Activating BRAF V600E Mutations Research Articles

Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease

UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.

Abstract P4-01-06: Genomic and transcriptomic analysis reveals known and novel resistance mechanisms to CDK4/6 inhibitors and sensitivity factors for the response to triplet therapy (palbociclib + everolimus + exemestane) in a phase I/IIb study in hormone-receptor positive (HR+)/HER2- metastatic breast cancer (MBC) after progression on a CDK4/6 inhibitor (CDK4/6i)

Abstract Background: Multiple studies in HR+/HER2- MBC have identified a variety of genomic resistance mechanisms to CDK4/6 inhibitors, but the complete landscape of resistance mechanisms is still being elucidated. A clinical trial evaluating the benefit of continued CDK4/6 blockade after disease progression on a prior CDK4/6i provides a unique setting to study the landscape of resistance to CDK4/6 inhibitors. Methods: We analyzed genomic data from a Phase I/II trial (NCT02871791) of triplet therapy: palbociclib (CDK4/6i) + everolimus (mTOR inhibitor) + exemestane (endocrine therapy) in patients (pts) with HR+/HER2− MBC who had progressed on prior CDK4/6i. For the phase IIa pts, a research tumor biopsy at baseline and serial research blood collection for circulating tumor DNA (ctDNA) analysis were mandatory. Additionally, when possible, we acquired tumor biopsies that preceded the patient's prior exposure to a CDK4/6i, which would allow us to identify acquired genomic resistance mechanisms to the prior CDK4/6i. The genomic data consisted of whole exome sequencing (WES) data from 23 tumor biopsies (19 pts) and 17 ctDNA samples (12 pts), and RNA sequencing (RNA-seq) from 27 tumors (22 pts). 4 pts had a biopsy or ctDNA sample at baseline and a biopsy that preceded their prior exposure to a CDK4/6i. WES data was used to identify mutations and copy number alterations, which was used to perform evolutionary analysis on the pts with multiple biopsies or ctDNA samples. RNA-seq data was used to make research-grade PAM50 calls and calculate gene expression signature scores. Results: For the baseline biopsy or ctDNA sample of most pts, we found genomic alterations in previously identified pathways and genes that could explain the tumor’s resistance to the prior CDK4/6i (16/19 pts) or to the prior endocrine therapies (17/19 pts). These pathways and genes include the PI3K/AKT/MTOR pathway (e.g. PTEN, AKT), the RAS/MAPK pathway (e.g. NF1), receptor tyrosine kinases (RTKs) (e.g. ERBB2, FGFR1), cell-cycle genes (e.g. RB1), and estrogen receptor signaling (e.g. ESR1, FOXA1). Two novel potential genomic resistance mechanisms in these pathways were identified: an activating MTOR T1977R mutation (PI3K/AKT/MTOR pathway) and an activating BRAF V600E mutation (RAS/MAPK pathway). Notably, the patient with the activating MTOR mutation responded to the triplet therapy (progression free survival of 8 months), consistent with prior work linking these mutations to sensitivity to everolimus. Evolutionary analysis revealed metastatic tumors with distinct lineages but derived from the same primary tumor (e.g. two lineages, one with activating ESR1 mutations and one with an activating MTOR mutation), some of which converged to activating the same pathway (e.g. two lineages with distinct activating ERBB2 mutations). Transcriptomic analysis found that activating mutations in ERBB2 and BRAF were correlated with the HER2-E PAM50 and that the expression signatures for MTOR and RTKs were correlated with clinical benefit to triplet therapy. Conclusions: Analysis of the genomic and transcriptomic data of baseline biopsies and ctDNA samples from NCT02871791 not only recapitulates genes and pathways previously implicated in resistance to endocrine therapy and CDK4/6i but also identified novel potential mechanisms of resistance including activating mutations in BRAF and MTOR. Evolutionary analysis demonstrates the complexity of resistance including both convergent and divergent paths to resistance. Integration of genomic and transcriptomic data may better identify pts likely to respond to CDK4/6i combinations. Citation Format: Jorge Gomez Tejeda Zanudo, Romualdo Barroso-Sousa, Esha Jain, Jorge Buendia-Buendia, Tianyu Li, Nabihah Tayob, Rebecca Rees, Alyssa Pereslete, Arlindo R. Ferreira, Daniel L. Abravanel, Karla Helvie, Ann H. Partridge, Beth Overmoyer, Eric P. Winer, Nikhil Wagle, Sara M. Tolaney. Genomic and transcriptomic analysis reveals known and novel resistance mechanisms to CDK4/6 inhibitors and sensitivity factors for the response to triplet therapy (palbociclib + everolimus + exemestane) in a phase I/IIb study in hormone-receptor positive (HR+)/HER2- metastatic breast cancer (MBC) after progression on a CDK4/6 inhibitor (CDK4/6i) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-06.

