Sarcoma Viral Oncogene Homolog B1 Research Articles

Bilateral Pterygium in a Paediatric Cancer Patient taking Vemurafenib: A Case Report

Introduction: Anticancer drugs with reactive oxygen species (ROS) inducing mechanisms, such as vemurafenib, have been shown to be effective against various types of cancer. However, the adverse effects of increased oxidative stress have the potential to damage not only cancer cells, but also normal cells such as those in eyes and potentially leading to ocular diseases including pterygium and cataract. Presentations: A nine-year-old patient presented with bilateral nasal pterygium and early bilateral cortical cataracts, which are uncommon for his age group. Patient has a background of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E positive pilocytic astrocytoma with anaplastic features, diagnosed at age 2.5 years old. Vemurafenib was commenced at the time of diagnosis. Conclusion: We suspect that these ophthalmic findings are associated with his prolonged treatment with vemurafenib which may be of a pathophysiological significance.

Association Between Gross Features and Coexistence of BRAFV600E and TERT Promoter Mutations in Papillary Thyroid Carcinomas: A Combined Analysis Incorporating Clinicopathologic Features.

Background: The coexistence of v-Raf murine sarcoma viral oncogene homolog B1 (BRAFV600E) and telomere reverse transcriptase promoter (TERT-p) mutations is considerably associated with aggressiveness and poor prognosis in papillary thyroid carcinoma (PTC). However, the association between gross findings and genetic alterations in PTC remains unknown. We aimed to investigate the association between clinicopathologic features, including macroscopic features, and the coexistent BRAFV600E and TERT-p mutations in patients with PTC. Methods: We retrospectively analyzed 375 cases of PTC surgically resected between January 2018 and October 2023 at a single institution, based on the presence of BRAFV600E and TERT-p double mutation. Clinicopathologic features, including gross features on the cut surface of tumors, were evaluated. Subsequently, the association between clinicopathologic features and mutation status was statistically examined. Cox proportional hazard models were used to analyze the impact of molecular pathological features on disease-free survival (DFS). Results: The BRAFV600E and TERT-p double mutation was identified in 78 (20.8%) patients among the PTC cases and was significantly correlated with shorter DFS. Multivariable analysis revealed that factors such as relatively older age (≥55 years) (odds ratio [OR] = 12.083, 95% confidence interval [CI] 4.498-32.456), larger tumor size (>2.0 cm) (OR = 2.722, CI 1.104-6.712), lobulated tumor margins (OR = 16.114, CI 3.155-82.296), papillary excrescences on the cut surface (OR = 17.573, CI 3.462-89.201), solid-cut surface (OR = 4.012, CI 1.084-14.849), minimal extrathyroidal extension (ETE) (OR = 4.156, CI 1.209-14.282), gross ETE (OR = 6.517, CI 1.734-24.490), and Ki-67 labeling index (LI) (≥5%, OR = 12.145, CI 4.354-33.877) were significantly associated with the double mutation. Conclusions: The BRAFV600E and TERT-p double mutation in PTC was significantly associated with relatively old age, larger tumor size, lobulated configuration in tumor margin, papillary excrescences on the cut surface, solid-cut surface, ETE, and high Ki-67 LI. These features are suggestive of the presence of the double mutation and should be analyzed at the molecular level in patients with PTC.

Clinicopathological features, response patterns, outcomes and BRAF status in patients with advanced acral melanoma: a preliminary Peruvian study.

Globally, acral melanoma (AM) is underrepresented in most clinical trials, being predominant in Caucasian populations. Latin America is a niche that needs to be explored. Therefore, this study aimed to determine the clinical features, response patterns, outcomes and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) status in Peruvian patients with advanced AM. We retrospectively reviewed the medical records of 19 patients with advanced AM who received immunotherapy (IO) in first- or subsequent-line therapy. The samples were analysed, and their mutational state was performed by deoxyribonucleic acid sequencing, focusing primarily on the most frequently mutated gene, BRAF. Descriptive statistics were used to assess the baseline characteristics. Overall survival was estimated using the Kaplan-Meier method. The median age was 64 years and 63.2% were men. Plantar was the site most frequently affected (84.2%). The most frequent stage was stage III (68.4%), with 26.4% receiving adjuvant therapy. The majority of cases exhibited a Breslow thickness of >4 mm (52%), a Clark level of IV/V (89.4%), and all patients presented ulceration and a high range of mitosis. During follow-up, all patients experienced recurrent advanced disease, with 52.6% developing visceral metastasis. Patients who received IO as first or subsequent line had an overall response rate (ORR) of 33.3%, and those who received it as first-line therapy had an ORR of 40%. Twenty-one percent of the patients harbored BRAF V6000E mutation and, showing an ORR of 50% compared to wild-type individuals (44.4%) after the first line of treatment. Our preliminary study reported that AM has poor clinico-pathological features and response rates to IO in Peruvian patients. However, those who received IO as a first-line treatment or harbored the BRAF mutation appeared to have a slightly better response than wild-type patients.

Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib)

BackgroundBRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors.MethodsCell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array.ResultsThe resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRβ receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion.ConclusionsIn summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

CDK4/6 inhibition to resensitize BRAF/EGFR inhibitor in patient-derived BRAF/PTEN-mutant colon cancer cells.

In v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant colorectal cancer (CRC), encorafenib-cetuximab has been established as standard second-line therapy, but not all patients respond and the duration of response is relatively short. Overcoming intrinsic or acquired resistance to BRAF/EGFR inhibitors is crucial for enhancing treatment outcomes in metastatic BRAF-mutated CRC. The aim of the study is to investigate the resistance mechanisms in BRAF-mutant CRC patient refractory to BRAF/EGFR targeted therapy. We established patient-derived cells (PDCs) from a patient with BRAF/PTEN-mutant metastatic colon cancer who progressed rapidly on encorafenib plus cetuximab. To explore potential treatment options for inherent resistance caused by simultaneous PTEN mutation in BRAF-mutated CRC, we conducted cell viability assays using PDCs treated with encorafenib-cetuximab in combination with a cyclin-dependent kinase-4 and 6 (CDK4/6) inhibitor. The patient's tumor had concurrent PTEN loss-of-function alteration at diagnosis and PDCs were generated from ascites after resistance to the BRAF/EGFR inhibitor. The PDCs were resistant to the encorafenib-cetuximab combination even at a high concentration of cetuximab (up to 500 µg/mL). Adding the CDK4/6 inhibitor, ribociclib, to encorafenib-cetuximab showed a synergistic effect in a proliferation assay. Ribociclib plus encorafenib-cetuximab represented a significantly lower expression of Ki-67 compared to the dual combination alone. An MTS assay showed that triplet therapy with ribociclib, encorafenib, and cetuximab suppressed cell viability more efficiently than the two-drug combinations. Investigating the combined effect of triplet therapy using the calculated combination index (CI) showed that ribociclib had a synergistic effect with encorafenib-cetuximab when applied to PDCs with a concurrent BRAF/PTEN mutation. Our results suggest that combining the CDK4/6 inhibitor with the BRAF/EGFR inhibitormight be a novel treatment strategy for concomitant BRAF and PTEN-mutant CRC.

Exosomal miR-19a derived from melanoma cell promotes the vemurafenib resistance of malignant melanoma through directly targeting LRIG1 to reactivate AKT and MAPK pathway

Exosomes derived from neighboring v-raf murine sarcoma viral oncogene homolog B1 inhibitor (BRAFi)-resistant melanoma cells mediate the formation of resistance in melanoma cells sensitive to BRAFi. The function and molecular mechanisms of exosomal miRNA in BRAFi resistance of melanoma have not been studied. We found that the expression of miR-19a in BRAFi resistant melanoma cells was significantly higher than that in sensitive cells, and miR-19a contributes to the resistance of melanoma cells to BRAFi by targeting immunoglobulin-like domains protein 1 (LRIG1). miR-19a was highly enriched in exosomes secreted from BRAFi resistant melanoma cells, and these exosomal miR-19a promote the spread of BRAFi resistant. The reactivation of Protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways is the main reason for the BRAFi resistant of melanoma cells. We demonstrated that exosomal miR-19a derived from melanoma cell promotes the formation and spread of BRAFi resistant in melanoma through targeting LRIG1 to reactivate AKT and MAPK pathway. Therefore, miR-19a may serve as a potential therapeutic target in melanoma patients with acquired drug resistance.

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

Genomic medicine advances for brain tumors.

