Harboring BRAF V600E Mutation Research Articles

P07.03.A COMPLETE LONG-LASTING RESPONSE TO MULTIMODAL THIRD LINE TREATMENT WITH NEUROSURGICAL RESECTION, CARMUSTINE WAFER IMPLANTATION AND DABRAFENIB PLUS TRAMETINIB IN A BRAFV600E MUTATED HIGH-GRADE GLIOMA

Abstract BACKGROUND BRAF mutations are drivers of oncogenesis in a variety of tumors and they can be successfully targeted with small molecule inhibitors. In primary central nervous system tumors, BRAFV600E mutations are most frequently found in pleomorphic xanthoastrocytoma, ganglioglioma, pilocytic astrocytoma and epithelioid glioblastoma. Dabrafenib is a potent and selective inhibitor of BRAF-mutant kinase that was approved, as monotherapy or in combination with trametinib (mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor), for unresectable or metastatic BRAF-mutated melanoma, advanced non-small cell lung cancer and anaplastic thyroid cancer harboring BRAFV600E mutation. MATERIAL AND METHODS In February 2016, a 21-year-old female was admitted to our Unit for seizures. A brain magnetic resonance imaging (MRI) revealed a frontal contrast enhanced lesion. Neurosurgical resection was performed. The histological analysis diagnosed anaplastic pleomorphic xanthoastrocytoma (PXA) WHO grade III. A volumetric modulated radiotherapy course was delivered for a total dose of 59,4 Gy in 33 fractions with concomitant and adjuvant temozolomide therapy (Stupp protocol). In October 2017, a brain MRI showed relapsing disease. Six courses of chemotherapy with procarbazine, lomustine and vincristine (PCV) were administered, and then neurosurgical partial removal and carmustine wafer implantation were performed. The histological analysis confirmed BRAF V600E mutated anaplastic PXA. RESULTS In April 2019 the 24-year-old female patient with BRAF mutated anaplastic PXA started her third-line therapy with dabrafenib. The well-known tumor residue was less evident on the subsequent MRIs performed every three/four months. Given the literature data of the most effectiveness and the best control of the side effects, in August 2020 the patient was put on combined treatment with dabrafenib plus trametinib. Since December 2021 the residual tumor has not been longer visible. Last MRI evaluation, performed on January 26th 2023, showed no recurrence of the disease, four years after BRAF inhibitor treatment beginning. CONCLUSION Dabrafenib plus trametinib is a promising new therapy for patients affected by BRAFV600E-mutant glioma, with high overall response and manageable toxicity. Due to very poor prognosis, there are a few described cases of high-grade glioma (HGG) patients in third-line therapy, showing complete response and for such a long time. The emergence of optimized sequencing strategies and targeted agents, including multimodal treatment and systemic therapy with dabrafenib plus trametinib, will continue to broaden personalized therapy in HGG and to improve patient outcomes.

Impact of BRAFV600E Mutation on Event-Free Survival in Patients with Papillary Thyroid Carcinoma: A Retrospective Study in a Romanian Population.

We aimed to evaluate the prognostic value of BRAFV600E mutation in a series of 127 papillary thyroid carcinoma (PTC) cases as a single factor, and in synergic interaction with other standard risk factors. BRAFV600E mutation was assessed by real-time PCR. Event-free survival (EFS) was calculated between the date of the first evaluation and the date of occurrence of an adverse event or the date of the last known status. The prevalence of BRAFV600E mutation was 57.2%. The Kaplan-Meier analysis showed a significant reduction of EFS among cases harboring BRAFV600E mutation compared to non-mutated cases (p = 0.010). In addition, BRAFV600E mutation was found to better predict adverse outcomes when associated with the following risk factors: age ≥ 55 years old (p < 0.001), male gender (p < 0.001), conventional (p = 0.005) and tall cell (p = 0.014) histology, tumor size > 40 mm (p = 0.001), extrathyroidal extension (p = 0.001), multifocality (p = 0.001) and lymph node metastasis (p < 0.001). In univariate analysis, a 3.74-fold increased risk for a reduced EFS (p = 0.018) was found for BRAFV600E-mutated cases, but no increased risk was further confirmed by multivariate analysis. Our results highlight that BRAFV600E mutation cannot be used alone as an independent predictive factor in PTC patients, but is prognostically valuable if integrated in the context of other clinicopathological risk factors.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion

BackgroundThe mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival.MethodsRSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses.ResultsIn line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing.ConclusionsCollectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

A phase 2 study of HLX208, a BRAFV600E inhibitor, in adult patients with Langerhans cell histiocytosis and/or Erdheim-Chester disease harboring BRAFV600E mutation.