Expression of chondroitin sulfate proteoglycan4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

Safety and Preliminary Efficacy Results from a Phase II Study Evaluating Combined BRAF and MEK Inhibition in Relapsed/Refractory Multiple Myeloma (rrMM) Patients with Activating BRAF V600E Mutations: The GMMG-Birma Trial

Safety and Preliminary Efficacy Results from a Phase II Study Evaluating Combined BRAF and MEK Inhibition in Relapsed/Refractory Multiple Myeloma (rrMM) Patients with Activating BRAF V600E Mutations: The GMMG-Birma Trial

An Animal Model Further Uncovers the Role of Mutant BrafV600E during Papillary Thyroid Cancer Development

Papillary thyroid carcinomas (PTCs) account for 90% of human thyroid cancer cases, which represent 1% of all cancer cases. They are likely to develop from papillary thyroid microcarcinomas (PTMCs), found in up to 36% of healthy individuals, due to rare progression events (0.01%). Although the prognosis of PTCs is excellent, 5% to 10% of tumors display an unfavorable outcome. About 45% of PTCs exhibit activating BRAFV600E mutations. Rats of the inbred BD strains postnatally exposed to the carcinogen N-ethyl-N-nitrosourea developed PTMCs, which closely resembled their human counterparts judging from their histology, size, and marginal tendency to progress. DNA sequencing revealed mutations in exon 15 of the Braf gene identical to the human BRAFV600E mutation in 82% of the cases. Predominantly a 50:50 ratio of wild-type to mutant Braf alleles was seen regardless of tumor size or animal age, indicating that the Braf mutation is an early, if not the initial, event in rat PTMC development. Surprisingly, most PTMCscarrying a confirmed BrafV600E mutation did not display BrafV600E protein expression. As the BrafV600Egene is supposed to be the driver in PTC development, down-regulation of expression should contribute to the low risk for progression of PTMC. This model system will enable further insights into the molecular mechanisms of PTMC initiation and progression to PTC, further translating into targeted tumor prevention strategies/therapies.

Overcoming acquired osimertinib-resistance in EGFR-mutant advanced non-small lung cancer mediated by activating BRAF V600E mutation.