Cancer genome profiling has revealed important genetic alterations that serve as prognostic indicators and guides for treatment decisions, enabling precision medicine. The shift to molecular diagnosis of brain tumors, as reflected in the 2021 World Health Organization Classification of Tumors of the Central Nervous System, is a crucial role for treatment decision-making. This review discusses the significance and role of cancer genome profiling in precision medicine for malignant brain tumors, particularly gliomas. Furthermore, we explore the progress in cancer genome analysis, focusing on cancer gene panel testing, integration of genomic information in brain tumor classification, and hereditary tumors. Additionally, we discuss the transformative effect of genomic medicine on early detection, risk assessment, and precision medicine strategies. The tumor mutational burden in brain tumors is considered low, but the application of molecular targeted drugs, such as isocitrate dehydrogenase inhibitors, v-raf murine sarcoma viral oncogene homolog B1 inhibitors, fibroblast growth factor receptor inhibitors, neurotrophic tyrosine receptor kinase, mechanistic target of rapamycin inhibitors, and anti-programmed death receptor-1 antibody drugs are promising for glioma treatment. We also discuss the future prospects of molecular targeted drugs.

Temporal trends and regional variability in BRAF and KRAS genetic testing in Denmark (2010-2022): Implications for precision medicine.

This study aims to evaluate the developments in the testing of Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations across different cancer types and regions in Denmark from 2010 to 2022. Using comprehensive data from the Danish health registries, we linked molecular test results from the Danish Pathology Registry with cancer diagnoses from the Danish National Patient Registry between 2010 and 2022. We assessed the frequency and distribution of KRAS and BRAF mutations across all cancer types, years of testing, and the five Danish regions. The study included records of KRAS testing for 30 671 patients and BRAF testing for 30 860 patients. Most KRAS testing was performed in colorectal (78%) and lung cancer (18%), and BRAF testing in malignant melanoma (13%), colorectal cancer (67%), and lung cancer (12%). Testing rates and documentation mutational subtypes increased over time. Reporting of wildtype results varied between lung and colorectal cancer, with underreporting in lung cancer. Regional variations in testing and reporting were observed. Our study highlights substantial progress in KRAS and BRAF testing in Denmark from 2010 to 2022, evidenced by increased and more specific reporting of mutational test results, thereby improving the precision of cancer diagnosis and treatment. However, persistent regional variations and limited testing for cancer types beyond melanoma, colorectal, and lung cancer highlight the necessity for a nationwide assessment of the optimal testing approach.

BRAF V600E Mutation in Malignant Melanoma-A Romanian Research Experience.

Background and Objectives: The most common mutation in malignant melanoma (MM) is the single-point mutation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) oncogene. Our study aims to evaluate BRAF V600E mutation, highlighting its frequency differences in primary versus metastatic MM. Materials and Methods: The study group comprised 133 patients diagnosed with MM in several county hospitals of the north-eastern region of Romania who have been assigned for investigation into BRAF V600E mutation in the private medical system. The material consisted of archived formalin-fixed paraffin-embedded (FFPE) blocks. BRAF V600E mutation was identified using the fully automated IdyllaTM BRAF mutation test system. Results: Out of the total of 133 cases, 78 cases were primary tumors, while 55 cases were metastatic MMs. Genetic analysis revealed the presence of BRAF V600E mutation in 66 cases (49.62%) and the wild-type genotype in 67 cases (50.37%). We found a statistically significant difference of the mutation frequency according to age (p = 0.0072). The mutated genotype was found in 45 cases out of 78 primary MMs (57.69%) and in 21 cases out of 55 secondary MMs (38.18%), with a statistically significant difference in favor of primary tumors (p = 0.0413). The correlations between the histopathological types, Clark's level, Breslow index, ulceration, and lymphovascular invasion, respectively, and the mutated genotype were not statistically significant. BRAF V600E mutation was identified in 15 out of 40 secondary tumors with lymph node location (37.5%) and in 6 out of 15 secondary tumors with another location (40%) without statistically significant differences between the mutation frequency and the location of the secondary tumors. Conclusions: Our results support MM high genetic heterogeneity, pointing out the relationship between BRAF V600E mutation and several clinicopathological characteristics, in primary and metastatic MMs, stressing the importance of BRAF testing implementation in Romania.

Is there a prognostic difference among stage I lung adenocarcinoma patients with different BRAF-mutation status?

