BRAF Rearrangements Research Articles

Tovorafenib: First Approval.

Tovorafenib (OJEMDA™) is a once-weekly oral, selective, brain-penetrant, type II RAF kinase inhibitor being developed by Day One Biopharmaceuticals, Inc., under a license from Takeda Oncology, for the treatment of paediatric low-grade glioma (pLGG) and solid tumours. Most pLGGs harbour alterations in the MAPK pathway, such as a BRAF mutation or BRAF fusion, which result in aberrant intracellular signalling. Tovorafenib is an inhibitor of mutant BRAF V600E, wild-type BRAF and wild-type CRAF kinases and BRAF fusions. In April 2024, tovorafenib received its first approval in the USA for the treatment of patients aged ≥ 6 months with relapsed or refractory pLGGs harbouring a BRAF fusion or rearrangement, or BRAF V600 mutation. It received accelerated approval for this indication based on the response rate and duration of response achieved in this population in the ongoing, pivotal, phase 2 FIREFLY-1 study. Clinical development of tovorafenib is underway in numerous countries worldwide. This article summarizes the milestones in the development of tovorafenib leading to this first approval for relapsed or refractory pLGG with an activating BRAF alteration.

LGG-26. A ROLE FOR BRAF FUSION PARTNERS IN ONCOGENIC SIGNALING

Abstract t was originally hypothesized that loss of BRAF N-terminal negative regulatory domains, which are deleted in the KIAA1549:BRAF rearrangement, results in constitutive activation of the BRAF kinase. This model suggests that BRAF fusion partners are dispensable for BRAF oncogenic signaling. Paradoxically, our data suggest expression of truncated BRAF is insufficient for transformation and identify specific domains in KIAA1549 that are necessary for transformation. These domains are critical for regulating subcellular localization of the fusion protein and highlight an aberrant pattern of cellular localization that is not observed with wildtype BRAF. Altered subcellular localization results in proteolytic cleavage of KIAA1549:BRAF, and we identify the protease responsible for this cleavage event. This protease is therapeutically tractable and clinically relevant inhibitors have been developed, presenting a MAPK-independent mechanism to target the fusion. We also highlight an unexpected role for rare fusion partners, including FAM131B, in BRAF activation. In total, these data suggest that BRAF fusion partners are not indispensable for transformation as was previously thought, presenting unexplored opportunities to therapeutically target pLGG tumors with BRAF rearrangements. We are currently working on the IP measures required to disclose the specific enzyme(s) at ISPNO.

Tovorafenib (Ojemda) for pediatric low-grade glioma.

Tovorafenib (Ojemda – Day One), a type II RAF kinase inhibitor, has received accelerated approval from the FDA for treatment of patients ≥6 months old with relapsed or refractory pediatric low-grade glioma harboring a BRAF fusion or rearrangement or a BRAF V600 mutation. Tovorafenib is the first systemic treatment to be approved in the US for pediatric low-grade gliomas with BRAF fusions. Accelerated approval of tovorafenib was based on response rates and duration of response.

Ciliated Muconodular Papillary Tumors of the Lung Harboring STRN::ALK Fusion: Case Report and Review of the Literature.

Ciliated muconodular papillary tumor (CMPT) is a rare pulmonary tumor, typically occurring in middle-aged and elderly individuals. The molecular mutation spectrum of CMPT remains insufficiently explored. Commonly known driver gene alterations include KRAS, BRAF, EGFR, and ALK rearrangement. This report details the clinicopathological features of 2 patients presenting with CMPT as pulmonary nodules during clinical examinations. Microscopic analysis revealed tumors with glandular or papillary structures, consisting of mucinous cells, ciliated columnar cells, and basal cells. Notably, both patients exhibited STRN::ALK fusion, a finding not previously associated with CMPT. STRN::ALK fusion serves as a target for therapy in various tumors, including non-small cell lung cancer, thyroid cancer, and colon cancer. Consequently, we conducted a review of relevant literature, summarizing the clinicopathological and molecular characteristics of CMPT to facilitate further research. Our insights enhance the understanding of this uncommon tumor and contribute to the expansion of its molecular alteration spectrum.