e20682 Background: There is growing insight in the mechanisms underlying resistance to the 3rdgeneration EGFR inhibitor osimertinib. Unlike resistance to 1stgeneration inhibitors, these mechanisms not necessarily lead to sequential targeted therapy approaches. Here we report on the treatment of two patients with acquired resistance to osimertinib with a new detected BRAF V600E mutation as resistance mechanism. Methods: We identified two patients with EGFR-T790M-mutant advanced NSCLC with progression on osimertinib and detection of a new BRAFV600E mutation in a tumor rebiopsy by next-generation sequencing (NGS). No other known resistance mechanism beside T790M loss in one patient was found. Osimertinib was discontinued and BRAF-targeted combination therapy with dabrafenib and trametinib at standard dose was initiated. We monitored the clinical course with sequential 18F-2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) / computed tomography (CT) assessing maximum standard uptake value (SUVmax), sequencing based liquid biopsies and tumor marker assessment. Results: Patient (1) with EGFR del19 (E746_A750del), preserved T790M mutation and acquired BRAF V600E mutation showed reduction in FDG uptake of 18% after 2 weeks of dabrafenib/trametinib that demonstrated a slight increase of 12% in a FDG-PET/CT scan 4 weeks thereafter and combination treatment has been continued. Patient (2) with EGFR del19 (E746_A750del), T790M loss and new BRAF V600E mutation showed continuous metabolic (+8% and + 39%, respectively) and morphologic progression after 2 and 4 weeks of dabrafenib and trametinib. A tumor rebiopsy showed no additional molecular changes. We changed the treatment to osimertinib and dabrafenib combination and observed an impressive metabolic response (-33%) after 2 weeks by FDG-PET/CT. Conclusions: BRAF V600E mutation has recently been described as a novel molecular resistance mechanism in osimertinib-resistant EGFR-mutant NSCLC. We describe one patient where combined BRAF/MEK inhibition with no additional EGFR-inhibition resulted in a preliminary feasible tumor control, but confirmatory CT staging is pending. In a second patient, co-inhibition of EGFR and BRAF pathway with osimertinib and dabrafenib was needed to overcome BRAF-mediated osimertinib resistance resulting in an impressive early tumor response that was not observed to either single-target inhibition of EGFR or BRAF. FDG-PET/CT was able to monitor tumor dynamics. Updated data will be presented.

A thyroid gland with over 30 foci of papillary thyroid carcinoma with activating BRAF V600E mutation.

SummaryMultifocal papillary thyroid carcinoma (PTC) is common and the number of tumor foci rarely exceeds ten. The mechanism of multifocal disease is debated, with the two main hypotheses consisting of either intrathyroidal metastatic spread from a single tumor or independent multicentric tumorigenesis from distinct progenitor cells. We report the case of a 46-year-old woman who underwent total thyroidectomy and left central neck lymph node dissection after fine-needle aspiration of bilateral thyroid nodules that yielded cytological findings consistent with PTC. Final pathology of the surgical specimen showed an isthmic dominant 1.5 cm classical PTC and over 30 foci of microcarcinoma, which displayed decreasing density with increasing distance from the central lesion. Furthermore, all malignant tumors and lymph nodes harbored the activating BRAF V600E mutation. The present case highlights various pathological features that support a mechanism of intraglandular spread, namely a strategic isthmic location of the primary tumor, radial pattern of distribution and extensive number of small malignant foci and BRAF mutational homogeneity.Learning points:Multifocal papillary thyroid carcinoma (PTC) is commonly seen in clinical practice, but the number of malignant foci is usually limited to ten or less.There is no clear consensus in the literature as to whether multifocal PTC arises from a single or multiple distinct tumor progenitor cells.Strategic location of the dominant tumor in the thyroid isthmus may favor intraglandular dissemination of malignant cells by means of the extensive lymphatic network.An important pathological finding that may be suggestive of intrathyroidal metastatic spread is a central pattern of distribution with a reduction in the density of satellite lesions with increasing distance from the dominant focus.PTCs originating from the isthmus with intraglandular metastatic dissemination behave more aggressively. As such, a more aggressive treatment course may be warranted, particularly with regard to the extent of surgery.

Efficacy and safety of dabrafenib (D) and trametinib (T) in patients (pts) with BRAF V600E–mutated biliary tract cancer (BTC): A cohort of the ROAR basket trial.