The data of the prognostic role of V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in early-stage lung adenocarcinoma (LUAD) patients is scarce. This study aimed to investigate the proportion, clinicopathological features, and prognostic significance of patients with stage I LUAD carrying BRAF mutations. We collected 431 patients with pathological stage I LUAD from cBioPortal for Cancer Genomics and 1604 LUAD patients tested for BRAF V600E and epidermal growth factor receptor (EGFR) mutations from Shanghai Pulmonary Hospital. Survival curves were drawn by the Kaplan-Meier method and compared by log-rank test. Cox proportional hazard models, propensity-score matching (PSM), and overlap weighting (OW) were performed in this study. The primary endpoint was recurrence-free survival (RFS). The proportion of BRAF mutations was estimated at 5.6% in a Caucasian cohort. BRAF V600E mutations were detected in six (1.4%) patients in Caucasian populations and 16 (1.0%) patients in Chinese populations. Two BRAF V600E-mutant patients were detected to have concurrent EGFR mutations, one for 19-del and one for L858R. For pathological stage I LUAD patients, BRAF mutations were not significantly associated with worse RFS than wild-type BRAF patients (HR = 1.111; p = 0.885). After PSM and OW, similar results were presented (HR = 1.352; p = 0.742 and HR = 1.246; p = 0.764, respectively). BRAF V600E mutation status also lacked predictive significance for RFS (HR, 1.844; p = 0.226; HR = 1.144; p = 0.831 and HR = 1.466; p = 0.450, respectively). In this study, we demonstrated that BRAF status may not be capable of predicting prognosis in stage I LUAD patients. There is a need for more data to validate our findings.

Splenic Lymphoma: A Series of Seven Cases

Splenic Lymphoma (SL) is a lymphoproliferative disorder characterised by involvement of the spleen, with or without bone marrow involvement. However, advancements in imaging modalities have led to more frequent diagnosis of isolated splenic lesions. There is always a dilemma in diagnosis as to whether the pathology truly originates from the spleen or if it is part of systemic involvement in Non-Hodgkin Lymphoma (NHL). Clinical manifestations vary widely, and treatment outcomes for SL depend on accurate diagnosis. However, prognosis still remains poor in these cases compared to systemic NHL. The present case series provide a comprehensive overview of patients diagnosed with SL, focusing on their clinicopathological characteristics, therapeutic details, prognosis and outcomes. Previously isolated case reports have been published, and there is still a lack of consensus on the management of this entity. The present case series included seven cases over a two-year period, from January 2022 to December 2023. In the current case series, authors have highlighted the heterogeneity and variability in the presentation of SL and how the clinical behaviour ranges from indolent to highly aggressive. Nevertheless, the presenting clinical, laboratory and pathological features of these diseases often display significant overlaps and pose confusion in arriving at an exact diagnosis. The authors emphasise how flow cytometry helps facilitate the diagnosis and systemic therapy is the modality of choice for treatment decided based on the histology type; however, outcomes still remain dismal, highlighting the unmet need to improve accurate diagnosis and explore newer treatment options. The novelty in this series lies in the detailed analysis and comparison of different subtypes of SL, a rare group of lymphomas primarily originating in the spleen. The present case series discusses the latest World Health Organisation (WHO) classification, which redefines the Hairy Cell Leukaemia (HCL) variant as Splenic B-cell Leukaemia/ Lymphoma with Prominent Nucleoli (SBLPN). Detailed molecular and genetic characteristics of various SLs are highlighted, such as the presence of the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) p.V600E mutation in HCL, Mitogen-activated Protein Kinase 1 (MAP2K1) and Immunoglobulin Heavy Variable 4-34 (IGHV4-34) mutations in the HCL-variant, and 7q distortion in Splenic Marginal Zone Lymphoma (SMZL). Emphasis is placed on the individualised treatment approach based on specific patient profiles, genetic markers and disease characteristics. The study underscores the need for further research and clinical trials to validate emerging therapies, such as the use of Rituximab plus Doxorubicin, Bleomycin, Vinblastine and Dacarbazine (R-ABVD) in Hodgkin lymphoma and combinations of novel agents in aggressive SLs like HCL-variant and CD20-negative B-cell NHL.

Repurposing Metabolic Inhibitors in the Treatment of Colon Adenocarcinoma Patient-Derived Models.

