BRAF Wt Research Articles

Plasma RAS dynamics and anti-EGFR rechallenge efficacy in patients with RAS/BRAF wild-type metastatic colorectal cancer: REMARRY and PURSUIT trials.

3518 Background: Rechallenge with anti-EGFR monoclonal antibody (EGFR mAb) showed certain activities in patients (pts) with RAS/ BRAF V600E wild-type (wt) metastatic colorectal cancer (mCRC), particularly in pts with negative plasma RAS (p RAS) mutation by circulating-tumor DNA (ctDNA) assay at ‘just before’ the rechallenge therapy. However, the efficacy is unknown in pts with RAS/ BRAF wt mCRC whose p RAS was converted to positive once during or after EGFR mAb. Therefore, we conducted REMARRY, a prospective longitudinal study to investigate the p RAS dynamics, and PURSUIT trials, a phase II trial to investigate the efficacy of EGFR mAb rechallenge in pts with p RAS wt just before rechallenge therapy. Methods: The eligibility criteria of REMARRY trial included RAS/ BRAF V600E wt mCRC; ECOG PS 0-1; CR or PR during EGFR mAb; and a refractory ≤ 2 months from the last administration of EGFR mAb. p RAS status by the BEAMing method was prospectively monitored at timepoints of progression on EGFR mAb and each subsequent therapy. The eligibility criteria of PURSUIT trial included enrollment in the REMARRY trial with confirmed p RAS wt prior to enrollment in PURSUIT trial; being refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; and ≥ 4 months of EGFR mAb-free interval. Study treatment was rechallenge with panitumumab + irinotecan (6 mg/kg + 150 mg/m2 q2wks). Primary endpoint was a confirmed objective response rate (ORR) according to RECIST v1.1. The required number of pts was 45, with a null ORR of 10%, an expected ORR of 25%, power of 85%, and one-sided α of 0.05. Results: Between May 2019 and May 2021, 183 pts with 343 timepoints (median, 2) were enrolled in REMARRY trial from 27 institutions, and 50 pts were enrolled in PURSUIT trial; median age, 68 years; left-sided primary, 44 pts; prior EGFR mAb, 1st/2nd/≥3rd lines was 28/6/16 pts; and p RAS status at progression on prior EGFR mAb, wt/mutant (mt)/unknown in 31/7/12 pts. Confirmed ORR and disease control rate were 14% (90% CI, 7.8%–23.9%) and 80% (95% CI, 67.0%–88.8%), respectively. In addition, 4 pts showed an unconfirmed PR. With a median follow-up time of 8.7 months, median progression-free survival was 3.6 months (95% CI, 3.0 – 4.7 months). The subgroup analysis showed a significantly higher confirmed ORR in pts with a longer EGFR mAb-free interval than a shorter one (> vs. < 365 days, 44.4% vs. 7.3%, p = 0.0037). Without any unexpected safety signals, 58.5% of pts had ≥ grade 3 adverse events. Of 31 patients with wt p RAS at progression on prior EGFR mAb, 5 had a confirmed response (ORR, 16%), whereas no response was observed in patients with 7 p RAS mt (ORR, 0%) (p = 0.25). Conclusions: The primary endpoint of confirmed ORR was not met; however, pts with p RAS wt at progression on prior EGFR mAb may benefit from rechallenge with, implying a lack of or worse response if p RAS becomes positive even once during or after EGFR mAb. Clinical trial information: REMARRY: UMIN000036424 PURSUIT: jRCTs031190096.

Design, synthesis and anticancer activity of novel 2-arylbenzimidazole/2-thiopyrimidines and 2-thioquinazolin-4(3H)-ones conjugates as targeted RAF and VEGFR-2 kinases inhibitors