187 Background: BTCs are rare, aggressive malignancies with poor prognoses. Treatment options and outcomes after first-line therapy are not well defined. Median progression-free survival (PFS) in second-line BTC is < 5 mo. Combined BRAF + MEK inhibition is efficacious in BRAF V600–mutated anaplastic thyroid cancer, melanoma, and lung cancer, but less so in BRAF V600E-mutated colorectal cancer. Activating BRAF V600E mutations have been reported in 0% to 20% of BTCs; thus, D (BRAF inhibitor) + T (MEK inhibitor) was evaluated as a treatment for pts with BRAF V600E–mutated BTC in the ROAR basket trial. Methods: In this phase II, open-label trial, pts with BRAF V600E mutations in 9 rare tumor types, including BTC, received D (150 mg BID) + T (2 mg QD) until unacceptable toxicity, disease progression, or death. Eligible pts had advanced or metastatic cancer and had been treated with ≥ 1 prior systemic therapy. The primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DOR), PFS, overall survival (OS), biomarker correlates, and safety. Results: Thirty-three pts with BTC had enrolled at data cutoff (January 3, 2018). BRAF V600E mutations were centrally confirmed in 30 of 33 pts with BTC (91%). Median age was 58 y, and 78% had received ≥ 2 prior lines of systemic therapy. 32 of 33 pts with BTC were evaluable. Investigator-assessed ORR was 41% (13/32; 95% CI, 24%-59%), with 6 of 13 responses ongoing at data cutoff, 7 of 13 pts (54%) had a DOR ≥ 6 mo. Median PFS was 7.2 mo (95% CI, 4.6-10.1 mo), and median OS was 11.3 mo (95% CI, 7.3-17.6 mo). Grade 3/4 adverse events in ≥ 3 pts were increased γ-glutamyltransferase (n = 3 [9%]) and decreased white blood cell count (n = 3 pts [9%]). Biomarker analyses demonstrate a heterogeneous genetic landscape, and suggest a higher baseline expression of MAPK pathway genes in the pts who did not respond to D + T. Conclusions: D + T demonstrated promising efficacy in pts with BTC, with a favorable safety profile. These pts should be considered for BRAF mutation analysis, and D + T should be considered for pts with BRAF V600E-mutated BTC. Clinical trial information: NCT02034110.

The Mutational Profile of Unicystic Ameloblastoma

BRAF V600E is the most common mutation in conventional ameloblastoma (AM) of the mandible. In contrast, maxillary AMs appear to harbor more frequently RAS, FGFR2, or SMO mutations. Unicystic ameloblastoma (UAM) is considered a less aggressive variant of ameloblastoma, amenable to more conservative treatment, and classified as a distinct entity. The aim of this study was to characterize the mutation profile of UAM (n = 39) and to compare it to conventional AM (n = 39). The associations between mutation status and recurrence probability were also analyzed. In the mandible, 94% of UAMs (29/31, including 8/8 luminal, 6/8 intraluminal, and 15/15 mural subtypes) and 74% of AMs (28/38) revealed BRAF V600E mutations. Among the BRAF wild-type cases, 1 UAM showed a missense SMO mutation (p.L412F), whereas 2 NRAS (p.Q61R), 2 HRAS (p.Q61R), and 2 FGFR2 (p.C383R) activating mutations were identified in AM. Of the 3 maxillary UAMs, only 1 revealed a BRAF V600E mutation. Taken together, our findings demonstrate high frequency of activating BRAF V600E mutations in both UAM and AM of the mandible. In maxillary UAMs, the BRAF V600E mutation prevalence appears to be lower as was shown for AM previously. It could therefore be argued that UAM and AM are part of the spectrum of the same disease. AMs without BRAF V600E mutations were associated with an increased rate of local recurrence (P = 0.0003), which might indicate that routine mutation testing also has an impact on prognosis.

Abstract 1839: Suppression of Spry1 sensitizes cutaneous melanoma to BRAF-targeted therapy