The effect of agonists on AMP-activated protein kinase (AMPK), mainly metformin and phenformin, has been appreciated in the treatment of multiple types of tumors. Specifically, the antitumor activity of phenformin has been demonstrated in melanomas containing the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation. In this report, we elucidated the synergistic antitumor effects of biguanides with metabolism inhibitors on colon tumors. Phenformin with 2-deoxy-D-glucose (2DG) inhibited tumor cell growth in cancer cell lines, including HT29 cells harboring BRAF- and p53-mutations. Biochemical analyses showed that two chemotherapeutics exerted cooperative effects to reduce tumor growth through cell cycle arrest, apoptosis, and autophagy. The drugs demonstrated activity against phosphorylated ERK and the gain-of-function p53 mutant protein. To demonstrate tumor regressive effects in vivo, we established patient-derived models, including xenograft (PDX) and organoids (PDO). Co-treatment of biguanides with chemotherapeutics efficiently reduced the growth of patient-derived colon models in comparison to treatment with a single agent. These results strongly suggest that significant therapeutic advantages would be achieved by combining AMPK activators such as phenformin and cancer metabolic inhibitors such as 2DG.

The Impact of BRAF Mutation Status on Survival Outcomes and Treatment Patterns among Metastatic Colorectal Cancer Patients in Alberta, Canada.

Colorectal cancer presents via multiple different clinical phenotypes that can arise from a variety of different genetic and molecular alterations. The aim of this study was to describe survival outcomes and treatment patterns of metastatic colorectal cancer (mCRC) patients by v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation status. The Alberta Cancer Registry was used to identify all patients >18 years old who had been diagnosed with mCRC in Alberta between 1 January 2017 and 31 December 2019 and had received at least one cycle of systemic therapy. Treatment patterns were compared between wild-type and mutant BRAF mCRC patients. Cox regression models and Kaplan-Meier curves were created to assess survival differences by both treatment pattern and BRAF status. A total of 488 patients were identified with mCRC, of which 42 (11.4%) were confirmed to have a BRAF mutation. The most common first-line treatment regimen was either capecitabine and oxaliplatin (CAPOX) or leucovorin calcium (folinic acid), fluorouracil, and oxaliplatin (FOLFOX). The median overall survival for mCRC patients was 20.01 months. Mutant BRAF patients had a median survival of 8.21 months compared to 20.03 months among those with wild-type BRAF. BRAF mutations among mCRC patients are associated with a considerably poor prognosis, reinforcing the need for clinical BRAF testing among newly diagnosed patients to better understand their prognosis.

Abstract C153: Rare BRAF mutations in menlanoma and beyond: Rationalizing the afficacy of B-raf inhibitors via HPC-based in silico/in vitro investigations

Abstract Melanoma is a highly aggressive type of cancer that affects the integumentary system and for which a family history of the disease, a fair complexion, the presence of a large number of moles, and skin exposure to natural/artificial ultraviolet (UV) radiation constitute established risk factors for malignancy development. The v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine protein kinase that plays a critical role in the RAS-RAF-MEK-ERK mitogen–activated protein kinase (MAPK) cell signaling pathway. Despite over 20 years of research into the regulation and function of the RAF proteins, it was only relatively recently that the BRAF isoform is mutated at a high frequency in different human cancers, identifying this kinase as another important oncogene along this pathway. FFocusing on melanoma, activating BRAF mutations were identified in 37%−53% of patients with the malignant form of this pathology, the most prevalent, missense kinase alteration being the replacement of the small, apolar residue valine (V) with the negatively-charged, phosphorylation mimicking glutamic acid (E) at position 600 (BRAFV600E) within the protein catalytic (i.e., ATP binding) site. It is interesting to note that BRAFV600E is not a UV-driven mutation; in fact, it may also be found in the molecular profile of atypical benign nevi; moreover, although BRAFV600E is the most frequent mutation (accounting for nearly 40% of all melanomas and in approximately 90% of BRAF-mutant melanomas), other more or less frequent activating variants in BRAF have also been reported, which include other aminoacidic substitutions at the same position (e.g., V600K/D/R), G469A/V/S, L597Q/R/S/V, A598V, and K601E/N/T, among others. Importantly, these mutations are also found in other cancers, e.g., thyroid cancer, colorectal cancer, and some brain malignancies including glioblastoma, pilocytic astrocytoma, and pediatric low-grade glioma. In this multidisciplinary work, we focused on some of these rare BRAF variants and studied their ability to interact with several, first-line FDA approved BRAF initbitors (BRAFis) from a structural, chemico-phisical and in vitro perspectives. To the purpose, we employed a combitation of in silico techniques, based on the use of high-performance computing (HPC), chemico-physical and spectroscopical techniques, and in vitro tests to verify the interactions between these mutated BRAF isoforms with the inhibitors. As a main point, we were able to determine the binding thermodinamics and kinetics of the selected BRAFis to the mutant BRAF proteins, and each BRAF/drug complex was characterized from a structural standpoint. All these information ultimately revelaed that some of the BRAF isoforms are able to effectively bind the corresponding BRAi, with consequent switch-off of the signaling patwhay, at least in vitro. The presented mutlidisciplinary protocol could be adoped in the future to predict the response of newly discovered BARF variants and/or variants of unknown significance towards current or newly-designed BRAFis. Citation Format: Sabrina Pricl, Erik Laurini, Domenico Marson, Gabriele Cavalieri. Rare BRAF mutations in menlanoma and beyond: Rationalizing the afficacy of B-raf inhibitors via HPC-based in silico/in vitro investigations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C153.