7574 Background: HLX208 is a novel BRAFV600E inhibitor, exhibiting excellent antitumor efficacy in preclinical studies. This phase 2 study aimed to evaluate the efficacy, safety, and pharmacokinetics of HLX208 in adult patients with Langerhans cell histiocytosis (LCH) and/or Erdheim-Chester disease (ECD) harboring BRAFV600E mutation. Methods: This was a single-arm, open-label, multicenter, phase 2 study. Patients with histologically confirmed LCH and/or ECD were enrolled and received oral administration of HLX208 at 450 mg twice every day. HLX208 was given in 28-day cycles until disease progression, experiencing intolerable toxicity, withdrawal of consent, initiation of new antitumor therapy, or death, whichever occurred first. The primary endpoint was the objective response rate (ORR) assessed by independent review committee (IRC) per PET Response Criteria in Solid Tumors (PERCIST) 1.0. Secondary endpoints included safety, other efficacy measures, and pharmacokinetics of HLX208. Results: As of January 15, 2023, 22 patients with LCH and/or ECD were enrolled. All patients have received at least one dose of HLX208 and were included in the current analysis. The median age was 39 (range 18–69) years. Nine (40.9%) patients were male. 12 (54.5%) patients were diagnosed with LCH, 9 (40.9%) were diagnosed with ECD, and 1 (4.5%) had combined LCH and ECD. 6 (27.3%) patients had single system multifocal disease, while 16 (72.7%) had multisystem disease. The median follow-up duration was 4.8 months (95% CI 2.7–5.7). Among the 10 efficacy evaluable patients, ORR (unconfirmed) assessed by IRC per PERCIST 1.0 was 90.0% (95% CI 55.5–99.7%) (9/10, including 2 complete metabolic response [CMR] and 7 partial metabolic response [PMR]), while that by investigators per PERCIST 1.0 was 100.0% (95% CI 69.2–100.0%) (10/10, including 3 CMR and 7 PMR). Disease control rate was 100.0% (95% CI 69.2–100.0%) as assessed by both IRC and investigators per PERCIST 1.0. The median duration of response, median progression-free survival, and median overall survival were not reached. Of the 22 patients who received the drug, 17 (77.3%) patients experienced treatment-emergent adverse events (TEAEs); 12 (54.5%) reported treatment-related adverse events (TRAEs), most commonly alanine aminotransferase increased (36.4%), aspartate aminotransferase increased (22.7%), γ-glutamyltransferase increased (18.2%), and blood lactate dehydrogenase increased (18.2%). Most TRAEs were grade 1 (27.3%) and 2 (18.2%). Only 2 (9.1%) patients experienced grade ≥3 TRAEs and 1 (4.5%) experienced treatment-related serious adverse event. There were no TEAEs that led to treatment discontinuation or death. Conclusions: In summary, HLX208 was safe and well tolerated, and showed promising efficacy in patients with LCH and/or ECD. Clinical trial information: NCT05092815 .

Secretome Screening of BRAFV600E-Mutated Colon Cancer Cells Resistant to Vemurafenib.

Patients with metastatic colorectal cancer (mCRC) carrying BRAFV600E mutation have worse response to chemotherapy and poor prognosis. The BRAFV600E inhibitor vemurafenib has shown modest efficacy as monotherapy in BRAF-mutated mCRC due to the development of resistance. The aim of this study was to conduct a comparative proteomics profiling of the secretome from vemurafenib-sensitive vs. -resistant colon cancer cells harboring BRAFV600E mutation in order to identify specific secretory features potentially associated with changes in the resistant cells' phenotype. Towards this aim, we employed two complementary proteomics approaches including two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry and label-free quantitative LC-MS/MS analysis. Obtained results pointed to aberrant regulation of DNA replication and endoplasmic reticulum stress as the major secretome features associated with chemoresistant phenotype. Accordingly, two proteins implicated in these processes including RPA1 and HSPA5/GRP78 were discussed in more details in the context of biological networks and their importance as potential secretome targets for further functional and clinical evaluation. Expression patterns of RPA1 and HSPA5/GRP78 in tumor tissues from colon cancer patients were also found in additional in silico analyses to be associated with BRAFV600E mutation status, which opens the possibility to extrapolate our findings and their clinical implication to other solid tumors harboring BRAFV600E mutation, such as melanoma.

Abstract CT031: A first-in-human, phase 1a/1b, open-label, dose-escalation and expansion study to investigate the safety, pharmacokinetics, and antitumor activity of the RAF dimer inhibitor BGB-3245 in patients with advanced or refractory tumors

