the median S/L ratio was below the median value across all tumor types and as high-burden otherwise. Interestingly, ctDNA detectability rates were significantly higher in high-burden tumors (27/52 vs 28/37, Fisher’s exact P-value = 0.03) suggesting that S/L ratios could define ctDNA detectability. Though anecdotal in evidence, LPWGS demonstrated CNAs distinctly present in primary and metastatic lesions and in at least one case, revealed ctDNA in plasma, 9 months prior to relapse. These findings suggest cfDNA may be more representative of tumor clonal composition and utilized for MRD detection. These findings affirm the clinical utility of liquid biopsy assays to diagnose and monitor a variety of pediatric solid and brain tumors. Clinical trials and larger cohorts are necessary for ctDNA guided risk and therapeutic stratification of pediatric cancers. Citation Format: Venkata Yellapantula, Eirini Christodoulou, Katrina O’Hollaran, Jianling Ji, Liya Xu, Jesse L. Berry, Anya Zdanowicz, Leo Mascarenhas, James Amatruda, Dejerianne Ostrow, Nicholas Chapman, Jason Chu, Mark Krieger, Peter Chiarelli, Pan Yachen, Moiz Bootwalla, Xiaowu Gai, Fariba Navid, Jaclyn A. Biegel. Diagnosis and monitoring of pediatric cancer patients using low-pass whole genome and targeted sequencing of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3322.

Spectrum of Melanocytic Tumors Harboring BRAF Gene Fusions: 58 Cases With Histomorphologic and Genetic Correlations

We report a series of 58 melanocytic tumors that harbor an activating fusion of BRAF, a component of the mitogen-activated protein kinase (MAPK) signaling cascade. Cases were diagnosed as melanocytic nevus (n = 12, 21%), diagnostically ambiguous favor benign (n = 22, 38%), and diagnostically ambiguous concerning for melanoma (n = 12, 21%) or melanoma (n = 12, 21%). Three main histopathologic patterns were observed. The first pattern (buckshot fibrosis) was characterized by large, epithelioid melanocytes arrayed as single cells or “buckshot” within marked stromal desmoplasia. The second pattern (cords in whorled fibrosis) demonstrated polypoid growth with a whorled arrangement of cords and single melanocytes within desmoplasia. The third pattern (spindle-cell fascicles) showed fascicular growth of spindled melanocytes. Cytomorphologic features characteristic of Spitz nevi were observed in most cases (n = 50, 86%). Most of the cases (n = 54, or 93%) showed stromal desmoplasia. Histomorphology alone was not sufficient in distinguishing benign from malignant melanocytic tumors with BRAF fusion gene because the only histopathologic features more commonly associated with a diagnosis of malignancy included dermal mitoses (P = .046) and transepidermal elimination of melanocytes (P = .013). BRAF fusion kinases are targetable by kinase inhibitors and, thus, should be considered as relevant genetic alterations in the molecular workup of melanomas. Recognizing the 3 main histopathologic patterns of melanocytic tumors with BRAF fusion gene will aid in directing ancillary testing.

Abstract 2014: Mosaic BRAF fusions are a recurrent cause of multiple congenital melanocytic naevi