Recent Advances in the Diagnosis, Pathogenesis, and Management of Myxoinflammatory Fibroblastic Sarcoma.

Myxoinflammatory fibroblastic sarcoma (MIFS) is an infiltrative, locally aggressive fibroblastic neoplasm of intermediate malignancy that typically arises in the distal extremities of middle-aged adults. It can histologically be confused with a number of benign and malignant conditions. Recently, high-grade examples of MIFS have been described. Immunohistochemistry plays a very limited role in the diagnosis of MIFS. Several genetic alterations have been identified in MIFS, including a t(1;10)(p22;q24) translocation with TGFBR3 and/or OGA rearrangements, BRAF rearrangement, and VGLL3 amplification. Although it appears that VGLL3 amplification is the most consistent alteration, the molecular pathogenesis of MIFS remains poorly understood. A wide resection is considered the standard treatment for MIFS. Radiotherapy may be a viable option in cases with inadequate surgical margins or cases where surgery is likely to cause significant functional impairment. The systemic treatment options for advanced or metastatic disease are very limited. This review provides an updated overview of the clinicoradiological features, pathogenesis, histopathology, and treatment of MIFS.

Non-canonical BRAF variants and rearrangements in hairy cell leukemia.

Hairy cell leukemia (HCL) is an uncommon mature B-cell malignancy characterized by a typical morphology, immunophenotype, and clinical profile. The vast majority of HCL patients harbor the canonical BRAF V600E mutation which has become a rationalized target of the subsequently deregulated RAS-RAF-MEK-MAPK signaling pathway in HCL patients who have relapsed or who are refractory to front-line therapy. However, several HCL patients with a classical phenotype display non-canonical BRAF mutations or rearrangements. These include sequence variants within alternative exons and an oncogenic fusion with the IGH gene. Care must be taken in the molecular diagnostic work-up of patients with typical HCL but without the BRAF V600E to include investigation of these uncommon mechanisms. Identification, functional characterization, and reporting of further such patients is likely to provide insights into the pathogenesis of HCL and enable rational selection of targeted inhibitors in such patients if required.

A novel GLCC1::BRAF fusion with independent MYCN and MYC amplifications in primary tumor and metastasis in pediatric pancreatic acinar carcinoma

Abstract Introduction/Objective Pediatric pancreatic acinar cell carcinoma (PACC) is a rare malignancy, comprising 5-15% of pediatric pancreatic tumors. BRAF rearrangement is found in 20-30% of PACC cases, most commonly an inversion in chromosome 7. We report a case of PACC with GLCC1::BRAF fusion. Methods/Case Report A 10-year-old male presented with six months of weight loss, back pain, and loose stools. Imaging demonstrated concentric soft tissue thickening around the celiac axis and superior mesenteric artery, clinically interpreted as vasculitis. Follow-up imaging showed extension of the soft tissue thickening, prompting biopsy of a periaortic lymph node showing metastatic PACC. Pancreaticduodenectomy revealed a 3.8 cm pancreatic neck mass with perineural invasion and metastatic deposits in multiple lymph nodes and retroperitoneal soft tissue. Fluorescence in situ hybridization of both specimens demonstrated BRAF gene rearrangement, with the partner identified as GLCC1 by next generation sequencing and fusion assays. Microarray on the lymph node demonstrated amplification of MYC on chromosome 8, but not in the resection specimen, which showed gain of chromosome 2 and amplification of MYCN. Results (if a Case Study enter NA) NA Conclusion The GLCC1::BRAF fusion has previously been reported once in a case of pheochromocytoma with peritoneal seeding. The MYC gene family members, c-MYC and MYCN, are involved many cancer types; amplifications are often associated with unfavorable prognosis in tumors such as neuroblastoma. However, the amplification of both genes in the same tumor at different loci has not been reported. The combination of the primary GLCC1::BRAF fusion with secondary amplification of MYC and MYCN is likely to drive the aggressive behavior and metastasis in this case of PACC. Treatment for PACC includes chemoradiation and BRAF inhibition. However, BRAF rearrangements without the V600E mutation show variable response to BRAF inhibitors. Potential alternatives include inhibitors to other targets within the BRAF signaling pathway. Further investigation is needed to determine the clinicopathologic impact of these novel genetic aberrancies.