FGFR blockade inhibits targeted therapy-tolerant persister in basal FGFR1- and FGF2-high cancers with driver oncogenes

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced anaplastic lymphoma kinase (ALK) fusion-positive DTP cell’s survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and fibroblast growth factor 2 (FGF2)-high protein expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs increased in patients with ALK fusion-positive non-small cell lung cancer with FGFR1- and FGF2-high mRNA expression at baseline. The combination of FGFR and targeted TKIs enhanced cell growth inhibition and apoptosis induction in basal FGFR1- and FGF2-high protein expressing cells with ALK-rearranged and epidermal growth factor receptor (EGFR)-mutated NSCLC, human epidermal growth factor receptor 2 (HER2)-amplified breast cancer, or v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma by preventing compensatory extracellular signal-regulated kinase (ERK) reactivation. These results suggest that a targeted TKI-induced DTP state results from an oncogenic switch from activated oncogenic driver signaling to the FGFR1 pathway in basal FGFR1- and FGF2-high expressing cancers and initial dual blockade of FGFR and driver oncogenes based on FGFR1 and FGF2 expression levels at baseline is a potent treatment strategy to prevent acquired drug resistance to targeted TKIs through DTP cells regardless of types of driver oncogenes.

Clinicopathological relevance of BRAF and SMO mutations in Chinese patients with ameloblastoma

Ameloblastomas are benign tumors associated with recurrence and morbidity. Mutations in the V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) and smoothened (SMO) genes have been identified in ameloblastomas. The mutation rate of BRAF in different regions and mutation sites of SMO remains controversial. This study assesses the relationship between these mutations and clinicopathological features of ameloblastoma in Chinese patients. Ameloblastomas were surgically removed from 30 patients and BRAF gene polymorphisms were detected using DNA sequencing analysis. We also examined the relationship between BRAF or SMO mutations and clinicopathological parameters. BRAF V600E mutation was detected in 18 of 30 ameloblastomas. No significant correlation was found between BRAF V600E mutation and age, sex, location, histologic type, capsular invasion, or tumor recurrence. Five cases carried an SMO T364N mutation in exon 5, whereas six carried an SMO S590 T mutation in exon 10. The SMO mutation rate in patients aged ≤ 20 years was significantly higher than that in patients aged > 20 years. SMO T364N and S590 T mutations were closely related to age and capsular invasion. These findings indicate that BRAF V600E, SMO T364N, and SMO S590 T mutations play important roles in the pathogenesis of ameloblastoma. Key highlights The BRAF V600E mutation was highly prevalent in Chinese patients with ameloblastoma. SMO T364N and S590 T mutations are closely related to age and capsular invasion. The BRAF V600E, SMO T364N, and SMO S590 T mutations play important roles in the pathogenesis of ameloblastoma.