In the current study, series of 2-arylbenzimidazole-thiopyrimidine and -thioquinazolin-4(3H)-ones conjugates 12a-d, 13a,b and 14a-l have been synthesized. All the synthesized compounds were tested in vitro for their anticancer activities against a panel of cancer cell lines at NCI - US and their growth inhibition (GI) % were determined at 10 µM. Compounds 14c and 14g-i were selected to be screened at the five dose assay and were found to exhibit GI50 values 1.1–30.0 µM. The benzimidazole-quinazolinone derivative 14c, in particular, showed potent anticancer activity against the tested cancer cell lines (GI50 of 1.3–4.2 µM). In addition, compounds 12a,b, 13a, 14a-e, 14g, 14i and 14j were selected to be tested against some cancer cell lines using MTT assay and the benzimidazole-quinazolinone 14g was found to have potent anticancer activities against melanoma (Mel-501 and A-375), breast (MCF-7), colon (HCT-116), prostate (PC-3), lung (A-549) and pancreas (Paca-2) cancer cell lines reporting IC50 values ranging between 0.1 and 6.2 µM. Moreover, the synthesized hybrids were tested in vitro on kinases; BRAF (wt), BRAF (V600E), CRAF and VEGFR-2. The benzimidazole-quinazolinone derivatives 14f,g revealed potent RAF kinases inhibitory activities on BRAF (wt), BRAF (V600E) and CRAF showing IC50 values 0.002–0.1 µM, whereas, the benzimidazole-quinazolinone derivatives 14i and 14k showed moderate VEGFR-2 inhibitory activity (IC50 = 20.60 and 6.14 µM, respectively). Moreover, the representative compounds 14g and 14i caused cell cycle arrest of A-375 melanoma cell line at G2/M phase and were found to induce late apoptosis. CRAF in the DFG-out inactive conformation homology modeling was first reported in this study and molecular docking studies on BRAF, CRAF and VEGFR-2 were also performed to investigate the binding modes of the target compounds and their interactions with the key amino acids; BRAF (Glu500, Cys531 and Asp593), CRAF (Glu393, Cys424 and Asp486) and VEGFR-2 (Glu885, Cys919 and Asp1046).

BRAF Modulates Lipid Use and Accumulation.

Simple SummaryBRAF is a serine/threonine kinase that is commonly mutated across cancers. The BRAF V600E mutation is targetable with kinase inhibitors; however, many patients eventually develop resistance. Recent evidence suggests that tumors harboring BRAF mutations may oxidize fatty acids for energy rather than utilizing aerobic glycolysis (the Warburg effect). Understanding the metabolism of cells harboring BRAF mutations may uncover targets to improve therapy response. We studied the effects of BRAF mutation and expression on metabolism. We found that cell expressing BRAF V600E were enriched with immunomodulatory lipids and have a metabolism that is distinct from cells expressing wild type BRAF. We also found that patients with melanoma who did not respond to BRAF-targeted therapy had plasma lipid profiles that were different from patients who responded to this therapy. Overall, our findings indicate that targeting lipid metabolism may be a potential alternative strategy to improve patient responses to BRAF-targeted therapies.There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.

Tumors carrying BRAF-mutations over-express NAMPT that is genetically amplified and possesses oncogenic properties

BackgroundNicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways.MethodsExploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose–response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself.ResultsThe first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT.ConclusionsOverall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.

Tumor mutational burden and somatic mutation status to predict disease recurrence in advanced melanoma.

Tumor mutational burden (TMB) has recently been identified as a biomarker of response to immune checkpoint inhibitors in many cancers, including melanoma. Co-assessment of TMB with inflammatory markers and genetic mutations may better predict disease outcomes. The goal of this study was to evaluate the potential for TMB and somatic mutations in combination to predict the recurrence of disease in advanced melanoma. A retrospective review of 85 patients with stage III or IV melanoma whose tumors were analyzed by next-generation sequencing was conducted. Fisher's exact test was used to assess differences in TMB category by somatic mutation status as well as recurrence locations. Kaplan-Meier estimates and Cox-proportional regression model were used for survival analyses. The most frequently detected mutations were TERT (32.9%), CDKN2A (28.2%), KMT2 (25.9%), BRAF V600E (24.7%), and NRAS (24.7%). Patients with TMB-L + BRAFWT status were more likely to have a recurrence [hazard ratio (HR), 3.43; confidence interval (CI), 1.29-9.15; P = 0.01] compared to TMB-H + BRAF WT. Patients with TMB-L + NRASmut were more likely to have a recurrence (HR, 5.29; 95% CI, 1.44-19.45; P = 0.01) compared to TMB-H + NRAS WT. TMB-L tumors were associated with local (P = 0.029) and in-transit (P = 0.004) recurrences. Analysis of TMB alone may be insufficient in understanding the relationship between melanoma's molecular profile and the body's immune system. Classification into BRAFmut, NRASmut, and tumor mutational load groups may aid in identifying patients who are more likely to have disease recurrence in advanced melanoma.

Clinical evolution after surgery of hepatic metastases of colorectal cancer according to genomic profile.