Abstract Background: BGB-3245 is a RAF dimer inhibitor with preclinical activity in MAPK-altered tumor models harboring BRAF V600 mutations, atypical BRAF mutations/fusions, and RAS mutations. This study is investigating the safety, pharmacokinetics, and preliminary antitumor activity of BGB-3245 in patients (pts) with advanced or refractory MAPK-altered solid tumors. Methods: Eligible pts were ≥18 yrs old with ECOG 0-1 and had solid tumors harboring MAPK pathway alterations. Dose-escalation and cohort-size decisions were made using the Modified Toxicity Probability Interval Design. The starting dose was 5 mg QD. Treatment emergent adverse events (TEAEs) were graded per NCI CTCAE v5.0. Tumor responses were assessed by investigators using RECIST v1.1. Results: As of 1 Sep 2022, 42 pts were treated across 6 cohorts (5-60 mg QD). The median age was 60 yrs; pts had received a median of 3 prior lines of treatment. All pts had TEAEs; 79% had treatment-related (TR) AEs. The most common TRAEs (≥ 10%) were rash acneiform (33%), rash maculopapular (24%), fever (17%), ALT elevations and nausea (both 12%). Gr ≥3 TRAEs were reported in 29% of pts, events in ≥ 2 pts included decreased platelet count and rash maculopapular (3 each), ALT and AST elevations, and fever (2 each). Dose reductions occurred in 5 pts: rhabdomyolysis (1), LVEF decreased (1), hand-foot syndrome (1), rash maculopapular (1), and liver function abnormalities (1). Dose interruptions due to AEs occurred in 60% of pts. Dose discontinuations occurred in 79% of pts, 57% due to disease progression or death, 21% due to AE. Dose limiting toxicities were observed at 10 mg, 40 mg, and 60 mg. 40 mg QD was determined as the MTD. PK results showed generally dose-proportional increases in exposure. Tmax was at ~2 h; BGB-3245 had a long terminal half-life and 7.4-fold average accumulation in exposure at steady-state. 79% of pts were efficacy evaluable. The disease control rate was 48% with 1 CR, 5 cPR, 2 uPR and 8 SD≥24 wks. Objective responders included BRAF V600E melanoma pts post-BRAF/MEK and checkpoint inhibitors (2; 1 CR, 1 cPR), 1 NRAS G12S melanoma and 1 NRAS Q61K melanoma (post checkpoint inhibitors), 1 BRAF V600E LGSOC (progressed on BRAF inhibitor), 1 BRAF V600E cholangiocarcinoma (progressed on BRAF/MEK inhibitors), 1 BRAF K601E/PIK3CA endometrial cancer, and 1 KRAS G12D appendiceal cancer. Preliminary analysis of circulating tumor DNA showed correspondence to clinical response. Conclusions: BGB-3245 has a manageable safety profile and a generally dose-proportional PK. Antitumor activity was observed in pts with no approved targeted therapy options. The safety and early efficacy profile of BGB-3245 supports further investigation in selected MAPK-altered tumors. Citation Format: Alison M. Schram, Vivek Subbiah, Ryan Sullivan, Rasha Cosman, Jia Liu, Eric I. Sbar, Thuy Hoang, Jiarong Chen, Mark Johnson, Vincent Amoruccio, Todd Shearer, Adeela Kamal, Jocelyn Lewis, Wenlin Shao, Badreddin Edris, Lusong Luo, Jayesh Desai. A first-in-human, phase 1a/1b, open-label, dose-escalation and expansion study to investigate the safety, pharmacokinetics, and antitumor activity of the RAF dimer inhibitor BGB-3245 in patients with advanced or refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT031.

Comparative analysis of adjuvant therapy for stage III BRAF-mut melanoma: A real-world retrospective study from single center in China.

BRAF V600 mutation is the most common oncogenic alternation in melanoma and is visible in around 50% of cutaneous and 10%-15% of acral or mucosal subtypes. Currently, immunotherapy with anti-PD-1 blockade and dual-targeted therapy with Dabrafenib plus trametinib (D + T) target therapy have been approved as adjuvant therapies for Stage III melanoma with BRAF V600 mutation. According to their phase III clinical trials, 3-year recurrence-free survival (RFS) is around 60% for both types of treatment. However, early disease control was slightly more effective with targeted therapy than immunotherapy. With different drug approval deadlines in China, anti-PD1 monotherapy, D + T combination, and Vemurafenib (V) monotherapy have all been used in real clinical practice as adjuvant settings for stage III BRAF-mut melanoma in recent years. We conducted this retrospective study to evaluate the efficacy of different treatments in the Chinese melanoma population. Patients who underwent radical surgery and were diagnosed as Stage III melanoma harboring BRAF V600 mutation by pathological report were retrospectively identified at Fudan University Shanghai Cancer Center from January 2017 to December 2021. Patients with mucosal melanoma, or with follow-up of <6 months, or receiving other adjuvant treatment were excluded. Pearson's chi-squared test or Fisher's exact test was performed for univariable analysis of the different adjuvant groups. Log-rank analysis was used to identify prognostic factors for relapse-free survival (RFS). Ninety-three patients with resected stage III melanoma with BRAF V600E mutation were identified in our study, including 25 patients receiving adjuvant anti-PD-1 immunotherapy (PD-1), 25 receiving adjuvant D + T, 23 receiving V, and 20 patients with observation-only (OBS). There were no statistical differences between treatment groups in baseline characteristics including age, gender, subtypes, primary thickness, ulceration, and nodal involvement. Median relapse-free survival (RFS) time was not reached in the D + T group, 15 months in the V group, 15 months in the PD-1 group, and 10 months in the OBS group, respectively. Compared to OBS, all three other groups showed a tendency to benefit from RFS, while only D + T achieved a statistical difference (p = 0.002). However, compared to D + T, anti-PD-1 monotherapy also showed significantly worse relapse control (p = 0.032). For Chinese stage III melanoma with BRAF mutation, both novel targeted therapy and immunotherapy showed potential benefits in relapse-free survival compared to observation only. Dual-targeted D + T therapy may still be the best choice for adjuvant therapy because anti-PD-1 monotherapy has failed to report equivalent efficacy in real-world practice.

Abstract 1173: Landscape of 4,506 pan-cancer samples harboring BRAFV600E mutations, and NTRK/RET Fusions from 137,401 adult patients with cancer: Clinical implications for tissue agnostic therapies