Abstract Background: About 50% of cutaneous melanoma (CM) harbor the activating BRAFV600E mutation, which exerts most of the oncogenic effects through the activation of the MAPK signaling pathways. BRAF inhibitors (BRAFi) have showed important clinical activities in CM, but their effectiveness is impaired by the emergence of an early drug resistance. Accordingly, a better understanding of the molecular basis of BRAFi resistance may provide insights useful to develop new therapeutic strategies in CM. Spry gene family includes four members (Spry1-4), which differ in their tissue distribution, activity and interaction partners, thus suggesting nonredundant functions. Although interactions between Spry proteins and several MAPK pathway components have been reported, the specific role of Spry1 in CM has yet to be defined. Methods: Bioinformatic analysis on data available on TCGA was performed using the pipeline suggested on the TCGA portal. Spry1 expression levels were measured in BRAFV600 mutant CM cell lines using quantitative real-time PCR (qRT-PCR) and Western blotting. CRISPR based strategy was used to knock out Spry1 gene in BRAFV600 mutant CM cell lines established in our Institution. Transfected cells were sorted into single cells and subsequently expanded. Spry1 knockout (Spry1KO) clones were validated by Sanger sequencing and Western blotting, respectively. Cell viabilities before and after BRAFi treatment were evaluated by MTT and clonogenic assays. Apoptosis was assessed by Annexin V/PI staining. Modulation of MAPK signaling pathways and apoptosis were examined by qRT-PCR and Western blotting. P53 nuclear translocation was evaluated through multispectral imaging flow cytometry analysis. Results: Specific bioinformatic analysis, using TCGA, was performed to provide more information regarding pathways associated with MAPK signaling. Our analysis revealed that Spry proteins are differentially expressed in CM. In line with these findings, Spry1 was found high expressed in a panel of CM cell lines with BRAFV600 mutation both at mRNA and protein level. Preliminary data demonstrated that the expression and/or phosphorylation of key proteins involved in MAPK signaling were modulated in Spry1KO clones with respect to the parental cell line. Furthermore, Spry1 inactivation associated with: i) enhanced activation and nuclear translocation of the tumor suppressor p53, and ii) decreased mRNA and protein levels of several antiapoptotic proteins. Of note, the treatment of Spry1KO clones with BRAFi reduced CM cell survival in clonogenic assays, and induced apoptosis in a dose- and time-dependent manner. Further studies are ongoing in order to determine the molecular changes involved in Spry1KO clones in response to BRAFi treatment. Conclusions: Our results suggest that Spry1 gene may exert oncogenic functions in CM and its upregulation can potentially contribute to BRAFi resistance. Citation Format: Giorgio Giurato, Francesca Colizzi, Aurora Rizzo, Debora Martorelli, Barbara Montico, Katy Mastorci, Dania Benedetti, Alessandro Weisz, Riccardo Dolcetti, Sigalotti Luca, Elisabetta Fratta. Suppression of Spry1 sensitizes cutaneous melanoma to BRAF-targeted therapy [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 1839.

Subependymal giant cell astrocytomas in Tuberous Sclerosis Complex have consistent TSC1/TSC2 biallelic inactivation, and no BRAF mutations.

Subependymal giant cell astrocytomas (SEGAs) are rare, low-grade glioneuronal brain tumors that occur almost exclusively in patients with tuberous sclerosis complex (TSC). Though histologically benign, SEGAs can lead to serious neurological complications, including hydrocephalus, intractable seizures and death. Previous studies in a limited number of SEGAs have provided evidence for a biallelic two-hit inactivation of either TSC1 or TSC2, resulting in constitutive activation of the mechanistic target of rapamycin complex 1 pathway. The activating BRAF V600E mutation is a common genetic alteration in low grade gliomas and glioneuronal tumors, and has been reported in SEGAs as well. In the present study, we assessed the prevalence of the BRAF V600E mutation in a large cohort of TSC related SEGAs (n=58 patients including 56 with clinical TSC) and found no evidence of either BRAF V600E or other mutations in BRAF. To confirm that these SEGAs fit the classic model of two hit TSC1 or TSC2 inactivation, we also performed massively parallel sequencing of these loci. Nineteen (19) of 34 (56%) samples had mutations in TSC2, 10 (29%) had mutations in TSC1, while 5 (15%) had no mutation identified in TSC1/TSC2. The majority of these samples had loss of heterozygosity in the same gene in which the mutation was identified. These results significantly extend previous studies, and in agreement with the Knudson two hit mechanism indicate that biallelic alterations in TSC2 and less commonly, TSC1 are consistently seen in SEGAs.