Abstract Congenital melanocytic naevi (CMN) are moles present at birth, and when multiple or very extensive can involve other organ systems as well as predisposing to melanoma. Some CMN patients develop a highly proliferative multinodular phenotype leading to chronic intense pruritus, and the causes for that specific phenotype progression are poorly understood. Genotypically CMN are mosaic single gene disorders. Thus far only two recurrent variants have been described as clearly causative: NRAS missense variants affecting codon 61 accounting for 68% of cases, and BRAF missense variants affecting codon 600 accounting for 7% of cases. Small numbers of cases of CMN have been reported to carry gene-fusions, and in 2 cases in the world literature (one BRAF-fusion and one RAF1-fusion) these have been shown to be clonal and therefore likely causal. We sought to address the issue of causation of the remaining 25%. From an initial large cohort study, skin biopsies from 19 patients were shown to be double wildtype for NRAS/BRAF and had sufficient tissue for further study after deep whole exome sequencing. These 19 samples then underwent transcriptome-wide paired-end RNA sequencing with bioinformatics analysis (STAR-Fusion v1.6 and Fusion Inspector v2.3) for gene fusion transcripts. 11/19 patients were found to have BRAFgene fusions, of which 7 had the multinodular proliferative phenotype. Fusions were confirmed on Sanger sequencing of the cDNA across the fusion junction, specifically demonstrated in 8 children from more than one separate skin lesion, confirming clonality. In the fusions identified, BRAF was fused to 11 different partner genes (GOLGA4, QKI, STRN3, AGAP3, MKRN2, PHIP, LCA5, EEA1, AKAP9, SEC31A, MIER3). This resulted in loss of the 5’ regulatory domain of BRAF but preservation of the kinase domain, such that expression was driven by the 5’ fusion partner. This structure follows the pattern of somatic BRAF-fusions reported previously in solid tumors including melanoma. Potential dimerization domains in the partner genes were identified in 9 cases. We identify here mosaic BRAF fusions as a recurrent cause of multiple CMN allowing genetic diagnosis in a further 15% of cases and linking this genotype to a highly proliferative pruritic phenotype. In vitro data from melanoma cell lines have suggested that higher expression levels of the fusion protein as well as dimerization domains in the partner gene correlate with resistance and/or paradoxical MAP-kinase activation after treatment with RAF and MEK inhibitors. This may have implications for the use of targeted therapies for attempted reduction of the nodular phenotype, or where melanoma arises in CMN patients caused by mosaic BRAF-fusions. Citation Format: Sara Barberan Martin, Satyamaanasa Polubothu, Alicia Bruzos, Neil Bulstrode, Gavin Kelly, Veronica Kinsler. Mosaic BRAF fusions are a recurrent cause of multiple congenital melanocytic naevi [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 2014.

LGG-07. Novel CRISPR/Cas9 induced KIAA1549:BRAF fusion model for preclinical studies of pediatric gliomas

BACKGROUND: Pediatric Low Grade Gliomas (pLGG) are the most common group of central nervous system (CNS) tumors in children and cause significant morbidities. Pilocytic astrocytoma (PA) is the most frequent pLGG. KIAA1549:BRAF fusion is a well-established oncogenic driver in PA. Oncogene induced senescence (OIS) has prevented establishing PA cultures for in vitro and in vivo studies. Here we look at a novel NIH-3T3 fibroblast model harboring KIAA1549:BRAF fusion gene via CRISPR/Cas9 somatic genome engineering technology. OBJECTIVES: Establishing that the CRISPR/Cas9 edited NIH-3T3 fibroblast model with the KIAA1549:BRAF fusion is valuable for in vitro and in vivo studies without early OIS. DESIGN/METHOD: CRISPR/Cas9 editing technology was used to establish a KIAA1549:BRAF fusion positive cell model. This cell model was studied in vitro with MEK inhibitor cobimetinib (GDC-0973) and using WST-1 viability assay, clonogenic assay, senescence β-galactosidase staining, and western blot. In vivo murine models with subcutaneous fusion positive NIH-3T3 fibroblast tumors were treated with GDC-0973. Survival studies and tissue studies were subsequently done. RESULTS: A fusion positive NIH-3T3 fibroblast model was successfully established. Increased BRAF cDNA expression and higher levels of p-ERK were observed. In vitro studies showed decreased viability with GDC-0973. Clonogenic assay showed qualitative and quantitative decreases in viable cells. P-ERK target inhibition was established without induction of senescence. In vivo studies demonstrated successful subcutaneous tumor implantation, therapy efficacy, and target inhibition. CONCLUSION: CNS tumors, most commonly pLGG, in children cause significant morbidities. KIAA1549:BRAF fusion is an oncogenic driver in PA. In vitro and in vivo studies are important for pre-clinical models. OIS has prevented establishing adequate fusion positive animal cell models. Here we have demonstrated a successful CRISPR/Cas9 edited fusion positive NIH-3T3 fibroblast model.