The importance of escalating molecular diagnostics in patients with low-grade pediatric brain cancer.

Pilocytic astrocytomas are the most common pediatric brain tumors, typically presenting as low-grade neoplasms. We report two cases of pilocytic astrocytoma with atypical tumor progression. Case 1 involves a 12-yr-old boy with an unresectable suprasellar tumor, negative for BRAF rearrangement but harboring a BRAF p.V600E mutation. He experienced tumor size reduction and stable disease following dabrafenib treatment. Case 2 describes a 6-yr-old boy with a thalamic tumor that underwent multiple resections, with no actionable driver detected using targeted next-generation sequencing. Whole-genome and RNA-seq analysis identified an internal tandem duplication in FGFR1 and RAS pathway activation. Future management options include FGFR1 inhibitors. These cases demonstrate the importance of escalating molecular diagnostics for pediatric brain cancer, advocating for early reflexing to integrative whole-genome sequencing and transcriptomic profiling when targeted panels are uninformative. Identifying molecular drivers can significantly impact treatment decisions and improve patient outcomes.

Unique Spectrum of Activating BRAF Alterations in Prostate Cancer.

Alterations in BRAF have been reported in 3% to 5% of prostate cancer, although further characterization is lacking. Here, we describe the nature of BRAF alterations in prostate cancer using a large cohort from commercially available tissue and liquid biopsies subjected to comprehensive genomic profiling (CGP). Tissue and liquid biopsies from patients with prostate cancer were profiled using FoundationOne CDx and FoundationOne Liquid CDx CGP assays, respectively. Tissue biopsies from non-prostate cancer types were used for comparison (n = 275,151). Genetic ancestry was predicted using a single-nucleotide polymorphism (SNP) based approach. Among 15,864 tissue biopsies, BRAF-activating alterations were detected in 520 cases (3.3%). The majority (463 samples, 2.9%) harbored class II alterations, including BRAF rearrangements (243 samples, 1.5%), K601E (101 samples, 0.6%), and G469A (58 samples, 0.4%). BRAF-altered prostate cancers were enriched for CDK12 mutations (OR, 1.87; 9.2% vs. 5.2%; P = 0.018), but depleted in TMPRSS2 fusions (OR, 0.25; 11% vs. 32%; P < 0.0001), PTEN alterations (OR, 0.47; 17% vs. 31%; P < 0.0001), and APC alterations (OR, 0.48; 4.4% vs. 8.9%; P = 0.018) relative to BRAF wild-type (WT) disease. Compared with patients of European ancestry, BRAF alterations were more common in tumors from patients of African ancestry (5.1% vs. 2.9%, P < 0.0001) and Asian ancestry (6.0% vs. 2.9%, P < 0.001). Activating BRAF alterations were detected in approximately 3% of prostate cancers, and most were class II mutations and rearrangements; BRAF V600 mutations were exceedingly rare. These findings suggest that BRAF activation in prostate cancer is unique from other cancers and supports further clinical investigation of therapeutics targeting the mitogen-activated protein kinase (MAPK) pathway.

Lack of Influence of Non-Overlapping Mutations in BRAF, NRAS, or NF1 on 12-Month Best Objective Response and Long-Term Survival after Checkpoint Inhibitor-Based Treatment for Metastatic Melanoma.