Oncologic relevance of genetic alterations in sporadic synchronous and solitary colorectal cancer: a retrospective multicenter study

BackgroundOncologic impact of genetic alteration across synchronous colorectal cancer (CRC) still remains unclear. This study aimed to compare the oncologic relevance according to genetic alteration between synchronous and solitary CRC with performing systematic review.MethodsMulticenter retrospective analysis was performed for CRC patients with curative resection. Genetic profiling was consisted of microsatellite instability (MSI) testing, RAS (K-ras, and N-ras), and BRAF (v-Raf murine sarcoma viral oncogene homolog B1) V600E mutation. Multivariate analyses were conducted using logistic regression for synchronicity, and Cox proportional hazard model with stage-adjusting for overall survival (OS) and disease-free survival (DFS).ResultsIt was identified synchronous (n = 36) and solitary (n = 579) CRC with similar base line characteristics. RAS mutation was associated to synchronous CRC with no relations of MSI and BRAF. During median follow up of 77.8 month, Kaplan–meier curves showed significant differences according to MSI-high for OS, and in RAS, and BRAF mutation for DFS, respectively. In multivariable analyses, RAS and BRAF mutation were independent factors (RAS, HR = 1.808, 95% CI = 1.18–2.77, p = 0.007; BRAF, HR = 2.417, 95% CI = 1.32–4.41, p = 0.004). Old age was independent factor for OS (HR = 3.626, 95% CI = 1.09–12.00, p = 0.035).ConclusionThis study showed that oncologic outcomes might differ according to mutation burden characterized by RAS, BRAF, and MSI between synchronous CRC and solitary CRC. In addition, our systematic review highlighted a lack of data and much heterogeneity in genetic characteristics and survival outcomes of synchronous CRC relative to that of solitary CRC.

BRAF Mutation Heterogeneity Detected Using Circulating Tumor DNA Sequencing in Synchronous Colon Cancer: A Case Report.

Synchronous colorectal cancer, which occurs in approximately 4.8-8.4% of all colorectal cancers, has a genetic profile with a higher rate of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and microsatellite instability-high than solitary colorectal cancer. However, little information is available on heterogeneity among tumor lesions because of difficulty in performing genetic tests in all lesions in clinical practice. A 44-year-old man presented with multiple recurrent lung metastases 42 months after the endoscopic resection of early stage synchronous ascending and sigmoid colon cancers. The genetic testing of sigmoid colon cancer tissue samples, their state being more advanced than that of ascending colon cancer, revealed a v-Ki-ras 2 Kirsten rat sarcoma viral oncogene homolog mutation (G13C) and BRAF wild type. However, the tumor was refractory to initial chemotherapy and rapidly progressed to new liver metastases. Therefore, we suspected that there may be biological heterogeneity between the primary sigmoid colon lesion and liver metastases. Next, we performed next-generation sequencing on circulating tumor DNA from the patient's plasma (Foundation One Liquid CDx®), which revealed the V600E mutation of BRAF, suggesting that there was genetic heterogeneity among the synchronized primary lesions, one of which was responsible for the chemo-refractory rapid-growing liver metastases. Genetic profiling with liquid biopsy at the time of recurrence and metastasis may be useful in patients with multiple synchronous cancers because there is less heterogeneity between primary and metastatic sites.

Anticancer effects of Mori radicis cortex, Mori ramulus, Angelica tenuissima, and Inonotus obliquus extract mixtures on B16F10 mur ine melanoma cells via up regulation of p53 and down regulation of Akt expression

Purpose: To investigate the anticancer effects of the extract mixture of Mori radicis cortex, Mori ramulus, Angelica tenuissima, and Inonotus obliquus extract (MRME), a new herbal preparation from Korea, on B16F10 cells.
 Methods: The MRME was extracted with water. Cell Counting Kit-8 (CCK-8) assays and 4′,6- diamidino-2-phenylindole (DAPI) staining were used to investigate the effect of MRME on cell proliferation and apoptosis in vitro. The expression of p53, p-p53, and phosphorylated Akt (p-Akt) were analyzed by western blotting. Wound-healing assay was used to analyze cell migration.
 Results: Based on CCK-8 and DAPI staining results, MRME inhibited the growth of B16F10 cells and induced apoptosis in B16F10 cells in a time- and dose-dependent manner (p < 0.05). MRME increased the expression of p53 and p-p53 and decreased the expression of p-Akt in a dose-dependent manner. MRME treatment inhibited cell migration and invasion (p < 0.05).
 Conclusion: Mori Radicis Cortex Mixture Extract exerts anticancer effect by inducing the expression of p53 and p-p53 and decreasing the expression of p-Akt, which may benefit patients with malignant melanoma when used in combination with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors.