Harboring BRAF V600E Mutation Research Articles

Abstract 4071: Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses

Abstract Introduction Tumor microenvironment plays an essential role in cancer development and progression. In the context of thyroid cancer (TC) it still represents a poorly investigated field. Recent data from in vitro and in vivo studies proposed a model of TC progression promoted by the converging action of thyroid tumor cells and cancer-associated fibroblasts (CAFs). According to this mouse model CAFs are recruited in tumor stroma where synthesize and deposit collagen (COL1A1) that is, in turn, cross-linked by the enzyme LOX, produced by the thyroid tumor cells. Collectively this coordinated action lead to matrix stiffness and TC progression. Interestingly, this process is reported to be specifically observed in murine thyroid tumors expressing BRAFV600E mutation, but not in those harboring RAS mutation. In this study we tested this cross-talk in human thyroid cancer tissues. Experimental procedures A series of thyroid specimens, comprising different tumor subtypes (PTC and PDTC) and histologic variants, was collected at our institution and investigated by immunohistochemical (IHC) analyses for the expression of α-SMA (marker for activated fibroblasts), collagen COL1A1 and LOX. Tumor samples were also screened for the most common genetic lesions reported in TC including BRAFV600E and N/H/KRAS mutations. Publicly available datasets of thyroid tumors and non-neoplastic thyroid controls, analyzed on Affymetrix HG-U133 Plus 2.0 array, were selected to investigate the expression profiles of α-SMA, COL1A1 and LOX genes. Robust Multi Array Average (RMA) normalization, Brain Array annotation and batch correction procedures were applied to obtain gene expression profiles across all samples. Results In IHC analyses α-SMA decoration was observed in the stroma of thyroid tumor tissues while was restricted to blood vessels in the adjacent non-neoplastic thyroid. Stromal α-SMA staining was not evenly distributed in the tumors but localized preferentially at the tumor invasive border, organized in peripheral structures as fibrous septa or capsule. In agreement with the proposed model, higher level of α-SMA staining were specifically observed in PTC tumors harboring BRAFV600E mutation compared with tumors harboring RAS mutation. IHC analyses on tissue serial sections confirmed the coordinated expression of α-SMA, COL1A1 and LOX; COL1A1 localized in tumor stroma and was closely correlated with α-SMA positive areas while LOX was expressed by thyroid tumor cells. The coordinated expression of the three genes was confirmed by meta-analysis of public datasets. Conclusions In this study we provide evidence of the coordinated presence of thyroid tumor cells, activated fibroblasts and proteins of extracellular matrix, such as COL1A1 and LOX, in the contest of human thyroid carcinoma. Citation Format: Emanuela Minna, Silvia Brich, Katia Todoerti, Silvana Pilotti, Federica Perrone, Nicholas Paielli, Luca Agnelli, Ilaria Bersani, Roberta Mortarini, Andrea Anichini, Paola Collini, Antonino Neri, Angela Greco, Maria Grazia Borrello. Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4071.

BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations.

Dabrafenib and trametinib are approved for the management of advanced non-small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 BRAF alterations in lung cancer. A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements. Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations (P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated. This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BRAF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

105 Assessing CEACAM1 blockade as a therapeutic strategy using a CRISPR-Cas9-based mouse melanoma model

Current standard of care for advanced-stage melanoma includes immune checkpoint blockade (ICB) and kinase inhibitors. Unfortunately, melanomas treated with kinase inhibitors typically develop resistance; therefore, ICB has become first line therapy due to more durable responses. However, currently available ICBs, such as anti-CTLA-4 and anti-PD-1/PD-L1, engender durable responses in only a subset of patients, demanding a better understanding of other immune checkpoints as potential therapeutic targets. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed on the surface of activated (but not naïve) T cells and on many types of tumor cells, has been found to contribute to tumor progression and immune evasion. We examine the relevance of CEACAM1 in a genetically engineered mouse melanoma model harboring BRAFV600E mutation, which represents 40-60% of human melanomas. In this model, melanomas are induced by activation of mutant BRAF and neonatal exposure to UV irradiation. We have detected high expression of CEACAM1 at RNA and protein levels in UV-BRAF melanomas. We hypothesize CEACAM1 is involved in immune evasion, and its inhibition alone or in combination with PD-1/PD-L1 and CTLA-4 blockade could be a new strategy to treat melanoma. To test our hypothesis, we developed an inducible CRISPR-Cas9 system to knockout CEACAM1 expression in UV-BRAF melanoma cells. We are injecting these cells into syngeneic immunocompetent mice to evaluate the effects of CEACAM1 knockdown on tumor growth, and will use immunodeficient mice to determine the implication of the adaptive immune system in our findings. Future preclinical studies will include the combination of anti-PD-L1 and anti-CTLA-4 treatments with CEACAM1 inhibition to elucidate potential synergistic effects of these ICBs. Overall, we have validated CEACAM1 expression in a mouse model representing a large cohort of human melanoma and created CEACAM1 knockout mouse melanoma cells for mechanistic studies. Amid current immunotherapies, blockade of CEACAM1 may offer a promising alternative treatment strategy for advanced melanoma.

Utility of BRAF VE1 Immunohistochemistry as a Screening Tool for Colorectal Cancer Harboring BRAF V600E Mutation.

BackgroundBRAF mutation has been recognized as an important biomarker of colorectal cancer (CRC) for targeted therapy and prognosis prediction. However, sequencing for every CRC case is not cost-effective. An antibody specific for BRAF V600E mutant protein has been introduced, and we thus examined the utility of BRAF VE1 immunohistochemistry for evaluating BRAF mutations in CRC.MethodsFifty-one BRAF-mutated CRCs and 100 age and sexmatched BRAF wild-type CRCs between 2005 and 2015 were selected from the archives of Asan Medical Center. Tissue microarrays were constructed and stained with BRAF VE1 antibody.ResultsForty-nine of the 51 BRAF-mutant CRCs (96.1%) showed more than moderate cytoplasmic staining, except for two weakly stained cases. Six of 100 BRAF wild-type cases also stained positive with BRAF VE1 antibody; four stained weakly and two stained moderately. Normal colonic crypts showed nonspecific weak staining, and a few CRC cases exhibited moderate nuclear reactivity (3 BRAF-mutant and 10 BRAF wild-type cases). BRAF-mutated CRC patients had higher pathologic stages and worse survival than BRAF wild-type patients.ConclusionsBRAF VE1 immunohistochemistry showed high sensitivity and specificity, but occasional nonspecific staining in tumor cell nuclei and normal colonic crypts may limit their routine clinical use. Thus, BRAF VE1 immunohistochemistry may be a useful screening tool for BRAF V600E mutation in CRCs, provided that additional sequencing studies can be done to confirm the mutation in BRAF VE1 antibody-positive cases.

FDA Approval Summary: Dabrafenib and Trametinib for the Treatment of Metastatic Non-Small Cell Lung Cancers Harboring BRAF V600E Mutations.

The approvals of dabrafenib and trametinib, administered concurrently, provide a new regimen for the treatment of a rare subset of non-small cell lung cancer (NSCLC) and demonstrate how drugs active for treatment of BRAF-mutant tumors in one setting predict efficacy and can provide supportive evidence for approval in another setting. The FDA also approved the first next-generation sequencing oncology panel test for simultaneous assessment of multiple actionable mutations, which will facilitate selection of optimal, personalized therapy. The test was shown to accurately and reliably select patients with NSCLC with the BRAF V600E mutation for whom treatment with dabrafenib and trametinib is the optimal treatment.

Prognostic Role of BRAFV600E Cellular Localization in Melanoma

Approximately half of cutaneous melanoma tissues harbor BRAFV600E mutations, resulting in a constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Nuclear-cytoplasmic transport machinery is dysregulated in neoplastic cells and alters the key regulatory proteins that can lead to tumor progression and drug resistance. The significance of nuclear localization of BRAFV600E has not been fully understood. We examined the clinical significance of intracellular localization of BRAFV600E in cutaneous melanoma. Immunohistochemical analysis of BRAFV600E was performed on formalin-fixed, paraffin-embedded specimens of cutaneous melanoma (n= 91). Staining intensity was graded in a blinded manner. Correlations to clinical factors were analyzed by Fisher's exact test and 2-tailed t-test. Localization of BRAFV600E was determined in melanoma cells, and we investigated their resistance to BRAFV600E-specific inhibitor according to nuclear localization in both invitro and invivo models. We included 91 patients, of whom 32% (29 of 91) had cytoplasmic BRAFV600E. Nuclear BRAFV600E was observed in 30% (27 of 91). Overall, BRAFV600E expression correlated with TNM stage (p=0.011), mitotic activity (p= 0.010), and ulceration (p= 0.045). Nuclear BRAFV600E expression correlated with overall clinical stage (p < 0.001), tumor size (p < 0.001), regional lymph node (p<0.017), depth of invasion (p= 0.005), Clark level (p < 0.001), mitotic activity (p<0.001), ulceration (p < 0.001), and margin status (p= 0.017). On a cellular level, BRAFV600E was identified in the nucleus, and its translocation was serum dependent. Our invitro and invivo data revealed sequestration of BRAFV600E in the cytosol-sensitized resistant cells to vemurafenib; nuclear retention of BRAFV600E was associated with aggressiveness and drug resistance. Nuclear localization of BRAFV600E is associated with melanoma aggressiveness. Further multi-institutional studies are warranted to confirm the clinical relevance of nuclear localization of BRAFV600E.

Vemurafenib.

The activating BRAF mutation V600E and related mutations in this codon are most important for the activation of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signalling pathway in melanoma. BRAF V600E mutations have been detected in ~40% of melanoma patients and BRAF V600K mutations in ~5% of melanoma patients. Activation of the MAPK pathway results in continuous stimulation of cell proliferation and inhibits programmed cell death. Vemurafenib (PLX4032) was developed as a low-molecular-weight molecule for the inhibition of the mutated serine-threonine kinase BRAF, and it selectively binds to the ATP-binding site of BRAF V600E kinase and inhibits its activity. The biochemical affinity of vemurafenib for mutated BRAF translates to potent inhibition of ERK phosphorylation and of cell proliferation exclusively in BRAF-mutant cell lines. In animal model experiments, it was demonstrated that vemurafenib achieved tumour regressions in cells harbouring the BRAF V600E mutation. The clinical trials with vemurafenib in unresectable metastatic melanoma in phases I, II and III for patients harbouring BRAF V600E mutations demonstrated all unexpected high objective response rates ranging between 50 and 80%. Median progression-free survival was prolonged from 2 months with dacarbazine to 7 months with vemurafenib, and median overall survival was, respectively, prolonged from 9 to 14months. A major problem remains in the development of resistance to vemurafenib treatment after several months in the majority of patients, and multiple resistance mechanisms have already been described. Under vemurafenib treatment, about 25% of patients developed cutaneous squamous cell carcinomas of the keratoacanthoma type with low invasive potential and without the occurrence of metastasis. The overall tolerability of the drug was quite good, and many patients remained on treatment for long times. As other solid tumours like papillary thyroid cancer, colorectal cancer, non-small-cell lung cancer and ovarian cancer likewise harbour BRAF mutation, vemurafenib is also tested in these entities. In future, combinations of vemurafenib with other kinase inhibitors and with immunotherapies will improve its therapeutic potential.

Rational Approaches for Combination Therapy Strategies Targeting the MAP Kinase Pathway in Solid Tumors.

Molecular characterization of oncogenic mutations within genes in the MAPK and PI3K/AKT/mTOR pathways has led to the rational development of targeted therapies. Combining BRAF and MEK inhibitors to target two steps in the MAPK pathway (vertical inhibition) is now standard of care in advanced-stage melanoma harboring BRAF V600 mutation. Encouraging results have been seen in several tumor types with the same mutation, including BRAF V600-mutant non-small cell lung cancer. Yet similar results in other tumors, such as colorectal cancer, have not been observed, highlighting the unique nature of different tumors. Furthermore, considerable cross talk occurs between signaling pathways, and cancer cells usually harbor multiple aberrations and/or develop compensatory mechanisms that drive resistance. Therefore, it is logical to target multiple pathways simultaneously (horizontal inhibition) by combining selective inhibitors or engineering multitargeted agents. Yet horizontal inhibition has proven to be a significant challenge, primarily due to dose-limiting toxicities. This review focuses on ongoing or completed clinical trials with combination targeted therapies for solid tumors and highlights the successes and ongoing challenges. Novel strategies to overcome these obstacles include new delivery technologies, combinations with emerging agents, and treatment schedule optimization. Mol Cancer Ther; 17(1); 3-16. ©2017 AACR.

Abstract IA15: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to &amp;gt;30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA15.

Dabrafenib in combination with trametinib in the treatment of patients with BRAF V600-positive advanced or metastatic non-small cell lung cancer: clinical evidence and experience.

Mutations in the BRAF oncogene are found in 2–4% of all non-small cell lung cancer (NSCLC) patients. The most common activating mutation present within the BRAF oncogene is associated with valine substitution for glutamate at position 600 (V600E) within the BRAF kinase. BRAF-targeted therapies are effective in patients with melanoma and NSCLC harboring BRAF V600E mutation. In both melanoma and NSCLC, dual inhibition of both BRAF and the downstream mitogen-activated protein kinase (MEK) improves response rates compared with BRAF inhibition alone. BRAF-MEK combination therapy (dabrafenib plus trametinib) demonstrated tolerability and efficacy in a recent phase II clinical trial and was approved by the European Medicines Agency and United States Food and Drug Administration for patients with stage IV NSCLC harboring BRAF V600E mutation. Here, in this review, we outline the preclinical and clinical data for BRAF and MEK inhibitor combination treatment for NSCLC patients with BRAF V600E mutation.

Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy

BackgroundCoexistence of enhancer of zeste homolog 2 (EZH2) and BRAF gene aberrations has been described in many cancer types. In this study, we aim to explore the coexistence status of BRAF V600E mutation and the copy number variation of EZH2 and explore the potential of this combination as a therapeutic target.MethodsA total of 138 cases of melanoma samples harboring BRAF V600E mutation were included, and EZH2 copy numbers were examined by QuantiGenePlex DNA Assays. Clinical pathological distinction between patient groups with or without EZH2 amplification (hereafter referred to as EZH2 gain) was statistically analyzed. The sensitivity of melanoma cell lines and patient-derived xenograft (PDX) models containing BRAF V600E mutation with or without EZH2 gain to vemurafenib (BRAF inhibitor), GSK2816126 (EZH2 inhibitor) and a combination of both agents was evaluated.ResultsIn our cohort, the coexistence rate of BRAF V600E mutation and EZH2 gain was up to 29.0%, and significant differences in overall survival and disease-free survival were found between no EZH2 copy number gain and gain groups (P = 0.038, P = 0.030), gain and high EZH2 copy number gain groups (P = 0.006, P = 0.010). Combination with BRAF and EZH2 inhibition showed better inhibitory efficacy in melanoma prevention compared with vemurafenib monotherapy. More importantly, this improved therapeutic effect was observed especially in melanoma cell lines and PDX models containing concurrently BRAF V600E mutation and EZH2 gain.ConclusionsCoexistence of BRAF V600E mutation and EZH2 gain is rather prevalent in melanoma. Our findings provided evidence for the feasibility of combination therapy with EZH2 and BRAF inhibitors in melanoma with concurrent BRAF V600E mutation and EZH2 gain.

Universal determination of microsatellite instability using BAT26 as a single marker in an Argentine colorectal cancer cohort.

Microsatellite instability (MSI) is a hallmark tool for Lynch syndrome (LS) screening and a prognostic marker for sporadic colorectal cancer (CRC). In regions with limited resources and scarce CRC molecular characterization as South America, the implementation of universal MSI screening is under debate for both its purposes. We sought to estimate the frequency of BAT26 in colorectal adenocarcinomas and to determine associated clinical and histological features. Consecutive patients from a CRC registry were included. BAT26 determination was performed in all cases; if instability was found, immunohistochemistry (IHC) and BRAF mutation analyses were done, as appropriate. Differences were assessed by chi-squared or Fisher's exact test, or by T test or Mann-Whitney. Multiple logistic regression was used to identify factors independently associated with BAT26-unstable tumors. We included 155 patients; mean age was 65.6 (SD 14.4) and 56.1% were male. The frequency of BAT26-unstable tumors was 22% (95% CI 15.7-29.3). Factors independently associated with BAT26-unstable tumors were right colon localization (OR 3.4, 95% CI 1.3-8.7), histological MSI features (OR 5.1, 95% CI 1.9-13.6) and Amsterdam criteria (OR 23.2, 95% CI 1.9-286.7). IHC was altered in 85.3% BAT26-unstable tumors and 70.6% lacked MLH1 expression; 47.8% of these harbored BRAF V600E mutation. We provide evidence to link the frequency of BAT26 to an increased diagnostic yield (up to 1.4-folds) of suspected LS cases in comparison to the revised Bethesda guidelines alone. In regions with limited resources, clinical and histological features associated with BAT26-unstable status could be useful to direct MSI screening in sporadic CRCs and may help guide clinical care and future research.