Abstract Background: Biomarker-driven trials have led to multiple FDA tissue-agnostic approvals. We aimed to explore the clinic-genomic landscape of alterations currently actionable by tissue-agnostic targeted therapies. Methods: We mined AACR Project GENIE to extract data about three molecular targets currently on the list of FDA tissue-agnostic approvals (RET fusions, NTRK fusions, and BRAFV600E mutations). Results: Among 137401 patients in GENIE database (153,834 samples), 4506 samples harbored BRAFV600E mutations (3%), 390 had NTRK fusions (0.3%), and 375 had RET fusions (0.2%) (Table 1). Cancers with BRAFV600E mutations included melanoma (n=1278; 28%), CRC (n=1120; 25%), thyroid cancer (n=845; 19%), glioma (n=359; 8%), NSCLC (n=300; 7%); and multiple other tumor types (n=1882; 42%). The most frequent co-occurring alterations were TERT and TP53. NTRK fusions were observed in breast cancer (n=42; 11%), glioma (n=37; 10%), thyroid cancer (n=37; 10%), soft tissue sarcomas (n=34; 9%), and NSCLC (n=32; 8%); among others (n=208; 53%). The most common fusion partners were ETV6, TPM3, and LMNA and most frequent co-occurring mutations were TP53 and TERT. RET fusions were seen in NSCLC (n=208; 56%), thyroid cancer (n=88; 24%), CRC (n=15; 4%), breast cancer (n=11; 3%), and gastroesophageal cancer (n=8; 2%); among others (n=45; 12%). Common fusion partners were were KIF5B, CCDC6, and NCOA4 and most frequent co-occurring alterations were TP53 and SETD2. Conclusions: BRAF, RET and NTRK alterations are prevalent across multiple tumors. Although rare, together they are found in around 3.4% of all human cancers. Further analysis of co-occurring TP53, TERT and SETD2 alterations and their impact on response to targeted therapies is warranted, in order to tailor personalized treatments for patients harboring these alterations. Table 1: Tissue spectrum of activity for anticancer drugs with tissue-agnostic approvals Approved Alteration Most Common Diagnoses Prevalence in Literature Prevalence in Disease Specific Analysis Approved Drug(s) Tissue Agnostic FDA Approval Year Tissue Agnostic ORR from Clinical Trials NTRK Fusions Breast Cancer 0.08-0.13% 0.3% Larotrectinib; Entrectinib 2018 (Larotrectinib); 2019 (Entrectinib) ORR: 75% and DOR: NR (Larotrectinib); ORR: 57% and 12mDOR: 45% (Entrectinib) Glioma 0.55% 0.4% Thyroid Cancer 2.22-2.28% 1.7% Soft Tissue Sarcoma 0.68-1.17% 0.8% NSCLC 0.16-0.23% 0.1% Salivary Gland Cancer 79.6-84.9% 3% CRC 0.26-0.35% 0.2% Gastroesophageal Cancer N/A 0.4% Pancreatic Cancer 0.30-0.34% 0.3% Melanoma 0.36-0.54 0.2% RET Fusions NSCLC 2% 0.9% Selpercatinib 2022 ORR: 44% and DOR: 24.5m Thyroid Cancer 5-10% 4.1% CRC &amp;lt;1% 0.1% Breast Cancer 0% 0.1% Gastroesophageal Cancer 0% 0.2% Carcinoma of Unknown primary 2% 0.1% Glioma n/a 0.04% Prostate Cancer 0% 0.1% Soft Tissue Sarcoma 0% 0.1% Bladder Cancer 0% 0.1% BRAF V600E Mutations Melanoma 26.1% 20.2% Dabrafenib + Trametinib 2022 ORR: 41% and 24mDOR: 44% CRC 10% 7.8% Thyroid Cancer 41% 39.7% Glioma 7% 3.9% NSCLC 1-2% 1.4% Mature B-Cell Neoplasms 1-100% 1.8% Histiocytosis 25.7% 18% Carcinoma of Unknown primary 3% 1.7% Ovarian Cancer 2% 0.9% Non-melanoma Skin Cancer 1% 3.4% NR=not reached; m=month; DOR: Duration of Response; ORR: Objective Response Rate Citation Format: Mohamed A. Gouda, Blessie E. Nelson, Lars Buschhorn, Adam Wahida, Vivek Subbiah. Landscape of 4,506 pan-cancer samples harboring BRAFV600E mutations, and NTRK/RET Fusions from 137,401 adult patients with cancer: Clinical implications for tissue agnostic therapies [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 1173.

Abstract 2693: Combining ulixertinib (ERK1/2 Inhibitor) with EGFR and BRAF inhibition yields significant efficacy in preclinical BRAFV600E mutant colorectal cancer models

Abstract The BRAFV600E mutation occurs in approximately 7% of colorectal cancer (CRC). BRAF plus EGFR inhibition (encorafenib with cetuximab) is an FDA approved treatment option for adult patients with metastatic CRC. Ultimately, patients develop resistance leading to disease progression. The addition of a MEK inhibitor, binimetinib, did not confer overall survival benefit. We hypothesized that addition of ERK inhibition would increase magnitude and duration of response to BRAF plus EGFR inhibition by overcoming MAPK-related acquired resistance mechanisms. ERK is the terminal master regulator kinase of the RAS-MAPK pathway, therefore, targeting this node is likely to be effective in the context of numerous mechanisms of acquired resistance that reactivate the MAPK pathway. Ulixertinib (BVD-523) is a first-in-class and best-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other therapeutics. Ulixertinib has demonstrated efficacy in patients with tumors harboring alterations within the RAS-MAPK pathway. The efficacy of ulixertinib, in combination with EGFR inhibition (cetuximab), plus BRAF inhibition (encorafenib), was assessed in CRC cell line derived xenograft models harboring BRAFV600E mutations. The triple combination resulted in superior tumor growth inhibition compared to dosing of any single agent or doublet. RNA sequencing was performed on treated tumor samples. Expression of the mutant BRAF allele was readily confirmed from RNA sequencing data. Gene expression analysis showed differential expression of MAPK pathway genes in triple combination treated groups versus single or doublet therapy. A complete response to ulixertinib in combination with cetuximab and encorafenib for the treatment of a patient with metastatic CRC has been reported (Stuhlmiller, Timothy Joseph, et al. "Updated clinical outcomes from ULI-EAP-100, an intermediate expanded access program for ulixertinib (BVD-523)." (2022): e15101-e15101). The patient was treated under the Expanded Access Protocol (NCT04566393) for compassionate use access to ulixertinib. Preclinical data and clinical complete response warrants further investigation of ulixertinib plus BRAF and EGFR inhibition. Citation Format: Deborah Knoerzer, Anupama Reddy, Jessica A. Box, Anna Groover, Brent Kreider, Martin Teresk, Caroline M. Emery. Combining ulixertinib (ERK1/2 Inhibitor) with EGFR and BRAF inhibition yields significant efficacy in preclinical BRAFV600E mutant colorectal cancer models [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 2693.