188 Background: Hepatic metastatic disease from colorectal cancer (CRC) is a significant clinical problem, as the liver is the dominant metastatic site for patients with CRC and 25% of patients will have liver affection at diagnosis. Due to improvement in systemic therapy and locoregional treatments in the last decade, survival has increased significantly. In this regard, it is key to be able to establish several prognostic factors that significantly influence survival. Methods: We carried out a retrospective analysis of 554 patients with mCRC treated at the Gregorio Marañon Hospital (Madrid, Spain) between January 2010 and 2021. We analyzed the clinical and molecular characteristics of patients undergoing liver metastasis surgery as first metastasis surgery together with relapse patterns to progression. Results: Out of our cohort of 554 patients with mCRC we identified 169 patients that underwent liver metastasis surgery as 1st surgery, achieving a media of survival of 56.38 months [95% CI, 44.21-73.78 months]. Regarding the clinical characteristics of the population, the majority were men (63,91%) and had a PS 0-1 (90,5%); and 46 patients (27,2%) were more than 70 years old. In relation to the location of the primary tumour, 46 patients (27,2%) had it n the right colon and 120 in the left colon (71,0%). And 43 patients (25,4%) had extrahepatic disease at the time of surgery. Regarding the biomakers, we indentified the following mutations: 68 mutated KRAS (40,2%), 5 mutated NRAS (2,9%), 9 mutated BRAF (5,3%), 13 mutated PI3K (7,6%), 1 HER2 amplification (0,5%) and 4 with IMS phenotype (2,3%). After the metastasis surgery, progression was mainly hepatic (50,3%), followed by pulmonary (24,8%), peritoneal (11,8%), lymph node (12%), bones (4,7%) and cerebral (1,1%), without having significant differences in relapse patterns at the statistics when analyzed by genomic profile. When analyzing progression-free survival (PFS) and overall survival (OS) according to the genomic profile, in the BRAF mutated vs BRAF WT population, no statistically significant differences were found, obtaining therefore an evident benefit. Furthermore, we found significant differences in OS for patients with right vs left primary tumour as well as for patients with extrahepatic involvement at the time of surgery. Conclusions: Patients undergoing sequential metastatic surgery have a long survival, so it is important to analyse patterns of relapse and clinical course. There is no evidence of significant differences in the progression patterns according to the mutational status of the mCRC; but selected patients with BRAF mutations may obtain benefit in PFS and OS with locoregional approaches to their liver disease.

Fibroblasts Influence Metastatic Melanoma Cell Sensitivity to Combined BRAF and MEK Inhibition.

Simple SummaryPreclinical 3D in vitro coculture models are known to be more complex systems than monolayer cell culture and mimic the physiological environment more closely. Three-dimensional dermal equivalents provide a relevant environment for cutaneous metastatic melanoma cells and are capable of modulating a cancer cell’s response to drugs. We showed that a combined targeted therapy (vemurafenib and cobimetinib) efficiently inhibits cell proliferation and induces apoptosis, especially in the 3D coculture model. A cancer-associated fibroblast population isolated from a cutaneous melanoma was also sensitive to the treatment but with no detectable induction of apoptosis. To better understand the complex crosstalk between melanoma cells and their microenvironment, we compared the influence of conditioned media obtained from healthy or cancer-associated fibroblasts on the response of metastatic melanomas to the drugs. Our data indicate that normal fibroblast supernatants potentialize the therapy’s efficiency, whereas cancer-associated fibroblast secretomes favor melanoma cell survival.The sensitivity of melanoma cells to targeted therapy compounds depends on the tumor microenvironment. Three-dimensional (3D) in vitro coculture systems better reflect the native structural architecture of tissues and are ideal for investigating cellular interactions modulating cell sensitivity to drugs. Metastatic melanoma (MM) cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt) were cultured as a monolayer (2D) or cocultured on 3D dermal equivalents (with fibroblasts) and treated with a BRAFi (vemurafenib) combined with a MEK inhibitor (MEKi, cobimetinib). The drug combination efficiently inhibited 2D and 3D MM cell proliferation and survival regardless of their BRAF status. Two-dimensional and three-dimensional cancer-associated fibroblasts (CAFs), isolated from a cutaneous MM biopsy, were also sensitive to the targeted therapy. Conditioned media obtained from healthy dermal fibroblasts or CAFs modulated the MM cell’s response differently to the treatment: while supernatants from healthy fibroblasts potentialized the efficiency of drugs on MM, those from CAFs tended to increase cell survival. Our data indicate that the secretory profiles of fibroblasts influence MM sensitivity to the combined vemurafenib and cobimetinib treatment and highlight the need for 3D in vitro cocultures representing the complex crosstalk between melanoma and CAFs during preclinical studies of drugs.

511TiP A phase III, multicenter, open-label, randomized study to assess the efficacy and safety of cetuximab plus capecitabine versus cetuximab as maintenance treatment following first-line induction treatment with FOLFOX and cetuximab in Chinese patients with RAS and BRAF WT mCRC

Considering the efficacy and safety of capecitabine and cetuximab as potent agent for maintenance therapy from previous studies, evaluation on the efficacy of cetuximab in combination with capecitabine as a potential maintenance regimen for RAS and BRAF wild-type mCRC patients is warranted. This abstract describes the design of this study.

Modern strategy for treatment of metastatic colorectal cancer as key to increasing life expectancy of patients with metastatic colorectal cancer without mutations in RAS genes

The review is devoted to the place of cetuximab in the treatment of metastatic colorectal cancer (mCRC) without mutations in the RAS (RAS wt) and BRAF (BRAF wt) genes, depending on the goals of therapy, as well as to the analysis of the inflence of various factors, including the localization of the primary tumor, on the effectiveness of treatment. Randomized clinical trials and meta-analyses conducted on their basis allow us to conclude that cetuximab in combination with an infusion doublet or triplet provides the maximum frequency of deep and early objective responses, regardless of the location of the primary tumor. The drug is superior in this parameter to both a single chemotherapy (CT) and a combination of CT with bevacizumab which is important in terms of achieving resectability in patients with potentially resectable metastases. For patients with left-sided localization of the primary tumor and RAS wt, cetuximab, prescribed in the 1st line, provides a reliable and clinically signifiant increase in life expectancy. Postponing the start of its use until 2–4 cycles of CT (until the result of a molecular genetic study is obtained) does not negatively affect the effectiveness of the 1st line of therapy for mCRC RAS wt, and with left-sided localization of the primary tumor, CT with delayed cetuximab exceeds the usage of CT with bevacizumab from the fist cycle for ORR, OS and PFS. The optimal duration of induction chemo-targeted therapy is 3–4 months (6–8 courses), after which it is advisable to switch to maintenance treatment with one cetuximab. The new mode of administration of cetuximab once every 2 weeks at a dosage of 500 mg/m 2 IV provides maximum convenience of its use.

Phase II Trial of Trametinib and Panitumumab in RAS/RAF Wild Type Metastatic Colorectal Cancer

IntroductionMEK inhibition may overcome resistance to EGFR inhibition in patients with RAS wildtype (wt) metastatic colorectal cancer (mCRC). We evaluated antitumor activity of trametinib (MEK1/2 inhibitor) with panitumumab (EGFR monoclonal antibody) in a phase II trial. MethodsPatients with KRAS, NRAS, and BRAF wt mCRC with prior 5-FU, irinotecan, oxaliplatin, +/- bevacizumab and no prior anti-EGFR therapy were treated with trametinib 1.5 mg oral daily and panitumumab 4.8 mg/kg IV every 2 weeks. Primary endpoint was clinical benefit rate (CB; CR, PR, or SD ≥24 weeks) by RECIST v1.1. A 2-stage minimax design was used. Serial plasma circulating free DNA (cfDNA) was collected and profiled using Oncomine Lung cfDNA assay. ResultsFourteen patients were enrolled from November 2015 to April 2019. CB rate was 38% (5/13) and median progression free survival (PFS) was 4.4 months (95% CI, 2.9-7.1). Confirmed overall response rate was 38% (5/13). Treatment-related AE (trAE) included acneiform rash (85%), diarrhea (62%), maculopapular rash (54%), mucositis (46%), and others. Dose modifications and interruptions of trametinib occurred in 69% and panitumumab in 54% of patients. The trial did not progress to stage II accrual due to tolerability and short duration of response. RAS or BRAF mutations cfDNA were detected in 3/13 patients (23%) before radiographic disease progression. ConclusionThe addition of trametinib to panitumumab led to a high rate of tumor shrinkage in RAS/RAF wt metastatic colorectal cancer, with poor tolerability due to a high incidence of skin toxicity. Median PFS was similar to panitumumab alone in historical control data.

Abstract 814: Identification of ERBB4 mutant alleles that function as tumor drivers

Abstract Introduction: ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are well-validated targets in a variety of solid tumors. In contrast, the roles that ERBB4 plays in human tumors are less well defined. Nonetheless, it is accepted that ERBB4 homodimers function as tumor suppressors and ERBB4 heterodimers function as oncogenes. Moreover, our in silico analyses of The Cancer Genome Atlas - Skin Cutaneous Melanoma dataset suggest that ERBB4 mutant alleles function as drivers of BRAF WT metastatic melanomas. Furthermore, ERBB2-ERBB4 heterodimers are associated with elevated tumor proliferation in pediatric ependymomas. Thus, here we describe model systems that we are using to identify ERBB4 gain-of-function (GOF) mutant alleles that stimulate the oncogenic activity of ERBB4-ERBB2 or ERBB4-EGFR heterodimers, as well as ERBB4 loss-of-function (LOF) mutant alleles that disrupt the tumor suppressor activity of ERBB4 homodimers. Experimental Procedures: In the 32D mouse myeloid cell line, ERBB4 homodimers inhibit cell proliferation, whereas ERBB4-ERBB4 heterodimers stimulate cell proliferation. Moreover, we have obtained seven human melanoma cell lines that possess WT BRAF. In these model systems we will express known GOF ERBB4 mutants (Y285C, D595V, Q646C, D931Y, K935I), known LOF ERBB4 mutants (V673I, K751M, G802dup, D861Y, R927Q, R1275W, Y1056F, Ct-b) and ERBB4 mutants found in melanoma samples (R106C, E452K, R711C, P759L, D813N, D861N, R992C). We will evaluate the effects of these ERBB4 mutants on cell proliferation, anchorage-independence, and motility. Data Summary: We anticipate that we will identify GOF ERBB4 mutant alleles that stimulate the oncogenic activity of ERBB4 heterodimers and LOF ERBB4 mutant alleles that disrupt the tumor suppressor activity of ERBB4 homodimers. By evaluating these mutants in the context of human melanoma cell lines, we anticipate that we will identify ERBB4 mutant alleles that function as bona fide tumor drivers. Conclusion: ERBB4 appears to be important to the genesis and/or progression of several types of cancer. However, the fact that ERBB4 functions as a context-dependent oncogene and tumor suppressor gene poses a unique challenge to the identification of driver mutant alleles. The development of the model systems that we present here is an invaluable step forward in the study of ERBB4 as a driver of cancer genesis and/or progression. Citation Format: Lauren Lucas, Vipasha Dwivedi, Joely Burgess, Connor Kelley, Elizabeth Knerr, Jessica Markham, Stephen Miller, David Riese. Identification of ERBB4 mutant alleles that function as tumor drivers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 814.

Abstract 1474: Antitumor activity of belvarafenib in melanoma brain metastasis and NRAS mutation melanoma models

Abstract Over the past decade, BRAF inhibitors, either alone or in combination with MEK inhibitors, have set a new milestone in the treatment of patients with BRAF V600 mutation melanoma, which accounts for about 50% of melanomas. More recently, anti-PD-(L)1 therapies such as nivolumab or pembrolizumab and atezolizumab have been approved as very useful treatments for patients with melanoma. Nevertheless, metastases to the brain in melanoma patients, which are highly aggressive and associated with poor outcomes, still require better treatment. Also, to date, no targeted therapy has been developed for patients with NRAS mutations, which account for about 20% of melanoma. Previously, we reported that belvarafenib (HM95573 or GDC5573) strongly inhibits the growth of various cancer cell lines harboring BRAF, NRAS or KRAS mutations and demonstrates remarkable anticancer efficacy in preclinical animal models. Belvarafenib, currently in Phase 1 clinical trials, is a potent and selective pan-RAF kinase inhibitor showing strong inhibition activities for BRAF WT, BRAF V600E and CRAF. Here, we present the results of preclinical model studies suggesting that belvarafenib could be effective in treating melanoma patients with metastases to the brain as well as NRAS mutation melanoma patients. First, belvarafenib was found to be able to accumulate in the brain of mice and rats following oral administration. The exposure of belvarafenib in the brain was similar to that in plasma (approximately 100% brain to plasma ratio). This level of brain penetration of belvarafenib differs significantly from currently approved BRAF inhibitors that are known to have low brain penetration. Belvarafenib showed excellent anti-tumor activity in an orthotopic brain tumor model using melanoma cells. Belvarafenib significantly increased the overall survival of mice implanted intracranially with BRAF V600E A375SM melanoma cells. Overall, our data demonstrated that belvarafenib has therapeutic potential to treat NRAS mutation melanoma patients. In fact, in the NRAS G13D K1735 murine melanoma cell syngeneic mice model, the combination of belvarafenib with atezolizumab (anti-PD-L1) significantly inhibited tumor growth and effectively induced infiltration of cytotoxic T-cells into tumor tissues. These effects of belvarafenib identified in the above preclinical studies need to be immediately warranted through appropriate clinical trials. Citation Format: Inhwan Bae, Yu-Yon Kim, Jisook Kim, Hyunjin Park, Taehyun Song, Joo-Yun Byun, Semi Lim, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. Antitumor activity of belvarafenib in melanoma brain metastasis and NRAS mutation melanoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1474.

Impact of laparoscopy on oncological outcomes after colectomy for stage III colon cancer: A post-hoc multivariate analysis from PETACC8 European randomized clinical trial

BackgroundIn colon cancer (CC), surgery remains the mainstay of treatment with curative intent. Despite several clinical trials comparing open and laparoscopic approaches, data on long-term outcomes for stage III CC are lacking. MethodsThis post-hoc analysis of the European PETACC8 randomized phase 3 trial included patients from 340 sites between December 2005 and November 2009, with long follow-up (median 7.56 years). Patients were randomly assigned to FOLFOX or FOLFOX+cetuximab after colonic resection. The surgical approach was left to the referring surgeon's discretion. ResultsAmong 2555 patients included, 1796 (70.29%) were operated on by open surgery and 759 (29.71%) by laparoscopy. The 5-year OS rate was better after laparoscopic resection (85.4%, 95%CI 82.5–87.7) than after open surgery (80.2%, 95%CI 78.2–82.0; p = 0.002). The 5-year DFS rate was also better after laparoscopy (p = 0.016). However, in multivariate analysis using a propensity matching, the surgical approach was not found to be an independent prognostic factor for OS or DFS. OS (p = 0.0243) and DFS (p = 0.035) were increased after laparoscopic surgery in KRAS/BRAF WT sub-group ConclusionWe showed that laparoscopic resection has comparable long-term outcomes to open surgery in patients with stage III CC. For those with RAS and BRAF WT CC, laparoscopic colectomy may favorably impact survival.

Differential gradients of efficacy of immunotherapy according to the sun-exposure pattern of the site of occurrence of primary melanoma: A multicenter prospective cohort study (MELBASE).

e21545 Background: The tumor mutational burden (TMB) is usually high in Cutaneous melanomas owing to ultraviolet radiation-induced oncogenesis. While a high TMB is a predictive biomarker of response to PD-1 inhibitors, it may be associated with the rise of resistant clones to targeted therapy over time. We hypothesized that the response pattern to immunotherapy and targeted therapy in advanced melanoma may vary depending on the sun-exposure profile of the site of primary melanoma. Methods: Patients were screened from MelBase, a French multicenter biobank dedicated to the prospective follow-up of unresectable stage III/IV melanoma. Within Melbase, all patients with a known cutaneous primary melanoma who received a first-line systemic treatment by immunotherapy or combined targeted therapy between January 2013 and November 2019 were included. Outcomes were progression-free survival (PFS ) and overall survival (OS). Results: 973 patients received either anti PD-1 monotherapy (n=466), anti CTLA-4 monotherapy (n=143), a combination of both (n=118), dabrafenib plus trametinib (n=187) or vemurafenib plus cobimetinib (n=59). Patients’ characteristics at treatment initiation were: male gender (62%), median age of 62 years old, BRAF WT (58%), AJCC stage IV (84%), brain metastases (18%), ECOG 0-1 (84%) and normal LDH (52%). Median follow-up was 15.5 months. The primary melanoma was located on chronically sun-exposed skin in 175 patients (G1: head neck), on intermittently sun-exposed skin in 615 patients (G2: trunk, arms, legs), and on sun-protected areas in 65 patients (G3: palms, soles, nails). Median PFS was significantly higher in G1 under anti PD-1 treatment (8,7 months vs 3,3 and 3,4 months for G2 and G3, respectively) (p=0.011), (19,2 months vs 8,1 and 6 months for G2 and G1) (p=0. although it is worthwhile to note that the number of G3 patients treated with targeted therapy was relatively low (n=8). PFS did not significantly differ between all groups under ipilimumab. Similarly, median OS was significantly higher in G1 receiving first-line anti PD-1 treatment (45,6 months vs 31,6 and 21,4 months for G2 and G3) (p=0.04), while OS was higher in G3 under targeted therapy (21,1 months vs 16,3 and 19,5 months for G2 and G1) (p=0.97). Conclusions: Our study suggests that first-line immunotherapy with anti PD-1 is particularly recommended for melanomas originating from chronically sun-exposed areas. On the other hand, melanomas originating from sun-protected areas may benefit more from first-line targeted therapy when possible (presence of BRAF mutation), but this finding needs to be confirmed by further research.

Phase II study of anti-EGFR rechallenge therapy with panitumumab driven by circulating tumor DNA molecular selection in metastatic colorectal cancer: The CHRONOS trial.

3506 Background: Despite advances in molecular segmentation of metastatic colorectal cancer (mCRC), beyond RAS status therapeutic actionability remains confined to the limited subgroups of ERBB2 amplified, BRAF mutated and MSI-H patients. Optimization of available treatments is therefore warranted. Rechallenge with anti-EGFR monoclonal antibodies is often empirically used with some benefit as late-line therapy. We previously found that mutant RAS and EGFR ectodomain clones, which emerge in blood during EGFR blockade, decline upon antibody withdrawal leading to regain drug sensitivity. Based on this rationale, we designed CHRONOS, a multicenter phase II trial of anti-EGFR therapy rechallenge guided by monitoring of the mutational status of RAS, BRAF and EGFR in circulating tumor DNA (ctDNA). To our knowledge, this is the first interventional clinical trial of liquid biopsy for driving anti-EGFR rechallenge therapy in mCRC. Methods: Eligible patients were PS ECOG 0-2 RAS/BRAF WT mCRC having first achieved an objective response and then progression in any treatment line with an anti-EGFR antibody containing regimen, displaying RAS, BRAF and EGFR ectodomain WT status in ctDNA at molecular screening after progression to the last anti-EGFR-free regimen. Clonal evolution in ctDNA was analyzed by ddPCR and next generation sequencing. Panitumumab 6 mg/kg was administered IV every two weeks until progression. The primary endpoint was objective response rate (ORR) by RECIST version 1.1 with independent central review. 27 total patients and 6 responses were required to declare the study positive (power = 85%, type I error = 0.05). Results: Between Aug 19, 2019 and Nov 6, 2020 52 patients were screened by liquid biopsy and 36 (69%) were negative in ctDNA for RAS/BRAF/EGFR mutations. Of these, 27 patients were enrolled in 4 centers. Median age was 64 years (range: 42-80). PS ECOG was 0/50%, 1/46%, 2/4%. Previous anti-EGFR was administered in 1st line in 63%, 2nd in 15% and > 2nd in 22%. Median number of previous treatments was 3. The primary endpoint was met, with 8/27 partial responses (PR) observed (2 unconfirmed) (ORR = 30%, 95% CI: 12-47%). Stable disease (SD) was obtained in 11/27 (40%, 95% CI: 24-59%), lasting > 4 months in 8/11. Disease control rate (PR plus SD > 4 months) was therefore obtained in 16/27 (59%, 95% CI: 41-78%). Median progression-free survival was 16 weeks. Median duration of response was 17 weeks (1 ongoing). Maximal grade toxicity was G3, limited to dermatological and occurring in 19% of patients. ctDNA dynamics were studied in all patients. Conclusions: Liquid biopsy-driven rechallenge with anti-EGFR antibodies leads to further objective responses in one third of patients. Genotyping tumor DNA in the blood to direct therapy can be effectively incorporated in the management of advanced CRCs. Clinical trial information: 2016-002597-12.

NRF2 DLG Domain Mutations Identified in Japanese Liver Cancer Patients Affect the Transcriptional Activity in HCC Cell Lines.

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.

Blood-Based Detection of BRAF V600E in Gliomas and Brain Tumor Metastasis.

Simple SummaryThe BRAF V600E mutation has been identified as a key driver in brain tumors and brain tumor metastasis. The ability to detect this mutation in a minimally invasive plasma assay offers advantages over traditional tissue-based biopsy for the disease diagnosis and monitoring. The aim of this study was to develop an assay for the detection of BRAF V600E in the plasma of patients with brain tumors and brain tumor metastasis. We demonstrate BRAF V600E detection using a novel plasma-based ddPCR assay. We detect the mutation in circulating nucleic acids in 4/5 patients with mutant gliomas and metastatic melanoma. We also show correlation between plasma BRAF V600E and clinical status. This proof of principle study is important in the context of application of liquid biopsy in plasma to the neuro-oncologic field. The assay may be useful as a diagnostic adjunct, prognostication tool, and method for monitoring of disease and treatment response.Liquid biopsy provides a minimally invasive platform for the detection of tumor-derived information, including hotspot mutations, such as BRAF V600E. In this study, we provide evidence of the technical development of a ddPCR assay for the detection of BRAF V600E mutations in the plasma of patients with glioma or brain metastasis. In a small patient cohort (n = 9, n = 5 BRAF V600E, n = 4 BRAF WT, n = 4 healthy control), we were able to detect the BRAF V600E mutation in the plasma of 4/5 patients with BRAF V600E-tissue confirmed mutant tumors, and none of the BRAF WT tumors. We also provide evidence in two metastatic patients with longitudinal monitoring, where the plasma-based BRAF V600E mutation correlated with clinical disease status. This proof of principle study demonstrates the potential of this assay to serve as an adjunctive tool for the detection, monitoring, and molecular characterization of BRAF mutant gliomas and brain metastasis.