NIS expression in thyroid tumors, relation with prognosis clinicopathological and molecular features.

Thyroid cancer therapy is based on surgery followed by radioiodine treatment. The incorporation of radioiodine by cancer cells is mediated by sodium iodide symporter (NIS) (codified by the SLC5A5 gene), that is functional only when targeted to the cell membrane. We aimed to evaluate if NIS expression in thyroid primary tumors would be helpful in predicting tumor behavior, response to therapy and prognosis. NIS expression was addressed by qPCR and immunohistochemistry. In order to validate our data, we also studied SLC5A5 expression on 378 primary papillary thyroid carcinomas from The Cancer Genome Atlas (TCGA) database. In our series, SLC5A5 expression was lower in carcinomas with vascular invasion and with extrathyroidal extension and in those harboring BRAFV600E mutation. Analysis of SLC5A5 expression from TCGA database confirmed our results. Furthermore, it showed that larger tumors, with locoregional recurrences and/or distant metastases or harboring RAS, BRAF and/or TERT promoter (TERTp) mutations presented significantly less SLC5A5 expression. Regarding immunohistochemistry, 12/211 of the cases demonstrated NIS in the membrane of tumor cells, those cases showed variable outcomes concerning therapy success, prognosis and all but one were wild type for BRAF, NRAS and TERTp mutations. SLC5A5 mRNA lower expression is associated with features of aggressiveness and with key genetic alterations involving BRAF, RAS and TERTp. Mutations in these genes seem to decrease protein expression and its targeting to the cell membrane. SLC5A5 mRNA expression is more informative than NIS immunohistochemical expression regarding tumor aggressiveness and prognostic features.

Abstract IA23: Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers

Abstract Personalized cancer medicine approaches, inhibiting kinases in tumors driven by defined genomic alterations, have demonstrated striking efficacy in many cancer types. However, the development of drug resistance is a major limitation to the efficacy of targeted therapies in oncology. Identifying and understanding the molecular mechanisms driving resistance may foster opportunities to develop therapeutic strategies to overcome resistance. For example, in the ~10% of colorectal cancer patients whose tumors harbor BRAF V600 mutations, we have found that feedback reactivation of MAPK signaling, often mediated by EGFR, underlies the relative insensitivity of these cancers to RAF inhibitors. The development of targeted therapy combination strategies to block feedback reactivation of MAPK signaling has led to marked improvements in response rates for these patients from ~5% to &amp;gt;30% over the past few years. However, in patients who respond to these targeted combinations, the rapid development of acquired resistance still limits clinical benefit. Acquired resistance in BRAF mutant colorectal cancer and in other molecularly-defined tumor types can be marked by the development of extensive molecular heterogeneity due to the selection of sub-clonal tumor cell populations, capable of growing under drug pressures, which poses significant diagnostic and therapeutic challenges. We will present data demonstrating how a single-lesion biopsy at disease progression to diagnose the mechanism of acquired resistance can vastly underrepresent the molecular heterogeneity of resistant tumor clones in an individual patient, and may fail to detect the existence of distinct but important resistance mechanisms that can drive mixed or lesion-specific responses and treatment failure to subsequent targeted therapy. By contrast, liquid biopsy approaches analyzing circulating tumor DNA have the potential to detect the presence of simultaneous resistance mechanisms residing in separate metastases in a single patient and to monitor the effects of subsequent therapies on specific sub-clonal tumor cell populations. These findings highlight the critical role of tumor heterogeneity in driving therapeutic resistance and how convergent targeted therapy strategies blocking common signaling nodes capable of overcoming multiple resistance mechanisms may be needed. Citation Format: Ryan B. Corcoran. Overcoming drug resistance and tumor heterogeneity in gastrointestinal cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA23.

BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAFV600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.

Abstract 2920: Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies

Abstract Introduction: Metastatic melanoma that harbor BRAFV600E mutations are treated with inhibitors that inhibit MAPK signaling downstream of BRAF. Although these treatments improve patient survival, the majority of tumors develop resistance. It is increasingly clear that the presence and emergence of cell subsets in the tumor microenvironment shapes treatment responses. Mass cytometry is a single cell platform capable of measuring &amp;gt;35 proteins on millions of tumor cells. Here, mass cytometry was used to characterize matched tumor samples obtained before, during, and after therapy in order to quantify the impact of therapy on tumor cell populations. Methods: Melanoma tumors from adults with metastases were biopsied or surgically resected from 12 patients with consent. Patients received two weeks of BRAFV600E inhibitor dabrafenib followed by two weeks of dabrafenib and MEK inhibitor trametinib. Tumors were enzymatically digested and cryopreserved with protocols for mass cytometry (Leelatian and Doxie et al., Cytometry B 2016). Samples were stained with a 30+ antibody panel focused on melanoma identity and markers of lineage and trafficking for leukocytes, fibroblast, and endothelial cells. Viable nucleated cells were identified by total histone H3 and rhodium dye exclusion. Expert biaxial gating and viSNE analysis were used to identify, computationally map, and track populations and their subsets over time. Tissue microarrays were also analyzed by histology to confirm findings observed by mass cytometry. Results: More than 99% of cells were characterized and fell into one of the four major populations. Melanoma phenotypes included loss of MHC class I and/or expression of one or more markers of identity including SOX10, SOX2, nestin, S100β, and MCAM. Significant decreases in nestin, MCAM, MHC I and S100β (p &amp;lt; 0.01, p &amp;lt; 0.05, p &amp;lt; 0.05, and p&amp;lt;0.05, respectively) were observed in cells after treatment. Decreases in nestin and S100β populations identified by mass cytometry were observed in histology analysis of TMAs from matched tumors. Analysis of pre- and post-therapy melanoma cells and BRAF mutated cell lines indicated pre-therapy tumor cells had phenotypes most similar to cell lines. Strikingly, 6hrs or 1 day of treatment of cell lines with dabrafenib and trametinib did not result in decrease in nestin expression. The nestin low post-therapy phenotype of melanoma tumor cells was present in all melanoma cell lines at a low abundance (average of 7.0% +/- 3.9% in cell lines, N = 7). Conclusions: Single cell analysis and longitudinal monitoring of patient’s revealed tumor ecosystems experience rapid changes in cellular diversity during targeted therapy. Lower nestin expression distinguished treated melanoma tumor cells. The in vitro results suggest that in vivo remodeling of the tumor microenvironment results from a shift in host-tumor cellular interactions and not a direct response to inhibitors. Citation Format: Deon B. Doxie, Allison R. Greenplate, Kirsten E. Diggins, Caroline E. Maier, Jeffrey A. Sosman, Mark C. Kelley, Jonathan M. Irish. Characterizing human melanoma treatment response in vivo using single-cell mass cytometry analysis of longitudinal tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2017-2920