BRAF v600E–mutant cancers treated with vemurafenib alone or in combination with everolimus, sorafenib, or crizotinib or with paclitaxel and carboplatin (VEM-PLUS) study

Combined BRAF + MEK inhibition is FDA approved for BRAF V600E-mutant solid tumors except for colorectal cancer. However, beyond MAPK mediated resistance several other mechanisms of resistance such as activation of CRAF, ARAF, MET, P13K/AKT/mTOR pathway exist among other complex pathways. In the VEM-PLUS study, we performed a pooled analysis of four phase one studies evaluating the safety and efficacy of vemurafenib monotherapy and vemurafenib combined with targeted therapies (sorafenib, crizotinib, or everolimus) or carboplatin plus paclitaxel in advanced solid tumors harboring BRAF V600 mutations. When vemurafenib monotherapy was compared with the combination regimens, no significant differences in OS or PFS durations were noted, except for inferior OS in the vemurafenib and paclitaxel and carboplatin trial (P = 0.011; HR, 2.4; 95% CI, 1.22–4.7) and in crossover patients (P = 0.0025; HR, 2.089; 95% CI, 1.2–3.4). Patients naïve to prior BRAF inhibitors had statistically significantly improved OS at 12.6 months compared to 10.4 months in the BRAF therapy refractory group (P = 0.024; HR, 1.69; 95% CI 1.07–2.68). The median PFS was statistically significant between both groups, with 7 months in the BRAF therapy naïve group compared to 4.7 months in the BRAF therapy refractory group (P = 0.016; HR, 1.80; 95% CI 1.11–2.91). The confirmed ORR in the vemurafenib monotherapy trial (28%) was higher than that in the combination trials. Our findings suggest that, compared with vemurafenib monotherapy, combinations of vemurafenib with cytotoxic chemotherapy or with RAF- or mTOR-targeting agents do not significantly extend the OS or PFS of patients who have solid tumors with BRAF V600E mutations. Gaining a better understanding of the molecular mechanisms of BRAF inhibitor resistance, balancing toxicity and efficacy with novel trial designs are warranted.

Papillary thyroid cancer organoids harboring BRAFV600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

BackgroundsPapillary thyroid cancer (PTC), which is often driven by acquired somatic mutations in BRAF genes, is the most common pathologic type of thyroid cancer. PTC has an excellent prognosis after treatment with conventional therapies such as surgical resection, thyroid hormone therapy and adjuvant radioactive iodine therapy. Unfortunately, about 20% of patients develop regional recurrence or distant metastasis, making targeted therapeutics an important treatment option. Current in vitro PTC models are limited in representing the cellular and mutational characteristics of parental tumors. A clinically relevant tool that predicts the efficacy of therapy for individuals is urgently needed.MethodsSurgically removed PTC tissue samples were dissociated, plated into Matrigel, and cultured to generate organoids. PTC organoids were subsequently subjected to histological analysis, DNA sequencing, and drug sensitivity assays, respectively.ResultsWe established 9 patient-derived PTC organoid models, 5 of which harbor BRAFV600E mutation. These organoids have been cultured stably for more than 3 months and closely recapitulated the histological architectures as well as mutational landscapes of the respective primary tumors. Drug sensitivity assays of PTC organoid cultures demonstrated the intra- and inter-patient specific drug responses. BRAFV600E inhibitors, vemurafenib and dabrafenib monotherapy was mildly effective in treating BRAFV600E-mutant PTC organoids. Nevertheless, BRAF inhibitors in combination with MEK inhibitors, RTK inhibitors, or chemotherapeutic agents demonstrated improved efficacy compared to BRAF inhibition alone.ConclusionsThese data indicate that patient-derived PTC organoids may be a powerful research tool to investigate tumor biology and drug responsiveness, thus being useful to validate or discover targeted drug combinations.

Implications of BRAF V600E mutation in gliomas: Molecular considerations, prognostic value and treatment evolution.