Relevance of detection of RAF fusion in pan-negative melanoma in routine practice.

e21500 Background: Pan-negative melanomas (i.e. without MAPK kinase pathway alteration and C-kit wild type) account for 30% of melanomas (according to the AACR GENIE database). In case of immunotherapy failure, therapeutic options are limited. Oncogene fusions represent a target of interest in many solid cancers (NSCLC, Cholangiocarcinoma, Glioma, GIST, pancreatic acinar carcinoma, thyroid, and prostate cancers). In melanoma, the frequency of oncogene fusion is not well documented and not routinely investigated. Methods: We conducted a single-center retrospective study. The objective was to determine the frequency of oncogene fusion detected by RNA sequencing (Archer Fusion pLex) performed in routine practice, in patients with advanced or metastatic pan negative melanoma. Data on clinical, pathological, and molecular characteristics and patient outcomes were collected. Analysis was carried out on the genetic material available for the diagnosis of the disease except for 1 patient who benefited from a new anatomopathological sample during an unfavorable evolution. In parallel, an extended molecular alteration search was performed using extended targeted NGS (OncoMine Comprehensive Assay panel). Results: We identified 48 patients with an advanced pan negative melanoma between January 2021 and January 2022 with a median age at diagnosis of 63 years. It was a cutaneous melanoma in 72,9 % (35/48) of the cases, a mucous melanoma in 14,5% (7/48) of the cases and a melanoma of unknown primary site in 12,5% (6/48) of the cases. The detection of fusion transcript was made in 89,5 % (43/48) of the cases. We identified 6 patients with a RAF fusion, including 4 BRAF gene fusion (MKLN1-BRAF, AGK-BRAF, SNX29-BRAF, PTPRJ-BRAF) and 2 RAF1 fusion (MAP4-RAF1, EFCC1-RAF1). Of the other molecular alterations, NF1 mutation was the most frequent molecular alteration identified (25 % (12/48) of patients). At lower frequencies, 6,8 % (3/48) of patients had a PI3K mutation, and 8,3 % (4/48) of patients had NOTCH, PTCH1, GNAQ mutations. Among the 6 patients with RAF fusions, all the patients initially received treatment with anti-PD1+/- anti-CTLA4 immunotherapy. After immunotherapy failure, 4 patients benefited from second-line targeted therapy (2 with BRAF and MEK inhibitors combination, 2 MEK inhibitors alone). One patient presented an objective imaging response, the other three patients have not yet benefited from reassessment imaging. Conclusions: In a population of pan negative melanoma, we detected 12,5 % (6/48) of RAF fusion. Fusion detection allowed the introduction of a second line of targeted therapy, in the absence of a validated therapeutic option in 66,6 % (4/6) of cases This study suggests the relevance of detecting RAF fusion in a selected population.

Pan-cancer analysis of BRAF alterations and tumor-specific mechanisms of activation.