Bevacizumab or standard chemotherapy in previously treated patients with metastatic colorectal cancer — a systematic review

Introduction. The BRAF V600E mutation ( BRAF mt) occurring in the metastatic colorectal cancer (mCRC) patients is associated with poorer prognosis, in comparison to the wild-type variant of the BRAF gene ( BRAF wt). Aim of this work was to assess the clinical efficacy of bevacizumab (BEVA) or standard chemotherapy (ChT) in the 2nd or further lines of treatment in mCRC BRAF mt population. Material and methods. MEDLINE/PubMed, Embase and Cochrane CENTRAL databases were systematically searched. The reference lists of relevant studies were also checked. Results. 6 eligible trials were identified: MOMA (BEVA ± ChT), allowing for limited overall survival (OS) assessment, WJOG 6210G (BEVA + FOLFIRI), RAISE and 20050181 (FOLFIRI), PICCOLO and Spindler 2013 (irinotecan monotherapy). None of those trials were designed for the treatment evaluation in BRAF mt population. Available evidence was restricted to limited analyses in small subgroups (from a few to several dozens of patients), occasionally comprising RAS gene mutation ( RAS mt) as well. Based on the identified studies, the comparison of BEVA ± ChT vs. ChT or among different ChTs in BRAF mt population was not feasible. In case of BEVA (MOMA), OS hazard ratio (HR) for BRAF mt vs. BRAF wt was 1.52 (95% CI: 0.79–2.89) with difference in medians equal to 12.1 months (19.2 vs. 31.3 months, respectivelly), and BRAF mt or RAS mt patients had median OS lower by 7.9 months and median progression free survival (PFS) by 3.0 months in WJOG 6210G trial. In case of ChT, median PFS was lower in BRAF mt by 12–67% (HRs range: 1.01–5.3), and median OS by 34–73% (HRs range: 1.05–5.00). Conclusions. Due to limited clinical evidence, assessment of further lines of treatment in BRAF mt mCRC patients is uncertain, however existing data consistently suggest lower effectiveness of BEVA ± ChT or ChT in BRAF mt, than in BRAF wt subgroup. Hopefully, combining anti-EGFR therapies with BRAF/MEK inhibitor is expected to improve prognosis of those patients.

NTRK fusion analysis reveals enrichment in Middle Eastern BRAF wild-type PTC.

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) are known oncogenic drivers in a broad range of tumor types. It recently gained attention as a predictor of targeted therapy since selective NTRK inhibitors are now approved in the US and Europe for patients with solid tumors harboring gene fusions. However, estimation of NTRK gene fusion/alteration frequency and its clinicopathological characteristics in papillary thyroid cancer (PTC) is limited, especially in a population with high incidence for PTC like Middle Eastern population. This study aims to characterize the NTRK gene fusion frequency and investigate the utility of pan-Trk immunohistochemistry (IHC) as predictor of NTRK fusion in a large cohort of Middle Eastern PTC. FISH analysis for NTRK gene fusions and pan-Trk IHC was performed on 315 Middle Eastern PTCs. Correlation of NTRK gene fusion and protein expression with clinicopathological markers and patient outcome were determined. In our cohort, 6.0% (19/315) patients showed NTRK gene fusions and were significantly associated with pediatric PTC (P = 0.0143), lymph node metastasis (P = 0.0428) and BRAF WT tumors (P < 0.0001). Pan-Trk IHC was positive in 9.2% (29/315) of cases and significantly associated with NTRK fusions, with a sensitivity of 73.7% and specificity of 94.9% in this cohort. This study confirms the presence of NTRK fusions in Middle Eastern PTC which is significantly enriched in BRAF WT as well as pediatric age group and proposes the usefulness of IHC to screen for PTC patients with NTRK fusion that might benefit from TRK inhibitors.

Epigenetic regulation of the Wnt-signaling pathway in CIMP-H BRAFV600E mCRC.

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p&lt; 0.0001 (FDR &lt; 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.