Non-overlapping somatic mutations in BRAF, NRAS, or NF1 genes occur in 85% of metastatic melanoma patients. It is not known whether these mutations affect immunotherapy outcome. Next-Gen sequencing of 324 oncogenes was performed in 73 metastatic melanoma patients. A retrospective review of immunotherapy outcome was performed. BRAF fusions/internal rearrangements, BRAF V600E, NRAS, NF1 mutations, and triple-negative genotypes occurred in 6.9%, 30.1%, 17.8%, 32.9%, and 12.3% of patients, respectively. Median potential follow-up was 41.0 months. Patients with BRAF fusion/rearrangement had decreased progression-free and overall survival (p = 0.015). The other genotypes each had similar progression-free and overall survival. Patients who achieved a complete best objective response at 12 months (n = 36, 49.3%) were found to have significantly improved survival compared those who failed to achieve remissions (n = 37, 50.7%, p < 0.001). The most important determinant of long-term survival was achievement of a complete response by 12 months following immunotherapy. PR and SD were not a stable type of response and generally resulted in progression and death from melanoma. Rare patients with BRAF fusions or rearrangements had decreased progression-free and overall survival following initial immunotherapy. Other BRAF, NRAS, or NF1 mutations were not associated with significant differences in outcome.

Liquid-biopsy detection of FGFR2 and other actionable rearrangements in GI malignancies.

4085 Background: Actionable rearrangements (RE) represent an emerging therapeutic target for GI malignancies. However, there remains uncertainty whether liquid biopsy (LBx) assays testing circulating tumor DNA (ctDNA) can detect RE with the same fidelity as tissue biopsy. Here we report the performance of ctDNA-based comprehensive genomic profiling (CGP) for the detection of RE leveraging an FDA-approved &gt;300-gene platform, with a focus on data from patients with GI malignancies. Methods: An institutional research database of clinical CGP results for tissue biopsy (TBx, FoundationOneCDx) and LBx (FoundationOneLiquid CDx) from patients with cancers of the GI tract was reviewed. Sensitivity for FGFR2 RE was studied in patients with cholangiocarcinoma (CCA) or carcinoma of unknown primary (CUP) and both TBx and LBx CGP available. Results: Across 7870 GI LBx, 1094 predicted pathogenic RE were detected in 826 cases (10%) including 283 oncogenic kinase RE, 686 inactivating RE, and 125 other gain-of-function RE. FGFR2 was the most frequently rearranged gene, enriched in CCA (4.4%, 37/833) and stomach (3.0%, 11/368). EGFR RE (38) occurred across 6 cancer types and included 8 fusions, 15 c-terminal truncations, 4 intragenic deletions/inversions, and 3 kinase domain duplications. BRAF RE (31) were detected among in colorectal (22) and pancreas (9) cases. LBx detected 44 exon 3 skipping (protein stabilizing) RE in CTNNB1 (beta-catenin) across all samples. Other frequent activating RE were detected in MYC (36), FGFR3, RET, ROS1 (21 each), and ALK (13). Potentially targetable inactivating RE were detected in BRCA1 (28), ARID1A (24), STK11 (22), PTEN (17), TSC2 (14), and BRCA2 (8). Focusing on CCA, FGFR2 RE were more prevalent in cases with elevated (≥1%) ctDNA tumor fraction (TF, 26/342, 7.6%), matching the prevalence in TBx (7.8%, 505/6,492) with a similar spectrum of fusion partners seen for both TBx and LBx (30% BICC1, 70% rare for both). In 13 CCA/CUP patients positive for FGFR2 RE by TBx, LBx detected 12 (92%) including 8 different fusion partners; the false negative case had TF &lt;1%. In 11 CCA LBx samples with FGFR2 resistance mutations detected, LBx CGP successfully detected a driver FGFR2 RE in 10 and driver C382R mutation in 1. Conclusions: Rearrangements are represented across GI malignancies, including many that result in known oncogenic drivers that may be targetable with available therapies. We observe reliable detection of FGFR2 fusions in ctDNA, in contrast to some prior publications. ctDNA represents a pragmatic analyte for detection of rearrangements and other actionable alterations, offering timely result return when tissue is inadequate or unavailable. [Table: see text]

Spectrum and implications of activating BRAF alterations in advanced prostate cancer (aPC).