e17005 Background: BRAF is a proto-oncogene that is altered in various cancers and is a druggable target. Particularly, the selective BRAF kinase inhibitors Dabrafenib and Vemurafenib are FDA approved for melanoma and thyroid cancer that harbor the activating BRAF V600 hotspot mutations. In pre-clinical studies, activated BRAF promotes tumor cell function across many additional cancer types in which therapeutics have not been considered. We propose that identifying BRAF-activated cancers will guide future pre-clinical investigations and potentially expand the usage of existing BRAF-targeting therapeutics. Methods: We utilized bioinformatics to investigate the molecular profiles of pan-cancer cohorts from two platforms based on distinct sequencing technology. This included a custom curated dataset (whole exome, 6 studies, 39 cancer types, n = 12,019) and GENIE9.1 (targeted gene panel, 62 cancer types, n = 78,619). Studies were included based on genomic modalities (copy number alterations, mutations, gene fusions). We cataloged the frequency and all types of BRAF genomic alterations pan-cancer. We also examined other genomic features, including tumor mutational burden (TMB) and fraction of the genome altered (FGA), and their association with types of BRAF alterations across cancer types. Results: In both platforms, we found that BRAF alterations were observed across over 80% of cancer types. Melanoma, thyroid, and colorectal tumors exhibited the greatest rates of mutations. Regarding gene body rearrangements, we noted endocrine-driven cancers including ovarian and metastatic prostate tumors (mPC) exhibited relative robust rates of amplification. In addition, thyroid carcinoma and mPC harbored recurrent BRAF gene fusion events. In mPC, we found that the BRAF gene fusions often included an N-terminus gene fragment from known androgen-target genes (TMPRSS2 and SND1). These fusion events represented a dysregulated hormone-driven mechanism of BRAF activation. We also found that hotspot missense mutations were cancer-type specific, including K601 hotspots in mPC, G469 hotspots in non-small cell lung cancers, and G466 hotspots in ovarian cancers. Lastly, BRAF-amplified endocrine tumors harbored a 1.5-fold increase in FGA medians, whereas BRAF-mutated lung and colorectal tumors exhibited a 1.6-fold increase in TMB medians. Conclusions: Our molecular analysis revealed that cancers accrued activated BRAF through cancer-type-specific and divergent genomic mechanisms. These molecular features provide genomic evidence to expand the application of BRAF-targeted therapies in additional cancer lineages, including mPC. BRAF mutations were also associated with genomic biomarkers, such as TMB, which may predict response to immune therapies. This suggests that BRAF alterations can be considered as an additional feature to aid therapy selection.

A pilot study of trametinib in combination with paclitaxel in the treatment of anaplastic thyroid cancer.

6088 Background: Anaplastic Thyroid Cancer (ATC) is a rare and highly aggressive tumor with extremely poor prognosis. Outside of the recent approval of dabrafenib/trametinib for BRAF mutant tumors, there are no other standard treatment available for metastatic ATC. The majority of ATC is driven through the MAPK pathway. Data in lung cancer suggested synergy with trametinib, a MEK inhibitor, and taxanes. Methods: In this pilot study we used Trametinib (2 mg) daily with Paclitaxel (80 mg/m2) administered weekly for the first 3 weeks out the 4 week-cycle. Restaging imaging was performed every 6-8 weeks (1.5-2 cycles). Eligible patients had ATC with baseline ECOG performance status ≤ 1 and were enrolled at Memorial Sloan Kettering Cancer Center. Prior treatment allowed. Prior brain metastases were allowed if treated and stable off of steroids. Primary objective was PFS at 6 months with the target of > 2 subjects out of 12 at the time point. Results: 12 patients (6 men and 6 women) were enrolled between 11/2017 and 10/2021. Seven (58%) had prior radiation to the neck; 4 (33%) had prior treatment (not including with radiation) for ATC; 1 (8%) had prior brain metastases. Three (25%) partial responses were reported, and five (41.67%) reported stable disease. Subjects with partial responses had a BRAF V600E mutation (1), BRAF fusion gene (1), and a RAS mutation (1). Median time on treatment was 10.5 weeks (3-47+ weeks). Median overall survival was 26 weeks (3-59+). Six-month progression free survival (PFS) was achieved in 3 patient (25%), one of whom remains on study. 2 patients discontinued treatment due to unacceptable toxicity. The most frequent adverse events observed (all grades) were anemia (75%), increased AST, diarrhea and leukopenia (all 50%). Grade 3/4 AEs included neutropenia (25%), with anemia, AST increased, febrile neutropenia, and lymphopenia, all 16.67%. Grade 4 reactions included lymphopenia (n = 2) and leukopenia (n = 1). Conclusions: Our target progression-free survival (PFS) at 6 months was observed on this study. The combination of trametinib and paclitaxel should be evaluated in a larger cohort of patients in the future. Clinical trial information: NCT03085056.