Efficacy of dabrafenib (D) and trametinib (T) in patients (pts) with BRAF V600E–mutated anaplastic thyroid cancer (ATC).

6023 Background: ATC is a rare, aggressive malignancy with a dismal prognosis. Median overall survival (OS) is < 6 mo. Combined BRAF and MEK inhibition is efficacious in BRAF V600–mutated melanoma and lung cancer. One-fourth of ATCs harbor activating BRAF V600E mutations; thus, D (BRAF inhibitor) + T (MEK inhibitor) was evaluated as a treatment for pts with BRAF V600E–mutated ATC. Methods: In this phase 2, open-label trial (NCT02034110), pts with BRAF V600E mutations in 9 rare tumor types, including ATC, received continuous D (150 mg BID) + T (2 mg QD) until unacceptable toxicity, disease progression, or death. Eligible pts had advanced or metastatic cancer with no standard-of-care treatment options. The primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DOR), progression-free survival (PFS), OS, and safety. We report data from the ATC cohort. Results: 16 pts with BRAF V600E–mutated ATC had evaluable data with a median follow-up time of 47 wk (range 4-120 wk). BRAF V600E mutations were centrally confirmed in 15/16 pts. Median age was 72 y; all 16 pts had undergone prior tumor radiation and/or surgery and 6/16 pts (38%) had received ≥1 prior line of systemic therapy. Investigator-assessed confirmed ORR was 69% (11/16; 95% CI, 41%-89%), with 7/11 responses ongoing at the time of data cut. The Bayesian estimate of ORR was 69% (95% credible interval, 47%-87%) with a 100% probability that this ORR exceeded the 15% historical RR. Median DOR, PFS, and OS were not estimable due to insufficient progression and death events. Kaplan-Meier estimates of DOR, PFS, and OS at 12 mo were 90%, 79%, and 80%, respectively. The safety population comprised 100 pts enrolled in 7/9 histologies. Among all pts, 92% had an AE. Common AEs of any grade for all histologies were fatigue (38%), pyrexia (37%), and nausea (35%). In the ATC cohort, the most common grade 3/4 events were hyponatremia (19%), pneumonia (13%), and anemia (13%). Conclusions: D+T combination therapy significantly improved outcomes in ATC with a favorable safety profile. This regimen represents a clinically meaningful therapeutic advance for pts with advanced/metastatic BRAF V600–mutated ATC. Clinical trial information: NCT02034110.

Absence of BRAF mutation in pheochromocytoma and paraganglioma.

Pheochromocytomas and Paragangliomas (PHEO/PARA) are rare endocrine tumors originating from the adrenal medulla. More than 20 genes are involved in the tumorigenesis of these tumors, but asubstantial part of the causative genetic events remains unexplained. Arecent study has reported the presence of the activating BRAF V600E mutation in PCC, suggesting arole for BRAF activation in tumor development. Other studies have not find this mutation. This study investigates the occurrence of the BRAF V600E mutation in these tumors.A cohort of 64 PHEO/PARA were screened for the BRAF V600E mutation using direct Sanger sequencing and QRT-PCR.All cases investigated displayed wild-type without V600E BRAF mutationTaken together with all previously screened tumors up to date, only 1 V600E BRAF mutation has been found among 427 PCCs. These findings imply that the V600E BRAF mutation is arare event in PHEO/PARA.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Response of metastatic glioma to vemurafenib.

Extraneural metastatic disease of glioma is rare and poses unique therapeutic challenges. Increasingly, the ability to sequence genetic alterations in tumors has allowed for the identification of common oncogenic signatures such as the activating BRAFV600E mutation and may be useful in therapeutic decision making. We report two patients with widespread aggressive gliomas whose tumors were found to express the BRAFV600E mutation and then responded robustly albeit transiently when exposed to vemurafenib. Although both patients succumbed to their disease, our results suggest that targeting BRAF might be appropriate for patients with aggressive gliomas that express this mutation.