220 Background: Activating genomic alterations (GA) in BRAF are rare in aPC and their impact on pathogenesis is poorly understood. However, emerging data suggest that these GA may represent clinically actionable targets (Fenor et al, Clin Transl Oncol, 2022). Using comprehensive genomic profiling (CGP) performed in a single commercial lab, we characterized the GA landscape of BRAF-activated tumors in aPC patients (pts). Methods: Tissue(N=15,864) and liquid (N=7,566) biopsies from aPC pts were profiled using FoundationOne CDx and FoundationOne Liquid CDx CGP assays, respectively. Each assay covers 324 cancer-related genes, including the full coding region of BRAF and introns 7-10, with additional sensitivity in exons 11-18 in the liquid assay. Activating GA were defined as hotspot missense mutations, in-frame indels in the kinase domain, or rearrangements (RE) that preserve the kinase domain. Tissue biopsies from non-aPC cancer types (N= 275,151) were used for comparison. Results: BRAF-activating GA were detected in 520/15,864 (3.3%) tissue biopsies: RE were the most common GA (243 samples, 1.5%), followed by K601E (101, 0.6%), and G469A (58, 0.4%). Median age of pts with BRAF-altered tumors was 69 (interquartile range 63-76), compared to 68 for BRAF wild-type (interquartile range 62-74). Rearrangement breakpoints occurred most frequently in intron 8 (37%), intron 9 (28%), intron 10 (20%), and intron 7 (13%). The most common RE were BRAF N-terminal truncations removing the auto-inhibitory domain (22%), SND1-BRAF fusions (13%), intragenic BRAF deletions of the auto-inhibitory domain (12%), and TMPRS22-BRAF fusions (5%). When studying cases with BRAF-activating GA compared to wild-type, we noted a larger proportion of CDK12 mutations (9.2% vs 5.2%, p=0.018), and a depletion of TMPRSS2-ERG fusions (11% vs 32%, p&lt;0.0001), PTEN GA (17% vs 31%, p&lt;0.0001), and APC GA (4.4% vs 8.9%, p = 0.018); alterations in AR occurred at similar rates (13% vs 13%, p=1.0). In liquid samples, overall BRAF GA were slightly less common (187/7566, 2.5%): 65 (0.9%) RE, 33 (0.4%) K601E, 12 (0.2%) G469A. Examining BRAF-altered samples across all cancer types, aPC had the highest proportions of RE (46%) and one of the lowest frequencies of V600E (0.1%). Conclusions: Activating BRAF alterations are detected in ~3% of aPC, with frequent BRAF-SND1 fusions. BRAF RE represent almost half of aPC BRAF GA, the highest fraction observed across a pan-tumor dataset. In addition, we detected a higher incidence of concurrent CDK12 GA and a lower relative frequency of concurrent PTEN, APC, and TMPRSS2-ERG GA. These findings suggest that genetic activation of BRAF in aPC pts may contribute to tumorigenesis and supports further clinical investigation of therapeutics targeting the MAPK pathway in this molecular subtype.

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.

Small Cell Lung Cancer Transformation following Treatment in EGFR-Mutated Non-Small Cell Lung Cancer.

EGFR-mutated lung adenocarcinoma patients who received tyrosine kinase inhibitors (TKIs) may initially respond to therapy, but over time, resistance eventually occurs. In a small population (5–10%), these patients can have a histological transformation to SCLC. Nine patients with EGFR-mutated lung adenocarcinoma who transformed to SCLC were evaluated at City of Hope. Patient clinical and pathology data, including multiple next-generation sequencing (NGS) results, clinical therapies, histology, and outcomes, were collected across multiple time points. Descriptive statistics were utilized to visualize and interpret the clinical therapeutic timeline and molecular transformation profiles for these patients. All patients received at least one line of EGFR TKI therapies prior to small cell lung cancer transformation, including erlotinib, afatinib, and osimertinib. Two patients also received chemotherapy prior to transformation (one with immunotherapy). The median months to small cell lung cancer transformation was 16 months, ranging from 4–49 months. The median overall survival (OS) was 29 months from diagnosis, with the minimum of 16 months and maximum of 62 months. The majority of patients had EGFR exon 19 deletion (n = 7, 77.8%), and no patients had a change of original oncogenic EGFR mutation over the different time points. Though a TP53 mutation was detected in eight patients (88.9%) either at the first biopsy or the subsequent biopsies, an RB1 alteration was only detected in one patient at presentation, and three patients upon subsequent biopsies (n = 4, 44.4%). Each patient had a unique molecular profile in the subsequent molecular testing post-transformation, but BRAF alterations occurred frequently, including BRAF rearrangement (n = 1), fusion (n = 1), and amplification (n = 1). Our results showed that EGFR-mutated lung adenocarcinoma to SCLC transformation patients have a unique histological, molecular, and clinical profile over multiple time points, with further heterogeneity that is not currently reported in the literature, and we suggest more work is required to better understand the molecular heterogeneity and clinical outcomes over time for this EGFR TKI resistance subtype.

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.

BRAF Rearrangements and BRAF V600E Mutations Are Seen in a Subset of Pancreatic Carcinomas With Acinar Differentiation.

Comprehensive genomic profiling has demonstrated that approximately 20% of pancreatic carcinomas with acinar differentiation harbor potentially targetable BRAF fusions that activate the MAPK pathway. To validate the above finding by BRAF break-apart fluorescence in situ hybridization (FISH) in a large series of pure acinar cell carcinomas (ACCs), evaluate tumors for the presence of BRAF V600E mutations, and compare clinicopathologic features of tumors with BRAF rearrangements with those without. Thirty cases of pure ACC and 6 cases of mixed acinar-neuroendocrine carcinoma (ACC-NEC) were retrieved. A break-apart FISH probe was used to detect BRAF rearrangements. Immunohistochemistry for BRAF V600E was performed. BRAF rearrangements by FISH were found in 6 of 36 cases (17%), 5 of which were pure ACC and 1 was a mixed ACC-NEC. Follow-up was available in 29 of 36 cases (81%). The median survival was 22 months for BRAF-rearranged cases and 16 months for BRAF-intact cases; the 2-year overall survival was 50% for BRAF-rearranged cases and 35% for BRAF-intact cases. No significant clinicopathologic differences were identified in cases with BRAF rearrangement compared with those without BRAF rearrangement. BRAF V600E mutation was identified in 2 of 34 cases (6%), both of which were pure ACC and were BRAF-intact by FISH. This study supports the finding that BRAF rearrangements are present in approximately 20% of cases and identified BRAF V600E mutations in approximately 5% of cases. These cases may benefit from targeted therapy.