The analysis of BRAF fusions in Chinese patients with glioma.

e14031 Background: KIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. KIAA1549-BRAF fusion has been considered to be a key variant of PA in WHO CNS5, however, fusions of BRAF with other partner genes leading to MAPK pathway activation, have been described rarely. Herein, we explore BRAF fusions profiles in Chinese glioma patients. Methods: Next generation sequencing of 131-gene profiling was performed to analyze BRAF fusions from 1955 Chinese brain tumor patients in 2019-2021. BRAF fusions were detected by following the standard operating procedure (SOP). We screened out the BRAF fusions, and calculated the mutation frequency and other rare BRAF gene fusions. Results: A total of 49 cases were detected with BRAF fusions, 36 cases were children (< 18 years old), and 13 cases were adults, with a mean age of 13 years (0-57 years). Interestingly, 39(80%) of them were KIAA1549-BRAF fusions, the most common fusion is between KIAA1549 exon 16 and BRAF exon 9 (exon 16–exon 9) followed by (exon 15–exon 9) and (exon 16–exon 11). And the other ten were rare fusions, including DENND2A-BRAF, MRPS33(Intergenic)- BRAF, BCAS1- BRAF, TMEM176B-BRAF, RNF130-BRAF, RIN2-BRAF, GNAI1-BRAF, EXOC4-BRAF, TNK2-BRAF. Most retained BRAF exons 9–18 (7/10, 70%). There was a significant statistical difference in age that the non- KIAA1549-BRAF fusions group was higher than the KIAA1549-BRAF fusions group (25 vs. 10, p < 0.005). Importantly, we identified new BRAF fusion variants resulting in RIN2-BRAF, EXOC4-BRAF, TNK2-BRAF and TMEM176B-BRAF rearrangements, which has not previously been reported in gliomas. Conclusions: We find new BRAF fusions in our 20% glioma patients. In addition, analysis of rare BRAF fusion may help guide diagnosis and develop potential targeted therapies of older PA.

BRAF Fusions in Histiocytic Disorders: Frequency and Clinical Characteristics

▪Background: Activation of extracellular-signal regulated kinase (ERK) is almost universal in histiocytic disorders and is driven by genomic alterations in the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K) pathways. While BRAFV600E mutations are well described in histiocytic disorders, less is known about BRAF fusions. The goal of our study is to describe the frequency and clinical characteristics of patients harboring BRAF fusions.Methods: We included patients with a diagnosis of histiocytic disorder seen at our institution from November 2016-June 2021. Only those who had adequate BRAF testing were included. The following were considered adequate BRAF testing: 1) unequivocally positive BRAF V600E immunostain with or without molecular confirmation; 2) successful multigene next generation sequencing (mostly Tempus â or FoundationOne â) if BRAF V600Eimmunostain was equivocal or negative. We also searched the literature for similar cases and compared BRAF fusions found in histiocytic disorders to those found in solid tumors and other hematologic conditions.Results: One hundred and twenty-six patients were included in this study. BRAF fusions were detected in 7 (6%) patients. The median follow-up for these patients was 1.4 years (95% CI: 0.4-not reached). The frequency according to disease subtypes is as follows: Erdheim-Chester disease (ECD; 2/46 [4%]), Langerhans cell histiocytosis (LCH; 1/41 [2%]), adult or juvenile xanthogranuloma (AXG/JXG; 4/7 [57%]), and histiocytic or Langerhans cell sarcoma (HS/LCS; 0/9 [0%]). The median age at diagnosis for our study cohort was 34 years (range, 7-81 years) and 5 were females. The clinical characteristics and nature of BRAF fusions of our 7 patients plus additional 13 cases from the literature are shown in Table 1. For the final cohort of 20 patients with BRAF-fusions, the median age was 16 years (range, 0.5-81 years) and most (56%) were females. The distribution by histiocytic subtypes is as follows: AXG/JXG (45%), LCH (40%), ECD (10%), HS/LCS (5%). Two patients received cobimetinib as frontline treatment in our cohort and achieved partial responses. Of these two patients, one completed 12 cycles (12 months) of cobimetinib and had a sustained PR while the other patient received 2 cycles before having to hold therapy due to intolerable adverse effects despite dose reduction. From literature review, another patient (LR-12) received cobimetinib as second line treatment and achieved complete response. We found 15 unique BRAF gene fusions with 4 being recurrent. Except for AGAP3-BRAF, none of these BRAF fusions in histiocytosis has been reported in solid tumors or hematologic conditions to date.Conclusions: BRAF gene fusions occur rarely (<5%) in histiocytic disorders. The only exception is AXG/JXG where more than half of the patients harbor BRAF gene fusions. These gene fusions are mostly distinct to histiocytosis and not found in solid tumors or other hematologic conditions. [Display omitted] DisclosuresBennani: Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board; Verastem: Other: Advisory Board.

