non-V600E Mutations Research Articles

Applying Precision to the Management of BRAF-Mutant Metastatic Colorectal Cancer.

Identifying the presence or absence of a BRAFV600E mutation is paramount for the management of patients with metastatic colorectal cancer (mCRC) as there are distinct predictive and prognostic implications, as well as unique therapeutic approaches for this molecular subtype. Traditional cytotoxic doublet chemotherapy has historically been ineffective for this poor prognostic group, thereby highlighting the critical need for novel targeted therapies to drive management. Unlike the early success achieved with BRAF-inhibitor monotherapy for patients with BRAFV600E-mutated metastatic melanoma, response rates were found to only be 5% in early-phase clinical trials for patients with BRAFV600E mCRC. A deeper understanding of predominant resistance mechanisms in BRAFV600E mCRC after exposure to BRAF inhibition has resulted in innovative combinatorial approaches targeting the mitogen-activated protein kinase (MAPK) pathway, revitalizing the treatment portfolio for these patients. Of note, in recent years non-V600 BRAF mutations have been appreciated as a distinct molecular subset in mCRC, representing 2-4% of patients with a unique clinical presentation and complex signaling biology. These mutations, referred to as "atypical" BRAF mutations, warrant individual clinical investigation and demand innovative drug development that leverages known signaling class biology. Here, we summarize the current molecular and clinicopathologic understanding of BRAFV600E mCRC, as well as the landmark clinical trials that have led to successful targeted therapy for this historically aggressive subtype of colorectal cancer. Additionally, we briefly describe the current understanding of patients with atypical BRAF mutations, highlighting the importance of continued research efforts to appropriately treat this evolving subset of BRAF mutations.

Single-nucleotide variants, tumour mutational burden and microsatellite instability in patients with metastatic colorectal cancer: Next-generation sequencing results of the FIRE-3 trial

BackgroundMolecular biomarkers and primary tumour sidedness guide treatment decisions in metastatic colorectal cancer. Comprehensive molecular profiling aims to identify targetable alterations and measure tumour mutational burden (TMB) to enable precision oncology. Material and methodsFoundationOne® next-generation sequencing identified single-nucleotide variants (SNVs), copy number alterations, high TMB (TMB-H) and high-grade microsatellite instability (MSI-H) in patients treated in the FIRE-3 trial. Data were correlated with objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). ResultsThree hundred seventy-three (49.6%) of 752 patients provided material for this analysis. Frequent SNVs included TP53, APC, KRAS, PIK3CA, BRAF, SMAD4 and FBXW7. KRAS, BRAF V600E and SMAD4 mutations were confirmed as prognostic biomarkers by logistic penalised regression for ORR. OS was significantly longer in patients with SMAD4 wild-type (WT) tumours than in those with SMAD4-mutated tumours (hazard ratio = 0.59 [95% confidence interval {CI} = 0.34–1.01], p = 0.05), with a higher probability of ORR [odds ratio, SMAD4 SNV versus WT = 0.32 [95% CI = 0.10–0.98], p = 0.05] when treated with cetuximab. MSI-H (30.0%, p = 0.03) and TMB-H (17.3%, p = 0.003) tumours were enriched by FBXW7 mutations. Numerically lower ORR, OS and PFS were observed in MSI-H tumours. ConclusionsRAS, BRAF V600E and SMAD4 mutations were identified as poor prognostic biomarkers in patients of the FIRE-3 trial, whereas improved outcome was observed for BRAF non-V600E mutation. SMAD4 mutation might provide predictive relevance for cetuximab efficacy. MSI-H tumours showed numerically lower ORR, OS and PFS.

Abstract 1092: A systematic analysis of BRAF mutations and their sensitivity to different BRAF inhibitors

Abstract The MAPK-ERK signaling cascade is among the most frequently mutated pathways in human cancer, with the BRAF V600 mutation being the most common alteration. FDA-approved BRAF inhibitors as well as combination therapies of BRAF and MEK inhibitors are available and provide survival benefits to patients with a BRAF V600 mutation in several indications. Yet non-V600 BRAF mutations are found in many cancers and are even more prevalent than V600 mutations in certain tumor types. As the use of NGS profiling in precision oncology is becoming more common, novel alterations in BRAF are being uncovered. This has led to the classification of BRAF mutations, which is dependent on its biochemical properties and affects it sensitivity to inhibitors. Therefore, annotation of these novel variants is crucial for assigning correct treatment. Using a high throughput method for functional annotation of MAPK activity, we profiled 151 different BRAF mutations identified in the AACR Project GENIE dataset, and their response to 4 different BRAF inhibitors- vemurafenib and 3 different exploratory 2nd generation inhibitors. The system is based on rapid synthesis of the mutations and expression of the mutated protein together with fluorescently labeled reporters in a cell-based assay. Our results show that from the 151 different BRAF mutations, ~25% were found to activate the MAPK pathway. All of the class 1 and 2 mutations tested were found to be active, providing positive validation for the method. Additionally, many novel activating mutations were identified, some outside of the known domains. When testing the response of the active mutations to different classes of BRAF inhibitors, we show that while vemurafenib efficiently inhibited V600 mutations, other types of mutations and specifically BRAF fusions were not inhibited by this drug. Alternatively, the second-generation experimental inhibitors were effective against both V600 as well as non-V600 mutations. Using this large-scale approach to characterize BRAF mutations, we were able to functionally annotate the largest number of BRAF mutations to date. Our results show that the number of activating variants is large and that they possess differential sensitivity to different types of direct inhibitors. This data can serve as a basis for rational drug design as well as more accurate treatment options for patients. Citation Format: Zohar Barbash, Dikla Haham, Liat Hafzadi, Ron Zipor, Shaul Barth, Arie Aizenman, Lior Zimmerman, Gabi Tarcic. A systematic analysis of BRAF mutations and their sensitivity to different BRAF inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1092.

Molecular Landscape of BRAF-Mutant NSCLC Reveals an Association Between Clonality and Driver Mutations and Identifies Targetable Non-V600 Driver Mutations.

Approximately 4% of NSCLC harbor BRAF mutations, and approximately 50% of these are non-V600 mutations. Treatment of tumors harboring non-V600 mutations is challenging because of functional heterogeneity and lack of knowledge regarding their clinical significance and response to targeted agents. We conducted an integrative analysis of BRAF non-V600 mutations using genomic profiles of BRAF-mutant NSCLC from the Guardant360 database. BRAF mutations were categorized by clonality and class (1 and 2: RAS-independent; 3: RAS-dependent). Cell viability assays were performed in Ba/F3 models. Drug screens were performed in NSCLC cell lines. A total of 305 unique BRAF mutations were identified. Missense mutations were most common (276, 90%), and 45% were variants of unknown significance. F468S and N581Y were identified as novel activating mutations. Class 1 to 3 mutations had higher clonality than mutations of unknown class (p < 0.01). Three patients were treated with MEK with or without BRAF inhibitors. Patients harboring G469V and D594G mutations did not respond, whereas a patient with the L597R mutation had a durable response. Trametinib with or without dabrafenib, LXH254, and lifirafenib had more potent inhibition of BRAF non-V600-mutant NSCLC cell lines than other MEK, BRAF, and ERK inhibitors, comparable with the inhibition of BRAF V600E cell line. In BRAF-mutant NSCLC, clonality is higher in known functional mutations and may allow identification of variants of unknown significance that are more likely to be oncogenic drivers. Our data indicate that certain non-V600 mutations are responsive to MEK and BRAF inhibitors. This integration of genomic profiling and drug sensitivity may guide the treatment for BRAF-mutant NSCLC.

A BRAF new world.

BRAF is a rare targetable mutation in non-small-cell lung cancer (NSCLC). Emerging evidence underlines that, rather than a single point mutation, BRAF genes present with a wide array of mutations, essentially in lung adenocarcinoma. Different BRAF mutations have divergent clinical and therapeutic implications, with a particular distinction between V600E and non-V600E mutations. The latter are at least as frequent in NSCLC as V600E, but lack any proven targeted therapy. In this paper, we briefly review the current literature and provide an update of scientific knowledge about different types of BRAF mutations in NSCLC.

Identification of actionable BRAF mutations and their genomic associations in advanced prostate cancer.

e17597 Background: Circulating tumor DNA (ctDNA) can be used to identify mutations and amplifications in patients with prostate cancer (PC). Identification of actionable mutations in BRAF may allow the use of targeted therapeutics not typically used in prostate cancer. Methods: We analyzed a ctDNA-based genomic profile database (Guardant Health) with 2274 unique advanced-stage patients, and a tissue-based genomic database (TCGA) with 3178 unique patients (73% locoregional, 27% metastatic). We compared the frequency and types of BRAF anomalies found in those datasets. We calculated the odds ratio of observing other genetic anomalies based on the BRAF mutation and amplification status. Finally, we utilized Principal Component Analysis (PCA) to study the utility of BRAF as a discriminator in identifying genetic subtypes of PC. Results: BRAF missense mutations in PC were found in 3.34% of the 2274 patients in the ctDNA cohort and in 1.1% of the 3178 patients in the TCGA cohort. Variants of Uncertain Significance (VUS) mutations were more commonly identified by ctDNA (53.8% of all mutations) vs TCGA (16.1% of all mutations). Biologically-significant mutations of all classes (I, II, III) were found in both datasets. BRAF mutations were mainly non-V600E and most mutations were clonal. Significant mutations common to both the ctDNA and TCGA cohorts include K601E, G469A, D594G, V600E, G466E, G446R, Q257K, I300V and R271H. Mutations unique to either ctDNA or TCGA datasets were found as well. BRAF mutations are highly associated with additional mutations in CDKN2A, TERT, and KRAS, as well as amplifications of CCNE1, KRAS, CDK6 (p &lt; 0.01). BRAF amplifications are highly associated with multiple other gene amplifications, and with AR mutations (p &lt; 0.01). Tumors with BRAF mutations and with BRAF amplifications are genetically distinct, and PCA indicates that BRAF status accounts for a significant amount of the difference. We described two patients that were treated with MEK targeted therapy with treatment response. Conclusions: 1. The use of ctDNA-based genomic analysis identified BRAF anomalies with a higher frequency than tissue-based techniques, but this is mostly due to higher numbers of VUS mutants found with ctDNA. Biologically significant BRAF mutations are mostly clonal and non-V600E mutations. 2. BRAF mutations and amplifications are associated with other specific gene anomalies. 3. ctDNA is a useful tool for identifying the genomic profile of PC in the clinical setting. 4. PC subjects with BRAF mutations identified by ctDNA may respond to MEK inhibitor therapy.

Clinical characteristics and treatment outcomes of 65 patients with BRAF-mutated non-small cell lung cancer (NSCLC).

e21745 Background: BRAF mutations are infrequently seen in non-small cell lung cancer (NSCLC) in Chinese population. We aimed to investigate the clinicopathologic characteristics and treatment outcomes of Chinese patients with NSCLC harboring BRAF mutations. Methods: We conducted a retrospective multicenter study in China of patients with NSCLC harboring BRAF mutations between Jan 2017 and Jul 2019. Results: A total of 65 patients treated in 22 centers were included, 54 harbored BRAF-V600E mutation and 11 had non-V600E mutations, including K601E, G469S, G469V, G469A, G596R, G466R and T599dup. No significant difference in age or gender was found between BRAF-V600E and non-V600E cases, while the majority of patients with non-V600E mutations were smokers (81.8%). Of the 18 patients with early-stage disease at diagnosis and underwent a resection, the median disease-free survival (DFS) was 43.2 months, 18.7 months and 10.1 months of stage I, II and IIIA patients (P = 0.07), respectively. In 46 patients with advanced-stage disease at data cutoff, disease control rate (DCR) and progression-free survival (PFS) of first-line anti-BRAF targeted therapy was superior than chemotherapy in patients harboring BRAF-V600E mutation (DCR, 100.0% vs. 70.0%, P = 0.027; median PFS, 9.8 months vs. 5.4 months, P = 0.149). Of 30 V600E-mutated patients who received anti-BRAF therapy during the course of disease, the median PFS of vemurafenib, dabrafenib, and dabrafenib plus trametinib was 7.8 months, 5.8 months and 6.0 months, respectively (P = 0.970). Median PFS were similar between V600E and non-V600E patients (5.4 months vs. 5.4 months, P = 0.825) to first-line chemotherapy. Nine patients were treated with checkpoint inhibitors, DCR and median PFS were 62.5% and 3.0 months (95%CI 2.9, 3.1), respectively. Conclusions: Our data demonstrated the clinical benefit of anti-BRAF targeted therapy in Chinese NSCLC patients harboring BRAF-V600E mutation. The value of immunotherapy and treatment selection among non-V600E population needs further study.[Table: see text]

Genomic landscape of malignant peripheral nerve sheath tumor (MPNST)-like melanoma.

e22072 Background: Malignant Peripheral Nerve Sheath Tumor (MPNST)-like melanoma is a rare entity wherein cutaneous melanoma mimics histomorphological features of MPNST. The genomic landscape of MPNST-like melanoma has not been previously described. We report the genomic findings of 6 patients diagnosed with MPNST-like melanoma. Methods: We identified consecutive patients with confirmed histological diagnosis of MPNST-like melanoma. Archival tissue was submitted for whole exome and transcriptome sequencing analysis and genomic findings were compared to publicly available data on the genomic landscape of both melanoma and limited genetic descriptions of MPNST. Results: Six patients diagnosed with MPNST-like melanoma between November 2002 and July 2018 had archival tissue available (8 samples). Mean age at diagnosis was 60.5 years (38-74). The mean tumor mutational burden (TMB) was 35.72 mutations/exon coding megabase, ranging from 4.27 to 86.36. NF1 alterations were found in 4 (67%) of cases: one had 3 truncating mutations (W696 + X1870_splice + R1590C) associated with NF1 amplification, one had a deep deletion and another two had truncating mutations (I1908L and W696 + A1530V). Two tumors had non-V600 BRAF mutations that predict alteration in kinase function. We observed deletions of cell cycle regulators: CDKN2A and CDKN2B in 33%; and ZNF331 (19q13.42) in 83% of cases. SERPINB4 (18q21.3), described previously as a tumor suppressor in oral squamous cell carcinomas, was deleted in all samples. The patient with NF1 amplification and truncating mutations received immunotherapy due to metastatic disease and achieved a complete response; his tumor had a TMB of 72.6. Another patient who died of metastatic melanoma, treated with chemotherapy in the pre-immunotherapy era, lacked any NF1 mutation but had a Q61K NRAS mutation; the tumor had a TMB of 15. The remaining patients were treated with surgical resection alone and continue on follow-up. Conclusions: MPNST-like melanoma shares some frequently altered genes in cutaneous melanoma but possesses an excess of NF-1 mutations or deletions, in keeping with its neural-like phenotype. The high frequency of SERPINB4 alterations suggests a critical tumor suppressor role in this subtype of melanoma, warranting further investigation. Additionally, cell cycle dysregulation caused by deletion of ZNF331 and CDKN2A/B may be important in MPNST-like melanoma carcinogenesis.

Prognostic effect of specific RAS/BRAF mutations in patients (pts) with metastatic colorectal cancer (mCRC).

4050 Background: Somatic mutations in KRAS, HRAS, NRAS (extended RAS) and BRAF have prognostic and predictive impact in pts with mCRC. We analyzed the prognostic impact of specific somatic mutations in extended RAS and BRAF. Methods: We retrospectively reviewed the electronic medical records of pts with mCRC at our institution who underwent comprehensive genomic profiling (CGP) utilizing the Foundation One assay. DNA was extracted from clinical specimens and CGP was performed on hybrid-capture, adaptor ligation-based libraries for up to 315 genes plus 47 introns from 19 genes frequently rearranged in cancer. BRAF mutations were classified as class I, II and III according to accepted nomenclature. Fisher’s exact test and Kaplan Meier estimates were used for statistical analyses. This project was approved by the Medical College of Wisconsin Institutional Review Board. Results: 273 pts were identified - median age at diagnosis was 57, 48% were male. Somatic mutations in extended RAS were found in 138 (50%) pts, majority being mutations in KRAS (46%). Among pts with KRAS mutations, codon 12, 13, 61 and 146 mutations accounted for 73%, 11%, 4% and 6% respectively while KRAS G12C mutations accounted for 9%. BRAF mutations were detected in 22 (8%) pts - BRAF V600E and non–V600E mutations accounting for 4.4% and 3.6% respectively. Among pts with BRAF mutations, 17 (77%) were kinase domain mutations, 16 of which could be further classified as class I (12/16), II (1/16) and III (3/16). Median overall survival (mOS) for the entire cohort was 26.4 months (mo). KRAS mutated pts had a mOS of 25.8 mo; pts with KRAS G12C mutation had a mOS of 23 mo compared to 27.1 mo for pts with other KRAS mutations (p &lt; 0.001).Pts with BRAF mutation had a mOS of 26.2 mo; pts with BRAF V600E mutation had a mOS of 14.1 mo compared to 30.6 mo for pts with BRAF non-V600E mutations (p = 0.1). Conclusions: The poor prognosis of pts with KRAS G12C and BRAF V600E mutations compared to pts with other KRAS and BRAF mutations merit further biologic characterization with functional assays. Individualized therapeutic strategies must be conceptualized for mCRC pts with specific RAS/BRAF mutations, considering their widely disparate prognosis and putative downstream signaling mechanisms. Dynamic molecular simulation to understand conformational changes in proteins associated with specific mutations will be pivotal to optimizing precision therapeutic strategies.

BRAF and DIS3 Mutations Associate with Adverse Outcome in a Long-term Follow-up of Patients with Multiple Myeloma.

Copy-number changes and translocations have been studied extensively in many datasets with long-term follow-up. The impact of mutations remains debated given the short time to follow-up of most datasets. We performed targeted panel sequencing covering 125 myeloma-specific genes and the loci involved in translocations in 223 newly diagnosed myeloma samples recruited into one of the total therapy trials. As expected, the most commonly mutated genes were NRAS, KRAS, and BRAF, making up 44% of patients. Double-Hit and BRAF and DIS3 mutations had an impact on outcome alongside classical risk factors in the context of an intensive treatment approach. We were able to identify both V600E and non-V600E BRAF mutations, 58% of which were predicted to be hypoactive or kinase dead. Interestingly, 44% of the hypoactive/kinase dead BRAF-mutated patients showed co-occurring alterations in KRAS, NRAS, or activating BRAF mutations, suggesting that they play a role in the oncogenesis of multiple myeloma by facilitating MAPK activation and may lead to chemoresistance. Overall, these data highlight the importance of mutational screening to better understand newly diagnosed multiple myeloma and may lead to patient-specific mutation-driven treatment approaches.

Clinicopathologic and Molecular Characteristics of Familial Adenomatous Polyposis-associated Traditional Serrated Adenoma.

Colorectal carcinogenesis in familial adenomatous polyposis (FAP) follows a conventional adenoma-carcinoma sequence. However, previous studies have also reported the occurrence of traditional serrated adenomas (TSAs) in patients with FAP. In the present study, we analyzed the clinicopathologic and molecular features of 37 TSAs from 21 FAP patients. Histologically, the majority of FAP-associated TSAs showed typical cytology and slit-like serration; however, ectopic crypt formation was infrequent. Next-generation sequencing and Sanger sequencing identified KRAS and BRAF V600E mutations in 18 (49%) and 14 (38%) TSAs, respectively. Somatic APC mutations were detected in 26 lesions (84% of analyzed cases). Three lesions had BRAF non-V600E mutations, and 2 of them had a concurrent KRAS mutation. Seven TSAs (19%) were associated with a precursor polyp, 6 with a hyperplastic polyp, and 1 with a sessile serrated lesion, and all of them showed the BRAF V600E mutation. Additional sequencing analysis of 4 TSAs with a precursor polyp showed that the BRAF V600E mutation was shared between the TSA and precursor components, but APC mutations were exclusive to the TSA component in all the analyzed lesions. None of the lesions showed the high CpG island methylation phenotype. These results indicate that FAP-associated TSAs frequently have KRAS or BRAF mutations, similar to sporadic cases, and second-hit somatic APC mutations are commonly involved in their tumorigenesis as in other FAP-associated tumors. Although progression to adenocarcinoma is likely rare, tumorigenesis via the serrated pathway occurs in patients with FAP.

Antitumor Effects of Pan-RAF Inhibitor LY3009120 Against Lung Cancer Cells Harboring Oncogenic BRAF Mutation.

The therapeutic strategy for patients with non-small-cell lung cancer (NSCLC) harboring the BRAF non-V600E mutation has not been established. LY3009120, a newly discovered pan-RAF inhibitor, has shown strong antitumor effects in cancers with various BRAF genotypes. This study investigated the antitumor effects of LY3009120 in NSCLC cells harboring the BRAF non-V600E mutation. We examined the antitumor effects of LY3009120 by MTS assay and flow cytometry. We analyzed the expression status of proteins by western blot. The mouse xenograft models were used for the in vivo experiments. LY3009120 suppressed BRAF-related downstream pathway molecules and induced cleavage of poly ADP-ribose polymerase in all examined NSCLC cell lines. LY3009120 also inhibited in vivo tumor growth in NSCLC cells harboring the BRAF non-V600E mutation. LY3009120 is a potent therapeutic agent for patients with BRAF non-V600E mutant NSCLC.

Clinical Characteristics and Treatment Outcomes of 65 Patients With BRAF-Mutated Non-small Cell Lung Cancer.

BRAF mutation is an oncogenic driver gene in non-small cell lung cancer (NSCLC) with low frequency. The data of patients with NSCLC harboring BRAF mutations is rare. We conducted a retrospective multicenter study in Chinese patients with NSCLC harboring BRAF mutations between Jan 2017 and Jul 2019. A total of 65 patients treated in 22 centers were included, 54 harbored BRAF-V600E mutation and 11 had non-V600E mutations, including K601E, G469S, G469V, G469A, G596R, G466R, and T599dup. Of 18 patients with early-stage disease at diagnosis and underwent a resection, the median disease-free survival (DFS) was 43.2, 18.7, and 10.1 months of stage I, II, and IIIA patients, respectively. In 46 patients with advanced-stage disease at data cutoff, disease control rate (DCR), and progression-free survival (PFS) of first-line anti-BRAF targeted therapy was superior than chemotherapy in patients harboring BRAF-V600E mutation (DCR, 100.0 vs. 70.0%, P = 0.027; median PFS, 9.8 vs. 5.4 months, P = 0.149). Of 30 V600E-mutated patients who received anti-BRAF therapy during the course of disease, median PFS of vemurafenib, dabrafenib, and dabrafenib plus trametinib was 7.8, 5.8, and 6.0 months, respectively (P = 0.970). Median PFS were similar between V600E and non-V600E patients (5.4 vs. 5.4 months, P = 0.825) to first-line chemotherapy. Nine patients were treated with checkpoint inhibitors, with median PFS of 3.0 months. Our data demonstrated the clinical benefit of anti-BRAF targeted therapy in Chinese NSCLC patients harboring BRAF-V600E mutation. The value of immunotherapy and treatment selection among non-V600E population needs further study.

Risk Stratification of Severely Dysplastic Nevi by Non-Invasively Obtained Gene Expression and Mutation Analyses

Background: Strategies to non-invasively detect cutaneous melanoma generally focus on differentiating melanoma from non-melanoma lesions. However, given the variabilities in practice and lack of guidelines, it is important for clinicians to understand how such strategies and technologies perform on borderline lesions of uncertain clinical behavior. Objective: To evaluate LINC and PRAME gene expression and the presence of somatic mutations in BRAF, NRAS, and/or TERT in severely dysplastic nevi (SDN) and to assess, how combining gene expression and mutation analyses may impact test performance.Materials and Methods: One hundred three eligible skin lesions clinically suspicious for melanoma were non-invasively sampled via adhesive patches to enable genomic analyses. Afterward, these same lesions were immediately surgically biopsied to enable comparisons of genomic analyses with histopathologic diagnoses rendered by a panel of three dermatopathologists. Twenty-three of these lesions analyzed were deemed borderline lesions of SDN histology by at least one dermatopathologist. RNA-based gene expression positivity by Pigmented Lesion Assay (PLA) analysis was defined by detectable levels of LINC and/or PRAME. DNA-based mutation positivity was defined as detection of somatic mutations in BRAF (non-V600E), NRAS, and/or the TERT promoter. Results: Adding TERT mutation analyses to PLA gene expression increases the test’s sensitivity to rule out melanoma from 93% to 97% in this study. In addition, 61% of PLA positive lesions that were not diagnosed as melanoma were found to have severe histologic atypia. PLA-positive lesions histopathologically diagnosed as melanomas harbored TERT mutations in 70% of cases while both SDN and non-melanoma lesions including nevi without severe histologic atypia harbored TERT mutations in 4% of cases. BRAF non-V600E and NRAS mutations were only found in the melanoma group and adding these mutations did not further enhance the test’s sensitivity. Conclusions and Relevance: PLA positivity increases with histologic atypia of pigmented skin lesions. Combining TERT mutation analyses with melanoma-associated gene expression provides additional genetic information to further non-invasively risk-stratify pigmented lesions. These findings furthermore support that pigmented lesions exist on a spectrum of genomic atypia that may prove useful in identifying borderline lesions beyond their morphological appearance.

Negative reactions of BRAF mutation-specific immunohistochemistry to non-V600E mutations of BRAF.

BRAF mutations are rare driver mutations in non-small cell lung cancer (NSCLC), accounting for 1%-2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non-V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non-V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation-specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non-V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non-V600E mutations, using BRAF mutation-specific IHC. None of the tumors with non-V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation-specific IHC is specific without any cross-reactions to non-V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.

BIG BANG study (EPOC1703): multicentre, proof-of-concept, phase II study evaluating the efficacy and safety of combination therapy with binimetinib, encorafenib and cetuximab in patients with BRAF non-V600E mutated metastatic colorectal cancer

BackgroundWhile the BRAF V600E mutation occurs in 5%–15% of metastatic colorectal cancer (mCRC), BRAF non-V600E mutations were recently reported to range from 1.6% to 5.1%. We have previously reported that BRAF non-V600E mutations could have a negative impact on efficacy outcomes as well as BRAF V600E mutation for antiepidermal growth factor receptor (EGFR) antibody treatment for pretreated patients with mCRC. Recently, simultaneous inhibitions of mitogen-activated protein kinase kinase (MEK), BRAF and EGFR exhibited relevant antitumour activities in patients with BRAF V600E mutant and also in BRAF non-V600E mutant but only in the preclinical model.Trial designThe BIG BANG (study is a multicentre, phase II study to assess the efficacy, safety and proof of concept of the combinations of binimetinib+encorafenib+cetuximab in patients with BRAF non-V600E mutated mCRC, identified by either tumour tissue (tumour tissue group) or blood samples (liquid biopsy group). Key eligibility criteria include Eastern Cooperative Oncology Group Performance Status of ≤1, mCRC with BRAF non-V600E mutant and RAS wild type, refractory or intolerant to at least one fluoropyrimidine-based regimen and no prior history of regorafenib, and no prior history of anti-EGFR antibody treatment (primary analysis cohort and liquid biopsy cohort) or refractory to prior anti-EGFR antibody treatment in patients with class 3 BRAF mutations (anti-EGFR antibody refractory class three cohort). Enrolled patients receive binimetinib (45 mg, two times per day), encorafenib (300 mg, once a day) and cetuximab (initially 400 mg/m2 and subsequently 250 mg/m2, once per week). The primary endpoint is the confirmed objective response rate in the primary analysis cohort.Trial registration numbersUMIN000031857 and 000031860.

Response to Anti-EGFR Therapy in Patients with BRAF non-V600-Mutant Metastatic Colorectal Cancer.

While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). Forty patients with oncogenic non-V600 BRAF-mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment.See related commentary by Fontana and Valeri, p. 6896.

Class(y) Dissection of BRAF Heterogeneity: Beyond Non-V600.

Different classes of BRAF mutations are present in colorectal and other cancers. Non-V600 mutations are rare; however, their detection rate will increase as the use of next-generation sequencing ramps up quickly in clinical practice. Different biochemical signaling pathways are active in non-V600 BRAF-mutant cancers and may affect treatment response.See related article by Yaeger et al., p. 7089.

Targeted Therapy in Advanced Melanoma With Rare BRAF Mutations.