P59.07 Mutation Profile of BRAF in Chinese Non-Small Cell Lung Cancer Patients

BRAF mutation is one of the most common driver gene mutations in non-small cell lung cancer (NSCLC) patients, especially in adenocarcinoma. BRAF alterations have been identified with the clinical application of next generation sequencing (NGS),however, mutation profile and co-occurring genomic alterations of BRAF have not been fully understood in Chinese non-small cell lung cancer patients. We enrolled 14703 Chinese NSCLC patients with confirmed histology subtype of adenocarcinoma, squamous carcinoma and large cell carcinoma. FFPE samples and matched peripheral blood or plasma were sequenced for NGS based 1021 cancer genes panel assay. Comprehensive genomic profiling including single nucleotide variants (SNV), short and long insertion and deletion (Indel), copy number variations (CNV) and gene arrangement/fusion were analyzed. 1.78% (261/14703) of Chinese NSCLC patients harbored at least one BRAF genomic alteration, which was mainly composed of patient with SNVs and Indels (97.3%, 254 patients), copy number variations (16.1%, 42 patients), and gene rearrangements (5.0%, 13 patients). None BRAF CNV was detected. Of the 42 patients carried CNV, 38 of them carried both CNV and BRAF SNV/Indel, whereas two of them carried both CNV and gene arrangement/fusion and two of patients carried aforementioned three types of alteration. BRAF V600E were the most common BRAF mutations, which accounted for 36% of all BRAF mutation, respectively. The most common resistant alteration G469A accounted for 11%. A total of nine types of BRAF rearrangement were detected from eight patients. AGK-BRAF were the most common BRAF rearrangement, which accounted for 36% of all BRAF rearrangement. Further analysis of co-occurring BRAF mutations revealed that of BRAF mutated Chinese NSCLC patients harbored both BRAF mutations and other driver gene mutation, such as EGFR, KRAS, PI3KCA. This study revealed BRAF variation in approximately 1.78% of Chinese NSCLC patients. BRAF V600E were the most common BRAF mutations, which accounted for 36% of all BRAF mutation. BRAF mutated Chinese NSCLC patients harbored both BRAF mutations and other driver gene mutation, such as EGFR, KRAS, PI3KCA.

Gene fusion characterisation of rare aggressive prostate cancer variants-adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma: an analysis of 19 cases.

To evaluate the molecular underpinnings of the rare aggressive prostate cancer variants adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma. We retrieved 19 tumours with one or more variant(s), and performed ERG immunohistochemistry, a next-generation sequencing assay targeting recurrent gene fusions, and fluorescence in-situ hybridisation (FISH) for ERG and BRAF. Divergent differentiation included: sarcomatoid carcinoma (n=10), adenosquamous carcinoma (n=7), and pleomorphic giant-cell carcinoma (n=7). Five patients had more than one variant. Four had variants only in metastases. ERG rearrangement was detected in nine (47%, seven via sequencing, showing TMPRSS2-ERG fusions and one GRHL2-ERG fusion, and two via FISH, showing rearrangement via deletion). ERG was immunohistochemically positive in the adenocarcinoma in eight of nine (89%) patients, but was immunohistochemically positive in the variant in only five of nine patients (56%, typically decreased). One patient had a false-positive ERG immunohistochemical result in the sarcomatoid component despite a negative FISH result. Two (11%) harboured BRAF fusions (FAM131A-BRAF and SND1-BRAF). ERG fusions are present in these rare prostate cancer variants with a frequency close to that in conventional prostate cancer (9/19, 47%). ERG immunohistochemistry usually detects rearrangement in the adenocarcinoma, but is less sensitive for the variant histology, with weak to negative staining. Adenosquamous and sarcomatoid variants can, particularly, occur together. Molecular assessment may be an additional tool in selected cases to confirm the prostatic origin of unusual tumours. The presence of two BRAF rearrangements suggests that this gene fusion may be enriched in this setting, as RAF kinase fusions have been previously reported in 1-2% of prostate cancers.