Spectrum of BRAF Mutations and Gene Rearrangements in Ovarian Serous Carcinoma.

Retrospective analysis of our institutional cohort of SBTs (n = 22), LGSCs (n = 119) and high-grade serous carcinomas (HGSCs, n = 1,290) subjected to targeted massively parallel sequencing was performed to identify cases with BRAF genetic alterations. Putative BRAF rearrangements were confirmed using targeted RNA sequencing and/or fluorescence in situ hybridization (FISH). BRAFV600E oncoprotein expression was assessed by immunohistochemistry on selected cases. BRAF somatic genetic alterations were identified in 29 of 1,431 (2%) serous tumors and included mutations (n = 24), gene rearrangements (n = 3), and amplification (n = 2). BRAF mutations were more frequent in SBTs (7 of 22; 32%) compared with LGSCs (11 of 119; 9%, P = .009) and HGSCs (6 of 1,290; 0.5%; P < .0001, SBT/LGSC v HGSC). The BRAF V600E hotspot mutation was most common (n = 16); however, other BRAF driver mutations were also detected (n = 8). BRAF mutations were often clonal or truncal in SBTs and LGSCs, but subclonal in most HGSCs. Pathogenic BRAF gene fusions were identified in LGSCs (n = 2) and HGSC (n = 1) and involved distinct fusion partners (AGK, MKRN1, and AGAP3). Three patients with BRAF-mutant LGSC were treated with targeted mitogen-activated protein kinase inhibitors, one of whom was maintained on therapy for over 3 years with clinical benefit. Recognition of BRAF alterations beyond V600E mutation in LGSC may have clinical implications for appropriate targeted therapy selection.

Gastrointestinal stromal tumors with BRAF gene fusions. A report of two cases showing low or absent KIT expression resulting in diagnostic pitfalls.

Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, rare cases have shown to be driven by gene fusions involving kinases, mainly involving NTRK3, and rarely BRAF or FGFR1. BRAF gene rearrangements have been described in only two patients to date, as separate case reports. In addition, BRAF V600E mutation is an uncommon but established oncogenic pathway in GIST. In this report, we describe two new GIST cases harboring novel BRAF fusion genes, arising in two young-adult women (37 and 40 years of age) in the small bowel and distal esophagus, both with a spindle cell phenotype. The small bowel GIST measured 2.8cm and showed a high cellularity and a mitotic rate of 20/50 HPFs, while the esophageal lesion measured 7cm and 1/50 HPFs. Immunohistochemically, both tumors showed diffuse reactivity for DOG1, while KIT/CD117 was weakly positive in the small bowel GIST and completely negative in the esophageal tumor. Based on these findings, the latter case was misinterpreted as a low-grade myxoid leiomyosarcoma, as it showed a myxoid stroma, reactivity for SMA and focal positivity for desmin. Archer FusionPlex revealed a fusion between BRAF with either AGAP3 or MKRN1 gene partners. Moreover, MSK-IMPACT DNA targeted sequencing confirmed both fusions but did not identify additional mutations. In one case with available material, the BRAF gene rearrangement was also validated by FISH. The recognition of BRAF fusion-positive GISTs is critical as it may be associated with a low level of KIT expression and may result in diagnostic challenges with significant impact on therapeutic management. The clinical benefit with KIT inhibitors, such as imatinib, remains to be determined.