Disseminated histiocytoses biomarkers beyond BRAFV600E: frequent expression of PD-L1.

The histiocytoses are rare tumors characterized by the primary accumulation and tissue infiltration of histiocytes and dendritic cells. Identification of the activating BRAFV600E mutation in Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH) cases provided the basis for the treatment with BRAF and/or MEK inhibitors, but additional treatment options are needed. Twenty-four cases of neoplastic histiocytic diseases [11 extrapulmonary LCH, 4 ECD, 4 extranodal Rosai-Dorfman disease (RDD), 3 follicular dendritic cell sarcoma (FDCS), 1 histiocytic sarcoma (HS) and 1 blastic plasmacytoid dendritic cell neoplasm (BPDCN)] were analyzed using immunohistochemical and mutational analysis in search of biomarkers for targeted therapy. BRAF V600E mutations were detected in 4/11 LCH and 4/4 ECD cases. A pathogenic PTEN gene mutation and loss of PTEN protein expression were identified in the case of HS. Increased expression of PD-L1 (≥2+/≥5%) was seen in 3/4 ECD, 7/8 LCH, 3/3 FDCS and 1/1 HS, with overall 81% concordance between 2 antibodies used in the study (SP142 vs. MAB1561 clone). These results show for the first time significant expression of the PD-L1 immune checkpoint protein in these disorders, which may provide rationale for addition of immune check-point inhibitors in treatment of disseminated and/or refractory histiocytoses.

Disseminated histiocytoses: Biomarkers beyond BRAFV600E.

e22207 Background: Histiocytoses are rare tumors characterized by the primary accumulation of monocytes, macrophages and dendritic cells. The most frequent subtypes are Rosai-Dorfman disease (RDD), Langerhans cell histiocytosis (LCH), Erdheim-Chester disease (ECD) and follicular dendritic cell sarcomas (FDCS). Clinical presentations range from localized to disseminated, life threatening disease. Identification of an activating BRAFV600E mutation in the majority of ECD and LCH cases provided the basis for the use of BRAF and MEK inhibitors in these patients. Responses have been reported with BRAF inhibitors, but no curative treatment has yet been developed. Thus, additional targeted therapies are needed. Methods: Twenty-two cases (M:F = 13:9, age range 2-65 years) of histiocytic diseases (4 RDD, 10 LCH, 4 ECD, 2 FDCS, 1 histiocytic sarcoma [HS] and 1 blastic plasmacytoid dendritic cell neoplasm [BPDCN]) were analyzed using immunohistochemical and molecular methods (cobas 4800 BRAFV600 Mutation Test and The...

Activating BRAF V600E mutation in aggressive pediatric Langerhans cell histiocytosis: demonstration by allele-specific PCR/direct sequencing and immunohistochemistry.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disease originating from cells characterized by antigen-presenting Langerhans cell phenotype. The clinical spectrum of LCH is highly variable including localized and disseminated forms mostly occurring in children. Recently, about 60% of LCHs were reported to carry the activating BRAF mutation V600E. In our retrospective study, we evaluated the occurrence and prognostic impact of the V600E mutation in formaldehyde-fixed, paraffin-embedded samples from 15 pediatric LCH cases treated at our institution. Allele-specific polymerase chain reaction (PCR) and direct sequencing were used to demonstrate the presence of V600E mutation, and immunohistochemistry (IHC) using the mutant protein-specific VE1 antibody clone was performed to confirm mutant BRAF protein expression. Eight of 15 (53.3%) cases proved to be BRAF mutants by any of the methods applied, with a single case showing a discrepancy (PCR negative/IHC positive). Four of the BRAF-mutant cases (50.0%) showed refractory disease and progressed to death within 43 months, whereas the remaining mutant cases were stable and responded well to therapy. Wild-type BRAF cases (7/15, 46.6%) with generally comparable initial presentation were all treated successfully. In conclusion, activating V600E BRAF mutation can be frequently demonstrated in pediatric LCH by both allele-specific PCR and IHC. Unfavorable risk cases potentially also responding to BRAF-inhibitory therapy can be identified by mutation testing using archival formaldehyde-fixed, paraffin-embedded tumor samples.