Gliomas are molecularly heterogeneous brain tumors responsible for the most years of life lost by any cancer. High-grade gliomas have a poor prognosis and despite multimodal treatment including surgery, radiotherapy, and chemotherapy, exhibit a high recurrence rate. There is a need for new therapeutic approaches based on precision medicine informed by biomarker assessment and BRAF, a key regulator of MAPK signaling pathway, influencing cell differentiation, proliferation, migration and pro-tumorigenic activity, is emerging as a promising molecular target. V600E, is the most frequent BRAF alteration in gliomas, especially in pediatric low-grade astrocytomas, pleomorphic xanthoastrocytoma, papillary craniopharyngioma, epithelioid glioblastoma and ganglioglioma. The possible application of BRAF-targeted therapy in gliomas is continuously growing and there is preliminary evidence of prolonged disease control obtained by BRAF inhibitors in tumors harboring BRAF V600E mutation. The possibility of introducing targeted therapies into the treatment algorithm represents a paradigm shift for patients with BRAF V600E mutant recurrent high-grade and low-grade glioma and BRAF routine testing should be considered in clinical practice. The focus of this review is to summarize the molecular landscape of BRAF across glioma subtypes and the novel therapeutic strategies for BRAF V600E mutated tumors.

IL-5 Targeted Therapies for Nervous System Tumors – Progress, Pitfalls, and Prospects

Abstract The molecular genetic understanding of nervous system tumors has expanded rapidly offering insight into potentially targetable oncogenic driver alterations. In monogenic tumor susceptibility syndromes such as tuberous sclerosis, neurofibromatosis type 1, and von Hippel Lindau syndrome targeted drugs have demonstrated efficacy and are now approved in the United States for the treatment of subependymal giant cell astrocytoma, plexiform neurofibroma, and hemangioblastoma, respectively. Some genetic alterations like BRAF V600E mutations or neurotrophic receptor tyrosine kinase (NTRK) fusions, occur across multiple cancers, including certain types of gliomas. Dabrafenib in combination with trametinib is approved for use in gliomas that harbor BRAF V600E mutations and larotrectinib is approved in gliomas that harbor certain NTRK fusions. In pediatric low-grade astrocytoma, one of the most common solid tumors of childhood, BRAF truncation-fusion events may render these tumors susceptible to tovorafenib, a type 2 RAF inhibitor, which is under study in clinical trials. Mutations in the isocitrate dehydrogenase (IDH) gene occur in the majority of low-grade and anaplastic gliomas, create specific metabolic vulnerabilities, and several IDH-mutant targeting strategies are under investigation. Glioblastoma is characterized by intratumoral molecular heterogeneity so strategies that target single genetic alterations may not be as effective as in other types of gliomas although a number of novel targeted strategies have been advanced to clinical trials. Other types of nervous system tumors such as medulloblastoma, meningioma, primary central nervous system diffuse large B-cell lymphoma, and craniopharyngioma harbor targetable genetic alterations and clinical trials are ongoing. Finally, metastatic brain tumors may harbor targetable genetic alterations that may differ from the primary site of cancer highlighting the importance of biopsy and molecular analysis of the brain metastases in order to enroll patients in clinical trials of targeted therapies.

Mutational Status and Clinical Outcomes Following Systemic Therapy with or without Focal Radiation for Resected Melanoma Brain Metastases

Brain metastases occur frequently in advanced melanoma and traditionally require surgery and radiation therapy. New evidence demonstrates that systemic therapies are effective for controlling metastatic melanoma brain metastases. This study evaluated outcomes after resection of melanoma brain metastases treated with systemic therapy, with or without focal radiotherapy. All patients received immunotherapy or BRAF/MEK inhibitors preoperatively or in the immediate 3 months postoperatively. Resection cavity failure, distant central nervous system progression, and adverse radiation effects were reported in the presence and absence of focal radiotherapy using the Kaplan-Meier method. Between 2011 and 2020, 37 resection cavities in 29 patients met criteria for analysis. Of lesions, 22 (59%) were treated with focal radiotherapy, and 15 (41%) were treated with targeted therapy or immunotherapy alone. The 12- and 24-month freedom from local recurrence was 64.8% (95% confidence interval [CI] 42.1%-99.8%) and 46.3% (95% CI 24.5%-87.5%), respectively, for systemic therapy alone and 93.3% (95% CI 81.5%-100%) at both time points for focal radiotherapy (P= 0.01). On univariate analysis, focal radiotherapy was the only significant factor associated with reduction of local recurrence risk (hazard ratio 0.10, 95% CI 0.01-0.85; P= 0.04). There were no significant differences in central nervous system progression-free survival or overall survival between patients who received systemic therapy plus focal radiotherapy compared with systemic therapy alone. BRAF mutation status was reviewed for either the brain metastasis (n= 9 patients, 31%) or the primary site (n= 20 patients, 69%), and patients harboring BRAFV600E mutations had worse progression-free survival (P= 0.043). Focal radiotherapy with systemic therapy for resected melanoma brain metastases significantly decreased resection cavity recurrence compared with systemic therapy alone. BRAF mutation status correlated with poorer outcomes.

Response to BRAF-targeted Therapy Is Enhanced by Cotargeting VEGFRs or WNT/β-Catenin Signaling in BRAF-mutant Colorectal Cancer Models.