BRAF/MEK inhibition is a standard of care for patients with BRAF V600E/K-mutated metastatic melanoma. For patients with less frequent BRAF mutations, however, efficacy data are limited. In the current study, 103 patients with metastatic melanoma with rare, activating non-V600E/K BRAF mutations that were treated with either a BRAF inhibitor (BRAFi), MEK inhibitor (MEKi), or the combination were included. BRAF mutation, patient and disease characteristics, response, and survival data were analyzed. Fifty-eight patient tumors (56%) harbored a non-E/K V600 mutation, 38 (37%) a non-V600 mutation, and seven had both V600E and a rare BRAF mutation (7%). The most frequent mutations were V600R (43%; 44 of 103), L597P/Q/R/S (15%; 15 of 103), and K601E (11%; 11 of 103). Most patients had stage IV disease and 42% had elevated lactate dehydrogenase at BRAFi/MEKi initiation. Most patients received combined BRAFi/MEKi (58%) or BRAFi monotherapy (37%). Of the 58 patients with V600 mutations, overall response rate to BRAFi monotherapy and combination BRAFi/MEKi was 27% (six of 22) and 56% (20 of 36), respectively, whereas median progression-free survival (PFS) was 3.7 months and 8.0 months, respectively (P = .002). Of the 38 patients with non-V600 mutations, overall response rate was 0% (zero of 15) to BRAFi, 40% (two of five) to MEKi, and 28% (five of 18) to combination treatment, with a median PFS of 1.8 months versus 3.7 months versus 3.3 months, respectively. Multivariable analyses revealed superior survival (PFS and overall survival) with combination over monotherapy in rare V600 and non-V600 mutated melanoma. Patients with rare BRAF mutations can respond to targeted therapy, however, efficacy seems to be lower compared with V600E mutated melanoma. Combination BRAFi/MEKi seems to be the best regimen for both V600 and non-V600 mutations. Yet interpretation should be done with care because of the heterogeneity of patients with small sample sizes for some of the reported mutations.

P2.16-08 Clinical Characteristics and Outcomes of Patients with BRAF Mutated Non-Small Cell Lung Cancer

BRAF mutation is a driver oncogene identified in 0.5-2% of non-small cell lung cancer (NSCLC) patients in China, and clinical data are relatively inadequate. This study assessed the clinical characteristics, course of disease and treatment outcomes in patients with BRAF mutated NSCLC in a real-world setting. Between Apr 1, 2017 and Feb 1, 2019, medical data of patients with NSCLC harboring BRAF mutation in our cancer center was retrospective collected. Patient characteristics and treatment outcomes were reviewed. Data cutoff was Apr 1, 2019. A total of 36 patients were identified. BRAF V600E was the most common (31/36, 86.1%), other BRAF molecular subsets were observed in 5 (13.9%) cases including K601E, G469V, G596R and D594N. The majority of patients with BRAF mutation were female (20/36, 55.6%) and non-smokers (20/36, 55.6%), all of them were adenocarcinoma, and median age at diagnosis was 56 (range, 33-79). Twenty-seven patients were recurrent or metastatic NSCLC at data cutoff, most of whom received chemotherapy (16/27, 59.3%) as first-line therapy with median progression-free survival (PFS) of 9.8 months (95%CI 0.0, 21.5) and disease control rate (DCR) of 68.8% (11/16). Eight patients received anti-BRAF targeted therapy (including dabrafenib, trametinib and vemurafenib) in the first-line and showed superior efficacy than those received chemotherapy (median PFS, 15.9 months [95%CI, 4.5, 27.3] vs. 9.8 months [95%CI, 0.0, 21.5], P = 0.183; DCR, 100% vs. 68.8%, P = 0.130). As for distinct molecular subsets, most patients with V600E mutation were female (19/31, 61.3%) and non-smokers (19/31, 61.3%), while four of five (80.0%) patients with non-V600E mutation were male and smokers. All of the 3 patients in non-V600E mutations subgroup with recurrent or metastatic disease received chemotherapy in the first-line, and achieved 2 of SD, 1 of PD. NSCLC with BRAF mutation was associated with specific characteristics, which were variable between molecular subsets. BRAF inhibitors should be considered firstly in treating patients with BRAF-mutated lung cancers. The prognosis value of non-V600E mutations and treatment selection needs more research.