Abstract 3163: Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma

Abstract Introduction RAF-MAPK pathway inhibition with BRAF inhibitors or the combination of BRAF and MEK inhibitors has become the mainstay of therapy of metastatic melanoma harboring BRAF V600 mutations with a response rate as high as 50-75 %. A minority of patients present with primary resistance while all patients develop secondary resistance. We explored a reversed translational approach using a novel multiplex kinase assay to first identify individuals not responding to therapy using baseline biopsy samples and second to understand the mechanism of resistance using patient-derived cell lines. Method Pre-dose tissues from 4 non-responders and 3 responders to vemurafenib monotherapy, all harboring the V600E mutation, were profiled for Serine-Threonine Kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of dabrafenib. For molecular studies three melanoma cell lines, all with BRAF V600E mutations, were made resistant to vemurafenib. Protein Tyrosine Kinase (PTK) and STK activities in lysates of sensitive or resistant cells were measured +/- dabrafenib, trametinib or both. Lysates were blotted against a selected panel of (phospho)proteins. Results In tissue lysates, concentration-dependent ex vivo inhibition with dabrafenib was stronger in the patients that were clinical responders than in non-responders. This difference in inhibition of STK activity by dabrafenib was confirmed in the cell lines that were made resistant against vemurafenib. IC50s for the sensitive cell lines were below 0.5 µM and varied from 10-21 µM for cell lines with acquired resistance. PTK activity was increased in resistant cell lines. STK activity was lower, but large differences exist among the cell lines. Common features of resistance were increased activity of receptor tyrosine kinases, Src family kinases and AKT signaling. These results were confirmed by Western blot analysis. Dabrafenib (10 µM) caused inhibition of STK activity while trametinib (10 µM) gave some activation. Dabrafenib (0.5 µM) gave strong inhibition of PTK activity, both in tissue and cell lines. Trametinib had hardly any inhibitory effect. Interestingly, the combination of dabrafenib and trametinib had an antagonistic effect on the STK activity and a synergistic effect on PTK activity, resulting in a stronger inhibition of kinase activity. Conclusion Resistance of tumors and cell lines to vemurafenib can affect kinase activity profiles as detected pre-dose using a multiplex kinase assay. Dabrafenib inhibition of PTK and STK activity is stronger in sensitive cell lines. The overall activation of kinases in a lysate upon MEK inhibition suggests MEK as suppressor of kinase activity. Resistance to MAPK pathway inhibitors could be linked to an increase in tyrosine kinase activity and AKT pathway activity. Biomarkers are needed to identify patients in need of combined MAPK pathway inhibition. Citation Format: Riet Hilhorst, Mohammed Krayem, Adrienne van den Berg, Fabrice Journe, Liesbeth Hovestad-Bijl, Tim van den Hooven, Rik de Wijn, Philippe Aftimos, Ghanem Ghanem, Rob Ruijtenbeek. Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2017-3163

Abstract 1225: A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors

Abstract The FDA approved RAF inhibitors vemurafenib and dabrafenib have elicited profound responses in melanoma patients with tumors harboring BRAFV600E mutation, but resistance limits their effectiveness. Furthermore, RAF inhibitors exhibit only modest efficacy in colorectal and thyroid BRAFV600E tumors and they are not effective in tumors harboring non-V600 BRAF mutations. While RAF/MEK inhibitor combinations demonstrate increased clinical efficacy, drug resistance develops as well, through similar mechanisms to those identified in RAF inhibitor monotherapy. This suggests that insufficient RAF inhibition limits treatment efficacy and indicates the need for more effective therapeutic approaches to potently and durably target RAF/ERK signaling. Although the unique biochemical properties of RAF inhibitors have been a topic of intensive investigation, a unified model of RAF inhibitor action has been lacking. Our biochemical and structural analysis in a panel of RAF inhibitors with different structural properties revealed that they exert their effects via the combined outcome of two distinct allosteric mechanisms. RAF priming and dimerization are the result of RAF/RAS-GTP interaction upon inhibitor binding to RAF and stabilization of the αC-helix in the IN (active) position. Resistance of the dimeric RAF to vemurafenib and dabrafenib is the result of stabilization of the αC-helix of the drug-bound protomer towards the OUT (inactive) position, a conformation that sterically prevents occupancy of both RAF protomers. Next generation RAF inhibitors that stabilize the αC-helix of RAF towards the IN position (αC-IN RAF inhibitors) allow occupancy of both RAF protomers and thus accomplish potent inhibition of the dimeric form of RAF. We further provide structural, biochemical and cell-based evidence that next generation αC-IN RAF inhibitors (such as MLN480, TAK632, LY3009120, AZ628 and others) will be more effective for the treatment of patients with tumors expressing non-V600 BRAF mutants and in BRAFV600E colorectal and thyroid tumors. However, αC-IN RAF inhibitors are predicted to have a low therapeutic window, since they inhibit dimeric wild-type BRAF also in normal cells. Together our findings provide a blueprint for the development of RAF inhibitors with defined biochemical properties and support therapeutic strategies in which relatively lower, non-toxic concentrations of αC-IN inhibitors will be administered in combination with currently approved RAF and MEK inhibitor regimens for the treatment of BRAFV600E melanoma, colorectal and thyroid cancers, as well as for tumors with BRAF mutations other than V600. Citation Format: Zoi Karoulia, Yang Wu, Tamer A. Ahmed, Qisheng Xin, Julien Bollard, Clemens Krepler, Xuewei Wu, Chao Zhang, Gideon Bollag, Meenhard Herlyn, James A. Fagin, Amaia Lujambio, Evripidis Gavathiotis, Poulikos I. Poulikakos. A unified model of RAF inhibitor action determines inhibitor activity in BRAF-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2017-1225