The fact that 10% of colorectal cancer tumors harbor BRAF V600E mutations suggested targeting BRAF as a potential therapy. However, BRAF inhibitors have only limited single-agent efficacy in this context. The potential for combination therapy has been shown by the BEACON trial where targeting the EGF receptor with cetuximab greatly increased efficacy of BRAF inhibitors in BRAF-mutant colorectal cancer. Therefore, we explored whether efficacy of the mutant BRAF inhibitor vemurafenib could be enhanced by cotargeting of either oncogenic WNT/β-catenin signaling or VEGFR signaling. We find the WNT/β-catenin inhibitors pyrvinium, ICG-001 and PKF118-310 attenuate growth of colorectal cancer cell lines in vitro with BRAF-mutant lines being relatively more sensitive. Pyrvinium combined with vemurafenib additively or synergistically attenuated growth of colorectal cancer cell lines in vitro. The selective and potent VEGFR inhibitor axitinib was most effective against BRAF-mutant colorectal cancer cell lines in vitro, but the addition of vemurafenib did not significantly increase these effects. When tested in vivo in animal tumor models, both pyrvinium and axitinib were able to significantly increase the ability of vemurafenib to attenuate tumor growth in xenografts of BRAF-mutant colorectal cancer cells. The magnitude of these effects was comparable with that induced by a combination of vemurafenib and cetuximab. This was associated with additive effects on release from tumor cells and tumor microenvironment cell types of substances that would normally aid tumor progression. Taken together, these preclinical data indicate that the efficacy of BRAF inhibitor therapy in colorectal cancer could be increased by cotargeting either WNT/β-catenin or VEGFRs with small-molecule inhibitors.

1657P Clinical and molecular profiles of radioiodine refractory differentiated thyroid cancer

At present, identification of radioiodine (RAI) refractory differentiated thyroid cancer (RAIR-DTC) is mainly based on the clinical characteristics of the lesions rather than the pathological changes. Here, we reported preliminary results of the genomic characteristics underlying RAIR-DTC. The formalin-fixed paraffin-embedded samples of 94 patients diagnosed as DTC were sequenced in a 24-gene commercial panel (ThyroLeadTM). Microsatellite instability (MSI) was detected with the MSIsensor2 tool (MSI-H was defined as score greater than 20%). 43 of them were identified as RAIR-DTC by manual inspection according to the 2015 American Thyroid Association guidelines. Among 94 patients, 73 patients (77.7%) had distant metastases, to the lungs in 93.3%, bone in 20.6%, both sites in 16.5%, brain in 6.9%, and liver in 1.4%. Overall, a higher rate of being RAIR-DTC is associated with older age at diagnosis (p=0.0059). In consistent with previous studies, it was found that BRAF V600E (p=0.046) and TERT promoter (p =0.0017) mutation were associated with worse RAI uptake. Of 43 RAIR-DTC cases, tumor from 25 patients (58.1%) harbored BRAF V600E mutations, 16 (37.2%) had TERT promoter mutation, 6 (14.0%) had TP53 mutation and 3 (7.0%) had KRAS mutation. Concomitant BRAF V600E and TERT promoter mutations were found in 2 patients, and all of them were in the RAIR stage. Meanwhile, 29 (67.4%) of RAIR-DTC cases harbored mutations in the RAS/RAF pathway. In clinical aspects, gene fusion is related to the younger age at diagnosis (standard deviation = 14, p＜0.001), while without statistical significance in indicating RAIR-DTC. Only 1 RAIR-DTC patient had an MSI-H result, and the rest exhibited microsatellite stability. Here we illustrated several factors associated with RAI uptake. Further stratification study is required to establish a more detailed profiling of RAI uptake, to reduce the overtreatment of RAIR-DTC.

Abstract IA25: Clinical trials and molecular therapies in iCCA

Abstract Cisplatin and gemcitabine (CisGem) has been the stadard of care for patients presenting with advanced iCCA for over a decade. Intensified (triplet) chemotherapy remains investigational except in Japan, where the CisGem and S1 is an option. Robust data has shown that 5FU-based, second-line, combination chemotherapy has modest activity in a molecularly-unselected population. An improved understanding of the biology underpinning iCCA has led to the identification of molecular alterations linked to targeted therapies. The most prominent alterations are mutations in IDH1 and FGFR2 fusions; both of these are addressed later in the session. This presentation will highlight other selected subgroups, including patients whose tumours are harbouring BRAF-V600E mutations, HER2 amplification/over-expression, and NTRK fusions as examples, although additional groups continue to emerge. Cholangiocarcinoma is recognised to have an underlying immune predisposition, in selected cases. Harnessing the immune response has previously provided limited signals of efficacy. However, the TOPAZ-1 study, a randomised phase III study has recently shown an incremental survival benefit in patients with biliary tract cancer with the addition of durvalumab to CisGem chemotherapy, including intrahepatic cholangiocarcinoma. No predictive factors are known to enrich for patients most likely to benefit from immunotherapy to date. Additional analysis of patient subgroups in TOPAZ-1 as well as the results of the KEYNOTE-966 phase III study (CisGem +/- pembrolizumab) are eagerly awaited. Citation Format: Juan W. Valle. Clinical trials and molecular therapies in iCCA [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA25.

Discovery of BRAF/HDAC Dual Inhibitors Suppressing Proliferation of Human Colorectal Cancer Cells.

The combination of histone deacetylase inhibitor and BRAF inhibitor (BRAFi) has been shown to enhance the antineoplastic effect and reduce the progress of BRAFi resistance. In this study, a series of (thiazol-5-yl)pyrimidin-2-yl)amino)-N-hydroxyalkanamide derivatives were designed and synthesized as novel dual inhibitors of BRAF and HDACs using a pharmacophore hybrid strategy. In particular, compound 14b possessed potent activities against BRAF, HDAC1, and HDAC6 enzymes. It potently suppressed the proliferation of HT-29 cells harboring BRAFV600E mutation as well as HCT116 cells with wild-type BRAF. The dual inhibition against BRAF and HDAC downstream proteins was validated in both cells. Collectively, the results support 14b as a promising lead molecule for further development and a useful tool for studying the effects of BRAF/HDAC dual inhibitors.