Abstract 817: Pancreatic acinar carcinoma - a rare entity with a targetable BRAF V600E mutation

Abstract Background: Pancreatic acinar carcinomas account for less than 1% of all pancreatic cancers and are considered genetically distinct entities form other pancreatic carcinomas. The pathogenesis of this malignancy is incompletely understood, and the efficacy of cytotoxic chemotherapy is not well defined especially in unresectable or metastatic disease. Methods: We identified one patient with pancreatic acinar carcinoma who was treated at our institution and was found to have a unique targetable pathogenic mutation on tumor sequencing. Results: A 64-year-old male patient was diagnosed with localized pancreatic acinar carcinoma during workup for epigastric pain. He underwent distal pancreatectomy with final pathology consistent with stage T3N0 acinar cell carcinoma without any adenocarcinoma component. He subsequently received six months of adjuvant chemotherapy with gemcitabine plus capecitabine, derived from evidence on pancreatic adenocarcinoma due to the size of his original tumor. Seven months after completion of adjuvant therapy, he was found to have local recurrence within the surgical bed and was treated with three cycles of systemic chemotherapy with FOLFIRINOX. During a following planned surgical resection, multiple liver lesions were seen intraoperatively, and surgery was aborted. Liver lesions were biopsied and hybrid capture next-generation sequencing was performed which revealed: a BRAF V600E gain of function mutation, CDKN2A loss as well as MYCN amplification. The patient declined further chemotherapy and his treatment was switched to Encorafenib (BRAF inhibitor) plus Binimetinib (MEK inhibitor). He attained a partial response to therapy at his first restaging CT scan after 2 months of therapy (-39% per RECIST 1.1). Both mass at the surgical bed as well as liver metastases were decreased in size. He tolerated treatment very well without significant adverse events (including no clinically significant rash). Conclusions: While BRAF rearrangements as described in the literature may occur in 15-23% of patients with acinar cell carcinoma of the pancreas, BRAF activating mutations and in particular the BRAF V600E substitution has not been reported. This particular substitution is commonly present in melanoma, hairy cell leukemia, Langerhans cell histiocytosis and less frequently in lung and colon cancer. A combination of BRAF and MEK inhibition may be used in patients with tumors harboring this activating mutation with variable efficacy across different histologies. In light of the rarity of acinar cell carcinomas of the pancreas and the lack of established systemic treatment guidelines, tumor sequencing should be considered if no other therapies are available. This is the first case reported in the literature of a patient with acinar cancer of the pancreas and BRAF V600E mutation where targeted therapy has shown an early response. Citation Format: Vaia Florou, Chris Nevala-Plagemann, Sean Mulvihill, Ignacio Garrido-Laguna. Pancreatic acinar carcinoma - a rare entity with a targetable BRAF V600E mutation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 817.

Highly Sensitive and Specific Molecular Test for Mutations in the Diagnosis of Thyroid Nodules: A Prospective Study of BRAF-Prevalent Population.

Molecular testing offers more objective information in the diagnosis and personalized decision making for thyroid nodules. In Korea, as the BRAF V600E mutation is detected in 70–80% of thyroid cancer specimens, its testing in fine-needle aspiration (FNA) cytology specimens alone has been used for the differential diagnosis of thyroid nodules until now. Thus, we aimed to develop a mutation panel to detect not only BRAF V600E, but also other common genetic alterations in thyroid cancer and to evaluate the diagnostic accuracy of the mutation panel for thyroid nodules in Korea. For this prospective study, FNA specimens of 430 nodules were obtained from patients who underwent thyroid surgery for thyroid nodules. A molecular test was devised using real-time PCR to detect common genetic alterations in thyroid cancer, including BRAF, N-, H-, and K-RAS mutations and rearrangements of RET/PTC and PAX8/PPARr. Positive results for the mutation panel were confirmed by sequencing. Among the 430 FNA specimens, genetic alterations were detected in 293 cases (68%). BRAF V600E (240 of 347 cases, 69%) was the most prevalent mutation in thyroid cancer. The RAS mutation was most prevalently detected for indeterminate cytology. Among the 293 mutation-positive cases, 287 (98%) were diagnosed as cancer. The combination of molecular testing and cytology improved sensitivity from 72% (cytology alone) to 89% (combination), with a specificity of 93%. We verified the excellent diagnostic performance of the mutation panel applicable for clinical practice in Korea. A plan has been devised to validate its performance using independent FNA specimens.