Dabrafenib in Patients with Braf V600E-Mutant Advanced Non-Small Cell Lung Cancer (Nsclc): a Multicenter, Open-Label, Phase Ii Trial (Brf113928)

ABSTRACT Aim: Activating BRAF V600E mutations in NSCLC are present in ≈1.5% of tumors, primarily adenocarcinomas, offering an opportunity to test targeted therapy for this disease. Methods: This single-arm, 2-stage design, Phase II study (NCT01336634) enrolled Stage IV BRAF V600E-mutant NSCLC patients (pts) determined by local laboratory testing. Pts received dabrafenib 150 mg twice daily. Primary endpoint was investigator-assessed overall response rate (ORR) per RECIST 1.1. Results: As of 30 April 2014, 84 pts (female 52%, median age 66 years [range 28–89], ECOG 0–1 86%, Asian 21%, never-smoker 37%, adenocarcinoma histology 96%) were enrolled from August 2011. Median duration on treatment was 4.3 months (range, 0.3–25.2) with 21 (25%) pts still on treatment. Six pts had not received any prior regimen for metastatic disease (first-line pts), 40 pts had one line and 38 pts had received ≥2 lines (range 2–9). ORR for 78 pts with ≥ one line of prior therapy (second-line plus pts) was 32% (n = 25, all partial responses [PRs]; 95% confidence interval [CI] 22–44%) and disease control rate (DCR) for >12 weeks was 56% (95% CI 45–68%), median duration of response (mDoR) was 11.8 months (95% CI 5.4–not reached) with 48% of responders progressed. Based on assessment by independent review committee (IRC), 64 second-line plus pts had measurable disease; ORR and DCR were 28% (n = 18; 95% CI 18–41%) and 52% (95% CI 39–64%) respectively, and mDoR has not been reached. Among the six first-line pts, three pts had PR, four pts had measurable disease based on IRC with three PRs. Most common (>25%) adverse events (AEs) were pyrexia (36%), asthenia (30%), hyperkeratosis (30%), decreased appetite (29%), nausea (27%), cough (26%), fatigue (26%) and skin papilloma (26%). Cutaneous squamous-cell carcinomas, including keratoacanthoma, were reported in 18%. Grade ≥3 AEs occurred in 45% with one event of grade 5 haemorrhage intracranial. Conclusions: Treatment of BRAF V600E mutated advanced NSCLC pts with dabrafenib demonstrated significant antitumor activity with durable objective responses and acceptable safety profile. Dabrafenib is the first drug of its class to show activity in a prospective trial of NSCLC with BRAF mutations. Disclosure: D. Planchard: has received personal fees for participating in advisory boards from AstraZeneca, BI, Lilly, Novartis, Pfizer, Roche and BMS; E. Quoix: has received honoraria for inclusion of patients into study from GSK; G.J. Riely: G.J.R.'s institution received a grant from GSK. G.J.R. received personal fees from Daiichi, Novartis and Abbott for consulting; F. Barlesi: has received a grant and personal fees from GSK; M.A. Socinski: received clinical trial support from GSK; C. Tucker: is an employee of GSK. C.T. owns stock in GSK; B. Ma: is an employee of GSK; B. Mookerjee: is an employee of GSK. B.M. owns stocks in Incyte Corporation and AstraZeneca; C.M. Curtis, Jr: is an employee of GSK. C.M.C.Jr owns stock in GSK; B.E. Johnson: has received personal fees from Roche/Genentech for being an expert witness; Roche/Genentech manufacture a competitor of dabrafenib (vemurafenib). B.E.J. has received personal fees from KEW Group for consultancy.All other authors have declared no conflicts of interest.