Abstract 2422: Migration of V600E-expressing cells is suppressed by ERK-dependent feedback inhibition of RAC1 and rescued in melanoma by secondary mutations

Abstract Oncogenic activation of ERK occurs in as many as 50% of human tumors and in the majority of melanomas. Activated BRAF V600E hyperactivates ERK signaling and causes ERK dependent dysregulation of proliferation. However, BRAF V600E alone is insufficient to drive melanoma formation in genetically engineered mice models. Moreover, 82% of benign nevi harbor BRAF V600E mutations. In contrast, in BRAF V600E/PTEN-/- GEMM models, rapid onset of aggressive and disseminated melanomas occurs. We show here that cell migration and invasion of melanocytes expressing BRAFV600E is depressed in vitro and in vivo and significantly accelerated by pharmacologic inhibition of ERK signaling or by deletion of PTEN. Upon ERK inhibition, cells lose their cell-cell junctions and become single cells with actin rich protrusions and a mesenchymal morphology. ERK pathway activation affects each step of the migration cycle, impeding cell spreading, assembly of stress fibers, and focal contact formation, all of which are associated with decreased mobility and invasion. Hyperactivation of ERK signaling by BRAF V600E has been shown to cause potent feedback inhibition of RTK signaling and wild type RAS. We now show that this is associated with feedback inhibition of RAC1, a RAS-like small GTPase required for cell migration. Moreover, ERK pathway inhibition reactivates RAC in an RTK-dependent manner. Consistent with these findings, expression of RAC1 P29S, an activating mutant, and overexpression of PREX2, an activator of RAC1, both rescue migration in BRAF V600E cells, as does loss of PTEN expression, which converges with RAC1 activation downstream. Taken together, these data suggest that whereas hyperactivation of ERK signaling by BRAFV600E dysregulates proliferation, ERK dependent feedback inhibition of RAC1 inhibits cell mobility and invasiveness and prevents full transformation. Selection of secondary genetic events that overcome or bypass RAC1 feedback is thus required for full transformation. In support of this view, RAC1 mutation, PTEN loss, or PREX2 amplification occurs in 38% of human melanomas. Thus, oncogene induced feedback may be deleterious and directs tumor evolution by causing selection of mutations that restore functions necessary for transformation Citation Format: Sunyana Gadal, Jacob A. Boyer, Hongyan Li, Ning Fan, Eric Chan, Yevgeniy Romin, Elisa De Stanchina, Rona Yeager, Marcus Bosenberg, Katia M. Todorova, Neal Rosen. Migration of V600E-expressing cells is suppressed by ERK-dependent feedback inhibition of RAC1 and rescued in melanoma by secondary mutations [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 2422.

LGG-06. Selumetinib in pediatric patients with non-neurofibromatosis type 1-associated, non-optic pathway (OPG) and non-pilocytic recurrent/progressive low-grade glioma harboring BRAFV600E mutation or BRAF-KIAA1549 fusion: a multicenter prospective Pediatric Brain Tumor Consortium (PBTC) Phase 2 trial

BACKGROUND: A greater understanding of the Ras-MAP kinase pathway in pediatric low-grade glioma (LGG) paired with the availability of selective inhibitors has enhanced the ability to target this pathway with therapeutic intent. METHODS: The PBTC conducted a multi-institutional phase II study (NCT01089101) evaluating selumetinib (AZD6244, ARRY-142886), a MEK I/II inhibitor, in children with recurrent/progressive LGG assigned to 6 strata and treated at a dose of 25 mg/m2/dose PO BID for up to two years. Here we present stratum 5 which enrolled children without NF1, non-OPG and non-pilocytic LGG harboring either a BRAFV600E mutation or BRAF-KIAA1549 fusion. RESULTS: Twenty-four of 25 children enrolled were eligible; 23 were evaluable for the primary radiologic response endpoint. Enrollment stopped early due to slow accrual and initiation of COG ACNS1931. The most common histologies were ganglioglioma (42%) and astrocytoma NOS (33%). Thirteen tumors (54%) had BRAF-KIAA1549 fusion; 11 (46%) had the BRAFV600E mutation. Five of 23 (22%) evaluable patients achieved a centrally confirmed partial response (PR), 12 (52%) had stable disease and 6 (26%) had progression with a 2-year progression-free survival of 75 + 9%. Four of 11 (36%) patients with a BRAFV600E mutation and 1/12 (8%) with a BRAF-KIAA1549 fusion achieved a PR. The 2-year PFS did not significantly differ between tumors with BRAFV600E mutation (82 + 12%) versus BRAF-KIAA1549 fusion (68 + 13%) (n=24, p=0.548). No patient remains on therapy. The most common attributable toxicities were grade 1/2 ALT/AST elevation, dry skin and leukopenia. Rare grade 3/4 toxicities included elevated CPK, rash, paronychia, fever, weight gain and sinus tachycardia. CONCLUSIONS: Despite lower than planned accrual, selumetinib met the design threshold for success in treating children with recurrent/progressive non-pilocytic, non-OPG LGG without NF1 that harbored the common BRAF aberrations. Ongoing phase 3 prospective studies will better determine the role of this agent in this population.