Clinical responses to ERK inhibition in BRAFV600E-mutant colorectal cancer predicted using a computational model

Approximately 10% of colorectal cancers harbor BRAFV600E mutations, which constitutively activate the MAPK signaling pathway. We sought to determine whether ERK inhibitor (GDC-0994)-containing regimens may be of clinical benefit to these patients based on data from in vitro (cell line) and in vivo (cell- and patient-derived xenograft) studies of cetuximab (EGFR), vemurafenib (BRAF), cobimetinib (MEK), and GDC-0994 (ERK) combinations. Preclinical data was used to develop a mechanism-based computational model linking cell surface receptor (EGFR) activation, the MAPK signaling pathway, and tumor growth. Clinical predictions of anti-tumor activity were enabled by the use of tumor response data from three Phase 1 clinical trials testing combinations of EGFR, BRAF, and MEK inhibitors. Simulated responses to GDC-0994 monotherapy (overall response rate = 17%) accurately predicted results from a Phase 1 clinical trial regarding the number of responding patients (2/18) and the distribution of tumor size changes (“waterfall plot”). Prospective simulations were then used to evaluate potential drug combinations and predictive biomarkers for increasing responsiveness to MEK/ERK inhibitors in these patients.

Efficacy and tolerability of vemurafenib in BRAF-mutant acral and mucosal melanoma.

e21017 Background: Vemurafenib has substantially impressive clinical efficacy in cutaneous melanoma (CM). However, compared with their cutaneous counterpart, acral melanoma (AM) and mucosal melanoma (MM) have distinct genetic patterns and worse prognosis. The efficacy and safety profiles of vemurafenib in AM and mm remain unclear. Methods: Clinical, pathological, and therapeutic data were collected and reviewed of patients (pts) with metastatic or unresectable BRAF-mutant AM and mm hospitalized and administrated vemurafenib during January 2011 and January 2017. Responses were evaluated by RECIST v1.1, survival data were analyzed by Kaplan-Meier survival curve, and adverse effects were assessed by CTCAE v4.0. Results: 24 pts were identified, 13 (54.2%) with AM, 11 (45.8%) with mm (1 patient without available radiology data). All 13 AM pts harbored BRAF V600E mutation (1 with simultaneous C-KIT E861K mutation); 10/11 (90.9%) mm pts carried BRAF V600E mutation (1 with simultaneous PDGFRA V824I mutation), 1/11 (9.1%) had BRAF D594N mutation. Median PFS were 5.4 (95%CI 3.5-8.7) and 4.5 (95%CI 1.5-15.0) months, median OS were 11.7 (95%CI 8.1-23.6) and 7.9 (95%CI 4.6-26.2) months; ORRs were 61.5% (8/13) and 40.0% (4/10), DCRs were 92.3% (12/13) and 90.0% (9/10) in AM and MM, respectively. Vemurafenib was well tolerated. The most common adverse effects (AEs) were hand-foot syndrome in AM and elevation of total cholesterol in MM, with the incidence of 23.1% (3/13) and 18.2% (2/11), respectively. Grade 3/4 AEs have not been observed. No patient discontinued vemurafenib because of treatment related toxicity. Conclusions: Vemurafenib yields substantial response in pts with AM and MM. Compared with in CM, vemurafenib has similar efficacy and safety profiles in BRAF-mutant AM and MM.